Conversion method for euphorbia lathyris phorbol derivative and use thereof for preparing antitumor drug

Abstract

The invention belongs to the technical field of the biological medicine, and relates to a microbial conversion method for a euphorbia lathyris diterpene compound. The method comprises the following steps: performing the microbial conversion to euphorbia lathyris phorbol and 7-hydroxy euphorbia lathyris phorbol by using Nocard's bacillus (i) Nocardia sp. ( / i) NRRL 5646, and preparing 3-carbonyl euphorbia lathyris phorbol and 7-acetoxyl euphorbia lathyris phorbol derivatives. Proved by the experiment, the polarity of the 3-carbonyl euphorbia lathyris phorbol in the derivative is reduced. Compared with a substrate, the derivative has the better cytotoxic activity. The derivative is an antitumor lead compound with development value.

Description

technical field

[0001] The invention belongs to the field of biopharmaceuticals, and relates to a method for transforming caperia diterpenoid compounds, in particular to using known microorganisms to perform microbial transformation of caperia diterpene alcohols and derivatives thereof to prepare caperia caperis with anticancer activity Diterpenoid derivatives. Background technique

[0002] It is disclosed in the prior art that the diterpene-type compound of Caperia spp. belongs to the natural product of the diterpene skeleton, and has obvious anti-tumor activity and anti-tumor multidrug resistance (MDR). The study of structure-activity relationship shows that the difference of substituents on the core can obviously change the antitumor activity and MDR activity of this kind of compound.

[0003] Some studies have disclosed that the diterpenoid compounds of Caperia chinensis were isolated for the first time from Euphorbialathyris L. (Tetrahedron Letters, 1971, 18: 1325-1328...

Examples

Embodiment 1

[0023] Example 1 Preparation of 3-carbonyl Capper diterpene alcohol (1)

[0024] The two-step activation method was used to activate the strains, and the obtained seed liquid was inoculated into 250 mL potato medium with a volume of 1 L Erlenmeyer flask at a volume ratio of 3%, and the shake flask was cultured at 28 °C and 180 rpm for 72 h. Add 4 mg / mL Capper diterpene alcohol ethanol solution to each bottle of activated bacterial liquid, and the final concentration is 0.1 mg / mL. After culturing under the same conditions for 72 h, 1.5 times the volume of ethyl acetate was added to extract three times (about 1200 mL), and the organic layers were combined. The organic layer with residual bacteria was suction-filtered through a Buchner funnel to obtain a clarified extract, and then the ethyl acetate was recovered under reduced pressure to obtain a fermentation broth extract;

[0025] The extract of the fermentation broth was dissolved in a small amount of ethyl acetate, mixed wi...

Embodiment 2

[0026] Example 2 Preparation of 7β-acetoxy Capper diterpene alcohol (2)

[0027] The two-step activation method was used to activate the strains, and the obtained seed liquid was inoculated into 250 mL potato medium with a volume of 1 L Erlenmeyer flask at a volume ratio of 3%, and the shake flask was cultured at 28 °C and 180 rpm for 72 h. Add 5 mg / mL ethanol solution of 7β-hydroxycapricorn diterpene alcohol to each bottle of activated bacterial liquid, and the final concentration is 0.1 mg / mL. After culturing under the same conditions for 72 h, 1.5 times the volume of ethyl acetate was added to extract three times (about 1200 mL), and the organic layers were combined. The organic layer with residual bacteria was pumped through a Buchner funnel to obtain a clarified extract, and then the ethyl acetate was recovered under reduced pressure to obtain a fermentation broth extract;

[0028] The extract of the fermentation broth was dissolved in a small amount of ethyl acetate, mi...

Embodiment 3

[0032] Example 3 Antitumor Activity Experiment of 3-Carbonyl Scaper Diterpene Alcohol (1)

[0033]Human breast cancer cells (MCF-7) and human colon cancer cells (Caco-2) were used as models. MCF-7 cell culture medium is RPMI-1640 medium containing 10% calf serum, and Caco-2 cell culture medium is DMEM medium containing 10% calf serum and 1% glutamic acid; cells are treated with 10% trypsin After digestion, the medium was blown to make a single cell suspension, and after counting, the cell concentration was adjusted to 10 5 cells / mL, the cells were seeded in a 96-well plate with a volume of 100 mL per well. After 37℃, 5% CO 2 After incubation for 24 h, the supernatant was discarded, and 200 mL of drug-containing medium was added to each group of cells, and 3 replicate wells were set up for each concentration; the experimental group was added with different concentrations of compound 1 and DMSO solution of Capper diterpene alcohol (100.0 , 80.0, 50.0, 40.0, 25.0, 20.0, 12.5, ...

technical field

[0001] The invention belongs to the field of biopharmaceuticals, and relates to a method for transforming caperia diterpenoid compounds, in particular to using known microorganisms to perform microbial transformation of caperia diterpene alcohols and derivatives thereof to prepare caperia caperis with anticancer activity Diterpenoid derivatives. Background technique

[0002] It is disclosed in the prior art that the diterpene-type compound of Caperia spp. belongs to the natural product of the diterpene skeleton, and has obvious anti-tumor activity and anti-tumor multidrug resistance (MDR). The study of structure-activity relationship shows that the difference of substituents on the core can obviously change the antitumor activity and MDR activity of this kind of compound.

[0003] Some studies have disclosed that the diterpenoid compounds of Caperia chinensis were isolated for the first time from Euphorbialathyris L. (Tetrahedron Letters, 1971, 18: 1325-1328...

Examples

Embodiment 1

[0023] Example 1 Preparation of 3-carbonyl Capper diterpene alcohol (1)

[0024] The two-step activation method was used to activate the strains, and the obtained seed liquid was inoculated into 250 mL potato medium with a volume of 1 L Erlenmeyer flask at a volume ratio of 3%, and the shake flask was cultured at 28 °C and 180 rpm for 72 h. Add 4 mg / mL Capper diterpene alcohol ethanol solution to each bottle of activated bacterial liquid, and the final concentration is 0.1 mg / mL. After culturing under the same conditions for 72 h, 1.5 times the volume of ethyl acetate was added to extract three times (about 1200 mL), and the organic layers were combined. The organic layer with residual bacteria was suction-filtered through a Buchner funnel to obtain a clarified extract, and then the ethyl acetate was recovered under reduced pressure to obtain a fermentation broth extract;

[0025] The extract of the fermentation broth was dissolved in a small amount of ethyl acetate, mixed wi...

Embodiment 2

[0026] Example 2 Preparation of 7β-acetoxy Capper diterpene alcohol (2)

[0027] The two-step activation method was used to activate the strains, and the obtained seed liquid was inoculated into 250 mL potato medium with a volume of 1 L Erlenmeyer flask at a volume ratio of 3%, and the shake flask was cultured at 28 °C and 180 rpm for 72 h. Add 5 mg / mL ethanol solution of 7β-hydroxycapricorn diterpene alcohol to each bottle of activated bacterial liquid, and the final concentration is 0.1 mg / mL. After culturing under the same conditions for 72 h, 1.5 times the volume of ethyl acetate was added to extract three times (about 1200 mL), and the organic layers were combined. The organic layer with residual bacteria was pumped through a Buchner funnel to obtain a clarified extract, and then the ethyl acetate was recovered under reduced pressure to obtain a fermentation broth extract;

[0028] The extract of the fermentation broth was dissolved in a small amount of ethyl acetate, mi...

Embodiment 3

[0032] Example 3 Antitumor Activity Experiment of 3-Carbonyl Scaper Diterpene Alcohol (1)

[0033]Human breast cancer cells (MCF-7) and human colon cancer cells (Caco-2) were used as models. MCF-7 cell culture medium is RPMI-1640 medium containing 10% calf serum, and Caco-2 cell culture medium is DMEM medium containing 10% calf serum and 1% glutamic acid; cells are treated with 10% trypsin After digestion, the medium was blown to make a single cell suspension, and after counting, the cell concentration was adjusted to 10 5 cells / mL, the cells were seeded in a 96-well plate with a volume of 100 mL per well. After 37℃, 5% CO 2 After incubation for 24 h, the supernatant was discarded, and 200 mL of drug-containing medium was added to each group of cells, and 3 replicate wells were set up for each concentration; the experimental group was added with different concentrations of compound 1 and DMSO solution of Capper diterpene alcohol (100.0 , 80.0, 50.0, 40.0, 25.0, 20.0, 12.5, ...

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