A kind of preparation method of triptolide derivative and its product and application
A technology for triptolide and derivatives, which is applied to the preparation of triptolide derivatives and their products and application fields, and can solve the problems of poor selectivity, many by-products, complicated processes and the like
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[0048] The present invention relates to a kind of preparation method of triptolide derivative, comprising the following steps:
[0049] Step 1: Take a transformed strain stored at 4°C and cultivated on a potato slant medium, place it in a constant temperature incubator at 25°C for 7 days, and blow off the spores on the slope with an appropriate amount of sterile water to make a single Adjust the concentration of the spore suspension to 107-108 / mL to obtain the seed spore liquid;
[0050] Step 2: Inoculate the spore liquid obtained in step 1 on the potato liquid medium with an inoculum amount of 100ml and 5mL, and cultivate it for 12 hours at a pH of 6.0, a temperature of 30°C±1°C, and a shaking speed of 200r / min; press 0.1 ~4ug inoculum, add various combined elicitors in different proportions, continue to cultivate for 12 hours, then add triptolide according to the inoculum of 1~10ug, and then continue to cultivate for 5 days, detect various triptolide derivatives every day W...
Embodiment 1
[0084] Example 1: Preparation of Triptolide Derivatives
[0085] 1. Preparation of 6α-hydroxytriptolide
[0086] Inoculate 5mL (108 cells / mL) spore liquid of Cunninghamella blakesleana AS3.970 in 100mL potato liquid medium, under the conditions of pH to 6.0, temperature 30°C±1°C, and shaking speed 200r / min After culturing for 12 hours, add methyl jasmonate (MeJA, 0.35mmol / mL), dexamethasone (DXM) and β-cyclodextrin at a weight ratio of (3:2:1) according to the inoculum size of 0.2ug (BCD, 0.51mmol / mL), after continuing to cultivate for 12h, add triptolide according to the inoculum amount of 5%, then continue to cultivate for 5 days, stop the cultivation, then centrifuge the filtrate at 8000r / min for 10min to remove the precipitate, The filtrate was extracted 3 times with an equal volume of ethyl acetate, and the extract was evaporated to dryness, dissolved in 2m L of acetone, and then separated by a chromatographic silica gel column. , 6:1, 4:1, 2:1, 1:1, 1:2, 1:4, 1:6, 1:8 ...
Embodiment 2
[0117] Embodiment two, pharmacodynamics evaluation experimental example
[0118] In the following experimental examples, the test sample is provided by the preparation method embodiment of the present invention, and the precursor compound triptolide is used as a positive control.
[0119] 1. The growth inhibitory effect of 12 compounds of the present invention on human lung cancer cell A549 cells cultured in vitro
[0120] Methods: Human A549 lung cancer cells were cultured in DMEM medium (Gibco, USA) containing 10% fetal bovine serum at 37°C, 5% CO 2 , tumor cells 0.7x10 4 / well was inoculated in a 96-well plate, and after 24 hours, the compound diluted with dimethyl sulfoxide (200uM) and PBS solution was added to make the final concentration of the culture medium 10 -4 、10 -5 、10 -6 、10 -7 、10 -8 M, after 72 hours of treatment, discard the culture medium, fix the cells with 10% cold trichloroacetic acid, stain with sulforhodamine B (SRB) solution, wash away the unbound...
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