Enantio-isopimarane diterpenoid with nerve cell protection activity as well as preparation method and application of enantio-isopimarane diterpenoid
An isopimarane, neurodegenerative technology, applied in the direction of organic active ingredients, medical preparations containing active ingredients, nervous system diseases, etc., can solve problems such as threats to human health and daily life, unclear etiology, incurable, etc. Achieving the effect of good nerve cell protection activity and inhibition of accumulation
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Embodiment 1
[0033] The preparation of embodiment 1 compound EP-1
[0034] The preparation process of compound EP-1 of the present invention is as follows:
[0035] (1) after pulverizing the dry root (16.5kg) of Euphorbia jingle, soak and extract with methanol at room temperature for 3 times, each time soaking with 40L methanol for 7 days, concentrating under reduced pressure to obtain 3.6kg of total extract;
[0036] (2) disperse the crude extract in water and extract with ethyl acetate to obtain ethyl acetate extract;
[0037] (3) Utilize macroporous resin to roughly segment ethyl acetate extract, and use methanol / water with volume ratio of 0:100, 30:70, 50:50, 80:20, 100:0 as mobile phase for gradient Elution, collect each elution fraction, carry out TLC detection with dichloromethane / ethyl acetate with a volume ratio of 5:1, combine and collect R f Component EPR-3 with a value of 0.5-0.6;
[0038] (4) The component EPR-3 is separated by MCI column chromatography, with a volume ratio...
Embodiment 2
[0047] The neuroprotective activity of embodiment 2EP-1
[0048] After digesting well-grown PC12 cells in the logarithmic growth phase, make a cell suspension, press 1×10 4 cells / mL were planted in a 96-well cell culture plate, with 100 μL medium per well, at 37°C, 5% CO 2 Cultivate for 24 hours under the condition of , absorb the old culture medium, set up the blank control group, the model group and the sample experiment group respectively, wherein the sample experiment group adds fresh culture medium containing the test sample (EP-1), and the final concentration of the sample is 50 μM, the blank control group and the model group were added with an equal volume of fresh medium. After 24 hours of pre-cultivation, the old culture medium was sucked away, and then fresh culture medium containing hydrogen peroxide was added to the model group and the sample experimental group respectively, the final concentration of hydrogen peroxide was 500 μM, and the same volume of culture me...
Embodiment 3
[0051] Embodiment 3 ROS index measurement
[0052] PC12 cells in good growth state were divided into 1×10 4 cells / mL were planted in a 6-well plate, with 2 mL of medium per well, at 37°C, 5% CO 2 Cultivate for 24 hours under the condition of 24h, suck away the old culture medium, set blank control group, model group and sample experiment group (low dose group, high dose group) respectively, wherein the sample experiment group adds the test sample (EP-1) Fresh culture medium, sample final concentration is 20 μ M (low dosage group), 40 μ M (high dosage group), blank control group and model group add the culture medium of same volume respectively. After 24 hours of pre-cultivation, the old medium was sucked away, and fresh medium containing hydrogen peroxide was added to the model group and the sample experimental group respectively, with a final concentration of hydrogen peroxide of 500 μM, and an equal volume of medium was added to the blank control group. Continue to incubat...
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