Method of extracting immunologic adjuvant component from cochinchina momordica seed
A technology of extract and immune adjuvant of wood turtle seed, applied in medical preparations containing active ingredients, organic active ingredients, antibody medical ingredients, etc., can solve the problem of low yield
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Embodiment 1
[0015] Preparation of the extract of the fruit of the wood turtle: 3000g of the fruit of the wood turtle, crushed, degreased with petroleum ether 3 times, 10.5L each time, a total of 601.5g of lipids were removed. The residue was reflux extracted with 70% ethanol at 60°C for 5 times, 12 L each time, and the extracts were combined. Concentrate under reduced pressure, and evaporate the solvent to obtain 175 g of brown solid, which is dissolved in 1.8 L of water, and the solution is centrifuged to precipitate (1600 g). Take the supernatant and pass it through a D101 adsorption macroporous resin column, first elute with water and 30% ethanol, and then elute with 60% ethanol, the amount of each eluent used is 2 times the column volume. Collect 60% ethanol eluent, and evaporate the solvent to obtain 26 g of light yellow powder, with a yield of 8.67 g / kg crude drug.
Embodiment 2
[0017] Analysis of the properties of the fruit of the fruit of the wood turtle: in order to confirm the properties of the fruit of the fruit of the plant, and whether it contains immune adjuvant active ingredients such as triterpene saponins, the following tests are carried out.
[0018] 1. Molish reaction: Prepare the components into a 0.05% aqueous solution, take 2 mL, add a few drops of 10% α-methylnaphthol ethanol solution, and then add 1 mL of concentrated sulfuric acid, a purple ring appears.
[0019] 2. Libermann-burchard reaction: Dissolve 10 mg of the component in 20 mL of n-butanol, take 2 mL, add 1 mL of glacial acetic acid without shaking, add 1 mL of concentrated sulfuric acid, the bottom of the test tube turns brown, and after standing for 3 to 4 hours, it becomes maroon.
[0020] 3. Foam reaction: Prepare the components into a 0.05% aqueous solution, take 2 mL, shake the test tube vigorously for 1 minute, foam appears, and after standing for 15 minutes, there is...
Embodiment 3
[0024] Experiment on the Immunological Adjuvant Activity of the Extract of Squilla Sinensis
[0025] Materials and methods: ICR mice were randomly divided into 3 groups (6 mice / group): (1) neck subcutaneous injection containing 10 μg ovalbumin (OVA); (2) OVA+50 μg extract A (prepared by the method of the present invention) ; (3) OVA+50 μg extract B (prepared according to the existing method of publication number: CN1748725). Two weeks later, the same method was used to boost the immunization once.
[0026] Indirect ELISA method for detection of anti-OVA IgG in serum: add 100 μl of coating solution (0.05mol / L carbonate buffer solution containing 5 μg OVA / ml) to each well of a 96-well ELISA plate, incubate at 4°C for 18 hours, wash with (PH7.4 phosphate buffer saline PBS) washed 3 times, 3min each time. Add 300 μl of PBS blocking solution (0.01mol / L, pH 7.4) containing 1% calf serum to each well, incubate at 37° C. for 1 hour, and wash with washing solution 3 times, 3 minutes ...
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