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A technology of earthworm protein and extraction method, applied in the field of protein extraction, can solve the problems of being susceptible to acid, alkali, ionic strength, temperature, affecting drug efficacy, instability of earthworm protein, etc., to improve purity, prolong time, prepare simple method effect
Active Publication Date: 2020-07-28
北京七清堂中医研究院
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Problems solved by technology
However, due to the instability of ground dragon protein, it is easily affected by acid, alkali, ionic strength, temperature, etc., so the traditional method of decocting it in water affects its efficacy.
Method used
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Embodiment 1
[0020] Embodiment 1 A kind of extraction method of earthworm protein
[0021] A method for extracting ground dragon protein, comprising the following steps:
[0022] 1) Take living earthworm, remove impurities, and pulverize after being treated with liquid nitrogen;
[0023] 2) Add 3 times the volume of absolute ethanol, soak at 0°C for 16 hours, centrifuge at 8000r / min for 8 minutes, and remove the supernatant;
[0024] 3) Add a perchloric acid solution with a concentration of 0.05%, adjust the pH value to 3.5, extract at 15°C for 20 minutes, centrifuge at a speed of 8000r / min for 8 minutes, and extract the supernatant;
[0025] 4) Add 50 mg of activated carbon to each milliliter of supernatant, stir and let it stand for 5 minutes, centrifuge at a speed of 8000r / min for 8 minutes, and extract the supernatant;
[0026] 5) Adjust the pH value of the supernatant to 5 with phosphate buffer, add sodium sulfate powder to make the saturation of sodium sulfate 40%, treat for 10 min...
Embodiment 2
[0028] Embodiment 2 A kind of extraction method of earthworm protein
[0029] A method for extracting ground dragon protein, comprising the following steps:
[0030] 1) Take living earthworm, remove impurities, and pulverize after being treated with liquid nitrogen;
[0031] 2) Add 4 times the volume of absolute ethanol, soak at 4°C for 24 hours, centrifuge at 12000r / min for 8 minutes, and remove the supernatant;
[0032] 3) Add a perchloric acid solution with a concentration of 0.1%, adjust the pH value to 4.5, extract at 20°C for 30 minutes, centrifuge at a speed of 12000r / min for 8 minutes, and extract the supernatant;
[0033] 4) Add 100 mg of activated carbon to each milliliter of supernatant, stir and let it stand for 10 minutes, centrifuge at a speed of 12000 r / min for 8 minutes, and extract the supernatant;
[0034] 5) Adjust the pH value of the supernatant to 6 with phosphate buffer, add sodium sulfate powder to make the saturation of sodium sulfate 55%, treat for 1...
Embodiment 3
[0036] Embodiment 3 A kind of extraction method of earthworm protein
[0037] A method for extracting ground dragon protein, comprising the following steps:
[0038] 1) Take living earthworm, remove impurities, and pulverize after being treated with liquid nitrogen;
[0039] 2) Add 3 times the volume of absolute ethanol, soak at 2°C for 20 hours, centrifuge at 10,000 r / min for 5 minutes, and remove the supernatant;
[0040] 3) Add a perchloric acid solution with a concentration of 0.075%, adjust the pH value to 4.0, extract at 18°C for 25 minutes, centrifuge at a speed of 10000r / min for 5 minutes, and extract the supernatant;
[0041] 4) Add 75 mg of activated carbon to each milliliter of supernatant, stir and let it stand for 8 minutes, centrifuge at a speed of 10,000 r / min for 5 minutes, and extract the supernatant;
[0042] 5) Adjust the pH value of the supernatant to 5.5 with phosphate buffer, add sodium sulfate powder to make the saturation of sodium sulfate 50%, trea...
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Abstract
The invention discloses an extraction method of earthworm protein. The extraction method comprises the following steps of 1), taking a living earthworm, removing an impurity, treating the earthworm by using liquid nitrogen, and then crushing the earthworm; 2), adding absolute ethyl alcohol into the earthworm, soaking the earthworm, carrying out centrifugation, and removing supernatant fluid; 3), adding a perchloric acid solution into an obtained first mixture, carrying out leaching and the centrifugation, and abstracting the supernatant fluid; 4), adding activated carbon into the supernatant fluid, agitating and standing an obtained second mixture, carrying out the centrifugation, and abstracting the supernatant fluid; 5), regulating the pH (potential of Hydrogen) value of the supernatant fluid to be 5 to 6 by using a phosphate buffer solution, adding sodium sulfate powder into an obtained third mixture, and carrying out deposition, so as to obtain an earthworm protein crude extract; 6), dissolving the earthworm protein crude extract by using 20mM of Tris-HCl buffer solution with the pH of 7.2, carrying out purification by using a DEAE (Diethylaminoethyl)-Sephadex ion exchange method, collecting an eluted component, and subjecting the eluted component to dialysis, concentration and freeze drying, so as to obtain the earthworm protein. A preparation method provided by the invention is simple, is short in period, is good in extraction effect, and is high in purity; the obtained earthworm protein can be used for prolonging a thrombin time and effectively decreasing the EOS (Eosinophil) sum in BALF (Bronchoalveolar Lavage Fluid) for a mouse.
Description
technical field [0001] The present invention relates to the field of protein extraction. More specifically, it relates to a method for extracting earthworm protein. Background technique [0002] Earthworm is one of the important animal medicinal materials. Earthworm protein is extracted from Earthworm, which contains collagenase, fibrinolytic enzyme, lumbrokinase, nucleic acid, trace elements and other components. Its molecular weight is 5000-10000, which is a short-chain small molecular substance. Modern studies have shown that ground dragon protein contains collagenase, fibrinolytic enzyme, nucleic acid, trace elements and other components, which have a wide range of effects on the blood coagulation system and fibrinolytic system in the body, which can significantly reduce the platelet adhesion rate and prolong the formation of thrombus in the body. It can dissolve blood clots in the body, increase cerebral blood flow, reduce cerebrovascular resistance, and improve the d...
Claims
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Application Information
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