Fresh earthworm extract, extraction method and application thereof, and fresh earthworm extract preparation

Abstract

The invention belongs to the technical field of biological extraction, and particularly relates to a fresh earthworm extract, an extraction method and application thereof and a fresh earthworm extract preparation. The extraction method comprises the following steps: soaking fresh earthworms in a buffer solution, homogenizing and filtering to obtain a crude enzyme solution; mixing the crude enzyme liquid with ammonium sulfate, taking precipitate, dialyzing, centrifuging, and collecting supernate; finally, the supernate is purified through an anion exchange column and a gel column in sequence, eluent is collected and dried, and the trehalose is obtained, and the buffer solution is a mixture of Tris hydrochloride and a trehalose solution with the volume ratio being (5-8): (1-2). According to the method disclosed by the invention, the yield of the fresh earthworm extract and the enzyme activity of lumbrukinase are obviously improved by utilizing the synergistic effect of the Tris hydrochloride and the trehalose solution, and the extraction of the high-quality fresh earthworm extract is realized. In the purification process, a 330 alkalescence epoxy anion exchange column and a gel column are selected, the resolution ratio of protein is high, the operation is simple, and industrial amplification production is easy.

Description

Technical Field

[0001] The invention belongs to the technical field of biological extraction, and in particular relates to a fresh earthworm extract, an extraction method and application thereof, and a fresh earthworm extract preparation. Background Art

[0002] Earthworm is one of the traditional Chinese medicines in my country. It has the effects of anticoagulation, promoting fibrinolysis and improving blood circulation. The effects of earthworm: clearing away heat, calming wind, calming asthma, unblocking meridians and diuresis. Modern pharmacological research has also proved that earthworm has a variety of pharmacological effects such as antipyretic, sedative, anticonvulsant, calming asthma and lowering blood pressure. For example, it can treat high fever and convulsions; treat asthma and asthmatic bronchitis, and treat hypertension. Earthworm has a hypotensive effect, is effective for primary hypertension, treats manic psychosis, treats rheumatoid arthritis and sequelae of stroke.

[0003] Clinically, its extract has a good anticoagulant effect, can reduce blood viscosity, inhibit thrombosis, and is mainly used to prevent and treat cerebral ischemic diseases and cardiovascular diseases. Previous studies have shown that the main components of earthworm anticoagulant active substances are earthworm fibrinolytic enzymes, earthworm kinase, earthworm collagenase and free amino acids.

[0004] Chinese invention patent publication number CN115869341A discloses an application of an earthworm extract in the preparation of an anti-fatigue product and medicines, health products and foods using the extract. The earthworm extract extracted from Eisenia fetida by an organic solvent reflux method in the invention is used independently to exert anti-fatigue effects. The earthworm extract of the invention can significantly prolong the weighted swimming time of mice, and can significantly reduce the blood lactic acid content and urea nitrogen content, while increasing the liver glycogen content, having a good anti-fatigue effect, and is suitable for the preparation of anti-fatigue drugs, health products or foods. However, the preparation process of the earthworm extract also uses organic solvents such as petroleum ether, chloroform and methanol, and the processing cost is high.

[0005] Another Chinese invention patent publication number CN114246885A discloses a preparation method and application of a ground dragon extract, and the specific steps are as follows: Step 1, fresh ground dragon processing: (1) take fresh ground dragon and rinse off the impurities on the surface of the ground dragon with clean water, and then put it into warm water to wash off the mucus on the surface of the ground dragon; (2) soak the ground dragon in salt water for 2-3 hours to purify the impurities in its body, and then rinse off the salt water on its surface with clean water; (3) cut the washed ground dragon into sections, lay it flat and dry it in the sun to make dried ground dragon. This invention uses petroleum ether, chloroform and methanol as organic solvents, and extracts and prepares ground dragon extract by reflux extraction. The bound lipids in the ground dragon are freed, and at the same time, its affinity with polar lipids such as phospholipids is increased, thereby effectively extracting all lipid substances, thereby improving the antihypertensive effect of the ground dragon extract. However, this preparation method not only uses organic solvents such as petroleum ether, chloroform and methanol, but also has high water treatment costs and a low yield of ground dragon extract.

[0006] Another Chinese invention patent publication number CN108354949A discloses a preparation method for earthworm extract and its application. The preparation method includes the steps of crude extraction by immersion in anhydrous ethanol, preliminary purification by MCI column, purification by dextran gel LH-20 and decolorization and deodorization by Soxhlet extraction. The method avoids the problem that the earthworm extract is easily bred by bacteria and produces secondary metabolites due to long-term immersion in water during the water extraction process, and has more effective ingredients; the separation effect is good, and the obtained earthworm extract is slightly yellowish and has no fishy smell when dissolved in water. It has strong activity and stable quality in terms of anti-oxidation, anti-melanoma cell and anti-inflammatory aspects, but the purification process of the preparation method is complicated, which is not conducive to industrial production.

[0007] In view of this, there is an urgent need in the art to develop a method for extracting fresh earthworm extracts with higher yield, simple process, no organic solvents and low cost. Summary of the invention

[0008] In view of the problems existing in the prior art, the present invention provides a fresh earthworm extract, an extraction method and application thereof, and a fresh earthworm extract preparation.

[0009] To achieve the above purpose, the technical solution adopted by the present invention is as follows:

[0010] A method for extracting fresh earthworm extract comprises the following steps:

[0011] (1) First, soak fresh earthworms in a buffer solution, homogenize, and filter to obtain a crude enzyme solution;

[0012] (2) Mixing the crude enzyme solution with ammonium sulfate, taking the precipitate, dialyzing, centrifuging, and collecting the supernatant;

[0013] (3) Finally, the supernatant is purified by anion exchange column and gel column in sequence, and the eluate is collected and dried to obtain:

[0014] Wherein, the buffer solution in step (1) is a mixture of Tris hydrochloride and trehalose solution in a volume ratio of 5-8:1-2.

[0015] More preferably, the buffer solution is a mixture of Tris hydrochloride and trehalose solution in a volume ratio of 6-8:1.5-2.

[0016] Further preferably, the buffer solution is a mixture of Tris hydrochloride and trehalose solution in a volume ratio of 4:1.

[0017] Preferably, the pH of the Tris hydrochloride is 7.4-7.8, and the mass concentration of the trehalose solution is 10-15%.

[0018] More preferably, the pH of the Tris hydrochloride is 7.6-7.8, and the mass concentration of the trehalose solution is 12-15%.

[0019] Further preferably, the pH of the Tris hydrochloride is 7.8, and the mass concentration of the trehalose solution is 15%.

[0020] Preferably, the mass volume ratio of the fresh earthworm and the buffer solution is 1kg:3-7L.

[0021] More preferably, the mass volume ratio of the fresh earthworm and the buffer solution is 1kg:5L.

[0022] Preferably, the soaking temperature in step (1) is 20-35° C., and the soaking time is 30-60 min.

[0023] More preferably, the soaking temperature is 25° C. and the soaking time is 40 min.

[0024] Preferably, the homogenization speed in step (1) is 8000-10000 rpm, and the homogenization time is 20-30 min.

[0025] More preferably, the homogenization speed is 10000 rpm and the homogenization time is 25 min.

[0026] Preferably, the filtration in step (1) uses an ultrafiltration membrane with a molecular weight cutoff of 30k-50k Da.

[0027] More preferably, the filtration uses an ultrafiltration membrane with a molecular weight cut-off of 30 kDa.

[0028] Preferably, the mass concentration of ammonium sulfate in the crude enzyme solution in step (2) is 30-40%.

[0029] More preferably, the mass concentration of ammonium sulfate in the crude enzyme solution is 35-40%.

[0030] Further preferably, the mass concentration of ammonium sulfate in the crude enzyme solution is 38%.

[0031] Preferably, the mixing temperature in step (2) is 2-5°C, the mixing time is 4-6 hours, and after the mixing is completed, centrifugation is required at 5000-8000 rpm for 15-25 minutes.

[0032] More preferably, the mixing temperature is 4° C. and the mixing time is 6 h.

[0033] Preferably, the dialysis process in step (2) comprises adding a phosphate buffer having a pH value of 7.6-7.8 to the precipitate to obtain a mixture, and then dialyzing the mixture in a phosphate buffer having a pH value of 7.6-7.8.

[0034] Preferably, the centrifugal speed in step (2) is 6000-8000 rpm, and the centrifugal time is 15-25 min.

[0035] More preferably, the centrifugal speed is 8000 rpm and the centrifugal time is 20 min.

[0036] Preferably, the anion exchange column in step (3) is a 330 weakly basic epoxy anion exchange column.

[0037] Preferably, the gel column in step (3) comprises a dextran gel column and a Q gel column.

[0038] Preferably, the model of the dextran gel column is Sephadex G-25 or Sephadex G-75.

[0039] Preferably, the model of the Q gel column is Q-Sepharose 6FF or Q-Beads 6FF.

[0040] Preferably, the purification process in step (3) comprises first filtering the supernatant through a 0.45 μm filter head to remove impurities, then loading 5-10 column volumes of the supernatant onto an anion exchange column and a gel column in sequence, and eluting with a 0.3-0.9 M sodium chloride solution.

[0041] Preferably, the eluate in step (3) needs to be concentrated by an ultrafiltration membrane with a molecular weight cutoff of 2k-3k Da, and the drying is freeze-drying or vacuum drying.

[0042] The present invention also provides a fresh earthworm extract obtained by the extraction method.

[0043] The present invention also provides a fresh earthworm extract preparation, comprising the fresh earthworm extract obtained by the above extraction method.

[0044] The present invention also provides the use of the fresh earthworm extract obtained by the extraction method in the preparation of medicines containing the fresh earthworm extract.

[0045] Compared with the prior art, the present invention has the following beneficial effects:

[0046] (1) The present invention provides a method for extracting fresh earthworm extract. Through effective pretreatment and utilizing the synergistic effect between Tris hydrochloride and trehalose solution, the yield of fresh earthworm extract and the lumbrokinase activity are significantly improved, thereby achieving the extraction of high-quality fresh earthworm extract.

[0047] (2) In the purification process, the present invention uses a 330 weakly alkaline epoxy anion exchange column and a gel column, which has high resolution for proteins and is easy to operate, thereby greatly improving the lumbrokinase activity in the fresh earthworm extract.

[0048] (3) Compared with the prior art, the extraction method of the present invention has the advantages of simple operation, low cost and reduced pollution, and is easy to scale up for industrial production. No harmful substances are added in the raw materials and operation process, so it is safe to use and has significant economic and environmental benefits. DETAILED DESCRIPTION

[0049] It is worth noting that the raw materials used in the present invention are all common commercially available products, among which bovine fibrinogen, lumbrokinase standard and bovine thrombin are all purchased from China Food and Drug Inspection Institutes.

[0050] Example 1

[0051] A method for extracting fresh earthworm extract, comprising the following steps:

[0052] (1) Soak 1 kg of fresh earthworms in 5 L of a buffer solution of Tris hydrochloride and trehalose solution (pH of Tris hydrochloride = 7.8, mass concentration of trehalose solution is 15%) at 25°C for 40 min. Then homogenize at 10,000 rpm for 25 min. Finally, filter with an ultrafiltration membrane with a molecular weight cutoff of 30 kDa to obtain a crude enzyme solution for later use;

[0053] (2) Mix the crude enzyme solution with ammonium sulfate so that the mass concentration of ammonium sulfate in the crude enzyme solution is 38%, and mix at 4°C for 6 hours. After mixing, centrifuge at 6000 rpm for 20 minutes, take the precipitate, add pH = 7.8 phosphate buffer to the precipitate to obtain a mixture, and then place the mixture in pH = 7.8 phosphate buffer for dialysis. Finally, centrifuge at 8000 rpm for 20 minutes and collect the supernatant;

[0054] (3) The supernatant was first filtered through a 0.45 μm filter to remove impurities, and then 8 times the column volume of the supernatant was loaded onto a 330 weak alkaline epoxy anion exchange column, a Sephadex G-25 column, and a Q-Sepharose 6FF gel column in sequence, and eluted with a 0.5 M sodium chloride solution. The eluate was collected and concentrated through an ultrafiltration membrane with a molecular weight cutoff of 3 kDa, and the concentrate was vacuum freeze-dried to obtain the product.

[0055] Example 2

[0056] A method for extracting fresh earthworm extract, comprising the following steps:

[0057] (1) Soak 1 kg of fresh earthworms in 6 L of a buffer solution of Tris hydrochloride and trehalose solution (pH of Tris hydrochloride = 7.6, mass concentration of trehalose solution is 12%) at 25°C for 50 min. Then homogenize at 10,000 rpm for 25 min. Finally, filter with an ultrafiltration membrane with a molecular weight cutoff of 40 kDa to obtain a crude enzyme solution for later use;

[0058] (2) Mix the crude enzyme solution with ammonium sulfate so that the mass concentration of ammonium sulfate in the crude enzyme solution is 36%, and mix at 4°C for 6 hours. After mixing, centrifuge at 7000 rpm for 18 minutes, take the precipitate, add pH = 7.8 phosphate buffer to the precipitate to obtain a mixture, and then place the mixture in pH = 7.8 phosphate buffer for dialysis. Finally, centrifuge at 7000 rpm for 18 minutes and collect the supernatant;

[0059] (3) The supernatant was first filtered through a 0.45 μm filter to remove impurities, and then 6 column volumes of the supernatant were loaded onto a 330 weakly alkaline epoxy anion exchange column, a Sephadex G-75 column, and a Q-Beads 6FF gel column in sequence, and eluted with a 0.6 M sodium chloride solution. The eluate was collected and concentrated through an ultrafiltration membrane with a molecular weight cutoff of 3 kDa, and the concentrate was vacuum freeze-dried to obtain the product.

[0060] Example 3

[0061] A method for extracting fresh earthworm extract, comprising the following steps:

[0062] (1) Soak 1 kg of fresh earthworms in 3 L of a buffer solution of Tris hydrochloride and trehalose solution (pH of Tris hydrochloride = 7.8, mass concentration of trehalose solution is 10%) at 20°C for 30 min. Then homogenize at 10,000 rpm for 20 min. Finally, filter with an ultrafiltration membrane with a molecular weight cutoff of 50 kDa to obtain a crude enzyme solution for later use;

[0063] (2) Mix the crude enzyme solution with ammonium sulfate so that the mass concentration of ammonium sulfate in the crude enzyme solution is 35%, and mix at 5°C for 4 hours. After mixing, centrifuge at 5000 rpm for 25 minutes, take the precipitate, add pH = 7.6 phosphate buffer to the precipitate to obtain a mixture, and then place the mixture in pH = 7.6 phosphate buffer for dialysis. Finally, centrifuge at 8000 rpm for 20 minutes and collect the supernatant;

[0064] (3) The supernatant was first filtered through a 0.45 μm filter to remove impurities, and then 5 times the column volume of the supernatant was loaded onto a 330 weak alkaline epoxy anion exchange column, a Sephadex G-25 column, and a Q-Beads 6FF gel column in sequence, and eluted with a 0.3 M sodium chloride solution. The eluate was collected and concentrated through an ultrafiltration membrane with a molecular weight cutoff of 2 kDa, and the concentrate was vacuum freeze-dried to obtain the product.

[0065] Example 4

[0066] A method for extracting fresh earthworm extract, comprising the following steps:

[0067] (1) Soak 1 kg of fresh earthworms in 7 L of a buffer solution of Tris hydrochloride and trehalose solution (pH of Tris hydrochloride = 7.4, mass concentration of trehalose solution = 10%) at 35°C for 60 min. Then homogenize at 8000 rpm for 30 min. Finally, filter with an ultrafiltration membrane with a molecular weight cutoff of 50 kDa to obtain a crude enzyme solution for later use;

[0068] (2) Mix the crude enzyme solution with ammonium sulfate so that the mass concentration of ammonium sulfate in the crude enzyme solution is 40%, and mix at 2°C for 6 hours. After mixing, centrifuge at 8000 rpm for 15 minutes, take the precipitate, add pH = 7.8 phosphate buffer to the precipitate to obtain a mixture, and then place the mixture in pH = 7.8 phosphate buffer for dialysis. Finally, centrifuge at 6000 rpm for 25 minutes and collect the supernatant;

[0069] (3) The supernatant was first filtered through a 0.45 μm filter to remove impurities, and then 10 times the column volume of the supernatant was loaded onto a 330 weak alkaline epoxy anion exchange column, a Sephadex G-75 column, and a Q-Sepharose 6FF gel column in sequence, and eluted with a 0.9 M sodium chloride solution. The eluate was collected and concentrated through an ultrafiltration membrane with a molecular weight cutoff of 3 kDa, and the concentrate was vacuum freeze-dried to obtain the product.

[0070] Comparative Example 1

[0071] The same as Example 1, the only difference is that the buffer solution in step (1) is Tris hydrochloride with a pH of 7.8.

[0072] A method for extracting fresh earthworm extract, comprising the following steps:

[0073] (1) Soak 1 kg of fresh earthworms in 5 L of Tris hydrochloride solution (pH of Tris hydrochloride = 7.8) at a volume ratio of 4:1 at 25°C for 40 min. Then homogenize at 10,000 rpm for 25 min. Finally, filter with an ultrafiltration membrane with a molecular weight cutoff of 30 kDa to obtain a crude enzyme solution for later use;

[0074] (2) Mix the crude enzyme solution with ammonium sulfate so that the mass concentration of ammonium sulfate in the crude enzyme solution is 38%, and mix at 4°C for 6 hours. After mixing, centrifuge at 6000 rpm for 20 minutes, take the precipitate, add pH = 7.8 phosphate buffer to the precipitate to obtain a mixture, and then place the mixture in pH = 7.8 phosphate buffer for dialysis. Finally, centrifuge at 8000 rpm for 20 minutes and collect the supernatant;

[0075] (3) The supernatant was first filtered through a 0.45 μm filter to remove impurities, and then 8 times the column volume of the supernatant was loaded onto a 330 weak alkaline epoxy anion exchange column, a Sephadex G-25 column, and a Q-Sepharose 6FF gel column in sequence, and eluted with a 0.5 M sodium chloride solution. The eluate was collected and concentrated through an ultrafiltration membrane with a molecular weight cutoff of 3 kDa, and the concentrate was vacuum freeze-dried to obtain the product.

[0076] Comparative Example 2

[0077] The same as Example 1, the only difference is that the buffer solution in step (1) is a trehalose solution with a mass concentration of 15%.

[0078] A method for extracting fresh earthworm extract, comprising the following steps:

[0079] (1) Soak 1 kg of fresh earthworm in 5 L of trehalose solution with a volume ratio of 4:1 (the mass concentration of the trehalose solution is 15%) at 25°C for 40 min. Then homogenize at 10,000 rpm for 25 min. Finally, filter with an ultrafiltration membrane with a molecular weight cutoff of 30 kDa to obtain a crude enzyme solution for later use;

[0080] (2) Mix the crude enzyme solution with ammonium sulfate so that the mass concentration of ammonium sulfate in the crude enzyme solution is 38%, and mix at 4°C for 6 hours. After mixing, centrifuge at 6000 rpm for 20 minutes, take the precipitate, add pH = 7.8 phosphate buffer to the precipitate to obtain a mixture, and then place the mixture in pH = 7.8 phosphate buffer for dialysis. Finally, centrifuge at 8000 rpm for 20 minutes and collect the supernatant;

[0081] (3) The supernatant was first filtered through a 0.45 μm filter to remove impurities, and then 8 times the column volume of the supernatant was loaded onto a 330 weak alkaline epoxy anion exchange column, a Sephadex G-25 column, and a Q-Sepharose 6FF gel column in sequence, and eluted with a 0.5 M sodium chloride solution. The eluate was collected and concentrated through an ultrafiltration membrane with a molecular weight cutoff of 3 kDa, and the concentrate was vacuum freeze-dried to obtain the product.

[0082] Comparative Example 3

[0083] The same as Example 1, the only difference is that in step (3), the 330 weakly basic epoxy anion exchange column is replaced by a DEAE-cellulose column (DEAE-Cellucose-52).

[0084] A method for extracting fresh earthworm extract, comprising the following steps:

[0085] (1) Soak 1 kg of fresh earthworms in 5 L of a buffer solution of Tris hydrochloride and trehalose solution (pH of Tris hydrochloride = 7.8, mass concentration of trehalose solution is 15%) at 25°C for 40 min. Then homogenize at 10,000 rpm for 25 min. Finally, filter with an ultrafiltration membrane with a molecular weight cutoff of 30 kDa to obtain a crude enzyme solution for later use;

[0086] (2) Mix the crude enzyme solution with ammonium sulfate so that the mass concentration of ammonium sulfate in the crude enzyme solution is 38%, and mix at 4°C for 6 hours. After mixing, centrifuge at 6000 rpm for 20 minutes, take the precipitate, add pH = 7.8 phosphate buffer to the precipitate to obtain a mixture, and then place the mixture in pH = 7.8 phosphate buffer for dialysis. Finally, centrifuge at 8000 rpm for 20 minutes and collect the supernatant;

[0087] (3) The supernatant was first filtered through a 0.45 μm filter to remove impurities, and then 8 times the column volume of the supernatant was sequentially loaded onto a DEAE-cellulose column (DEAE-Cellucose-52), a Sephadex G-25, and a Q-Sepharose 6FF gel column, and eluted with a 0.5 M sodium chloride solution. The eluate was collected and concentrated through an ultrafiltration membrane with a molecular weight cutoff of 3 kDa, and the concentrate was vacuum freeze-dried to obtain the product.

[0088] Comparative Example 4

[0089] The same as Example 1, the only difference is that in step (3), only the sample is loaded onto Sephadex G-25 and Q-Sepharose 6FF gel columns.

[0090] A method for extracting fresh earthworm extract, comprising the following steps:

[0091] (1) Soak 1 kg of fresh earthworms in 5 L of a buffer solution of Tris hydrochloride and trehalose solution (pH of Tris hydrochloride = 7.8, mass concentration of trehalose solution is 15%) at 25°C for 40 min. Then homogenize at 10,000 rpm for 25 min. Finally, filter with an ultrafiltration membrane with a molecular weight cutoff of 30 kDa to obtain a crude enzyme solution for later use;

[0092] (2) Mix the crude enzyme solution with ammonium sulfate so that the mass concentration of ammonium sulfate in the crude enzyme solution is 38%, and mix at 4°C for 6 hours. After mixing, centrifuge at 6000 rpm for 20 minutes, take the precipitate, add pH = 7.8 phosphate buffer to the precipitate to obtain a mixture, and then place the mixture in pH = 7.8 phosphate buffer for dialysis. Finally, centrifuge at 8000 rpm for 20 minutes and collect the supernatant;

[0093] (3) The supernatant was first filtered through a 0.45 μm filter to remove impurities, and then 8 times the column volume of the supernatant was loaded onto Sephadex G-25 and Q-Sepharose 6FF gel columns in sequence and eluted with 0.5 M sodium chloride solution. The eluate was collected and concentrated through an ultrafiltration membrane with a molecular weight cutoff of 3 kDa, and the concentrate was vacuum freeze-dried to obtain.

[0094] Comparative Example 5

[0095] The same as Example 1, the only difference is that in step (3), only the sample is loaded onto a 330 weakly basic epoxy anion exchange column.

[0096] A method for extracting fresh earthworm extract, comprising the following steps:

[0097] (1) Soak 1 kg of fresh earthworms in 5 L of a buffer solution of Tris hydrochloride and trehalose solution (pH of Tris hydrochloride = 7.8, mass concentration of trehalose solution is 15%) at 25°C for 40 min. Then homogenize at 10,000 rpm for 25 min. Finally, filter with an ultrafiltration membrane with a molecular weight cutoff of 30 kDa to obtain a crude enzyme solution for later use;

[0098] (2) Mix the crude enzyme solution with ammonium sulfate so that the mass concentration of ammonium sulfate in the crude enzyme solution is 38%, and mix at 4°C for 6 hours. After mixing, centrifuge at 6000 rpm for 20 minutes, take the precipitate, add pH = 7.8 phosphate buffer to the precipitate to obtain a mixture, and then place the mixture in pH = 7.8 phosphate buffer for dialysis. Finally, centrifuge at 8000 rpm for 20 minutes and collect the supernatant;

[0099] (3) The supernatant was first filtered through a 0.45 μm filter to remove impurities, and then 8 times the column volume of the supernatant was loaded onto a 330 weakly alkaline epoxy anion exchange column and eluted with a 0.5 M sodium chloride solution. The eluate was collected and concentrated through an ultrafiltration membrane with a molecular weight cutoff of 3 kDa, and the concentrate was vacuum freeze-dried to obtain the product.

[0100] Test Example 1

[0101] The yields and lumbrokinase activities of the fresh earthworm extracts prepared in Examples 1-4 and Comparative Examples 1-5 were calculated respectively, and the data results are shown in Table 1.

[0102] Among them, the reference for enzyme activity test method is: Zhang Li, Zhao Jun, Zhao Shanyu. Research on improving the quality standard of Longxinsu capsules [J]. Chinese Journal of Biochemical Pharmaceutics, 2012, 33(04): 414-416. "Research on improving the quality standard of Longxinsu capsules", the specific test method is as follows:

[0103] (1) Prepare standard solution

[0104] Fibrinogen solution: Take an appropriate amount of bovine fibrinogen and use borax buffer (pH7.8) to prepare a solution containing 2 mg of coagulable protein per 1 mL. Thrombin solution: Take bovine thrombin and use borax buffer (pH7.8) to prepare a solution containing 40 BP units per 1 mL. Agar with a mass concentration of 0.5%: Accurately weigh 0.5 g of agar, add 100 mL of borax buffer (pH7.8), and then boil in a water bath to dissolve. Preparation of standard solution: Take the lumbrokinase standard and dilute it with physiological saline according to the labeled potency to make two solutions with concentrations of 12500U and 25000U per 1 mL.

[0105] (2) Preparation of test solution

[0106] The fresh earthworm extracts prepared in Examples 1-4 and Comparative Examples 1-5 were prepared with physiological saline to a mass concentration of 0.3 mg / mL, subjected to ultrasonic extraction for 10 min, centrifuged, and the supernatant was taken as the test solution.

[0107] (3) Preparation of fibrin agar plates

[0108] Take a sterilized culture dish (90 mm in diameter), pipette 8 mL of fibrinogen solution into the dish, quickly pipette 2 mL of 0.5% agarose solution and 0.5 mL of thrombin solution into the dish, mix thoroughly, and place horizontally for 0.5 h.

[0109] (4) Take no less than 4 fibrin plates, take 10 μL of different standard品 solutions and test品 solutions respectively, and spot them on the same plate (the high and low doses are spotted diagonally). Cover the plate and incubate it at 37 °C for 18 h. Take it out, stain the spots formed after fibrinolysis with 0.25% safranin staining solution, measure the two vertical diameters (long and short diameters) of the spots with a vernier caliper respectively, calculate the area based on the average diameter, convert the lysed circle area into logarithm, and calculate the enzyme activity of lumbrokinase.

[0110] Table 1 Test data of the fresh earthworm extract prepared in Examples 1-4 and Comparative Examples 1-5

[0111] serial number Yield of fresh earthworm extract (%) Lumbrokinase activity (U / mg) Example 1 2.2 48200 Example 2 2.0 46320 Example 3 2.1 47240 Example 4 2.1 48080 Comparative Example 1 1.1 38400 Comparative Example 2 0.7 36540 Comparative Example 3 2.8 34130 Comparative Example 4 2.9 37220 Comparative Example 5 3.1 36400

[0112] Test Example 2

[0113] 1. Test sample: The fresh earthworm extract capsules prepared in Example 1 of the present invention and Comparative Examples 1-5.

[0114] Preparation of fresh earthworm extract capsules: Weigh 100 g of the fresh earthworm extract prepared in Example 1 and Comparative Examples 1-5, 150 g of hydroxypropyl cellulose, 76 g of talc, 30 g of magnesium stearate, 46 g of sodium carboxymethyl starch, and 1200 g of sucrose respectively, place them in a high-speed stirring device, stir at 2000 rpm for 20 min to mix evenly. Add 150 g of ethanol with a mass concentration of 50%, stir at 2000 rpm for 10 min, then granulate at 30000 rpm for 2 min, vacuum freeze-dry, pass through a 50-mesh sieve, and fill it into the capsule shell to obtain the fresh earthworm extract capsules (0.1 g per capsule).

[0115] 2. Experimental animals: 80 SD rats, weighing 250-300 g, provided by the Experimental Animal Department of Kunming Medical University, production license number: SCXK[Yunnan]2005-0008, breeding and management conditions: SPF-class animal laboratory, room temperature 20±2 °C, relative humidity 60-70%.

[0116] 3. Modeling method: Anesthetize the rats with ether, fix them supine, clip the hair and disinfect the skin, longitudinally cut about 2 cm of the neck skin, separate the internal carotid artery, common carotid artery and external carotid artery, clamp the external carotid artery and the proximal end of the common carotid artery respectively, and clamp another vascular clamp outside the distal end. After shaking the embolization agent evenly, insert a 0.25 mL syringe into the common carotid artery at a dose of 0.03 mg / 100 g of rats, open the distal end, and inject the embolization agent with the vascular clamp. Then, clamp the distal end of the common carotid artery, pull out the needle, and bond the needle hole with medical glue. After 1 min, release the vascular clamps at the distal end, proximal end of the common carotid artery and the common carotid artery in turn to restore blood flow, clean the wound, and suture the skin to obtain the cerebral infarction model.

[0117] 4. Experimental method: SD rats were divided into 8 groups, namely, sham operation group, blank group, Example 1 and comparative examples 1-5, with 10 rats in each group. Except for the rats in the sham operation group, the rats in the other groups were subjected to animal modeling, and the modeling operation of the blank group remained unchanged, only physiological saline was used instead of embolic agent and injected into the internal carotid artery.

[0118] The rats in Example 1 and Comparative Examples 1-5 were orally administered with a syringe 2 mL of 1 mg / mL aqueous solution of fresh earthworm extract capsule powder starting from the day of modeling, and the administration was continued for 9 days; the sham operation group and the blank group were given an equal amount of normal saline. After 9 days, the rats were anesthetized, and the right common carotid artery was bled in a heparin test tube. The high shear whole blood viscosity, medium shear whole blood viscosity, low shear whole blood viscosity and plasma viscosity were detected using Tianhai automatic blood rheology analyzer (MVIS-2035). SPSS23.0 statistical software was used to process the data, and P<0.05 was considered statistically significant.

[0119] 5. Experimental results: As shown in Table 2, after taking fresh earthworm extract capsules, the high shear whole blood viscosity, medium shear whole blood viscosity, low shear whole blood viscosity and plasma viscosity of rats in Example 1 and Comparative Examples 1-5 were lower than those in the blank group, among which, the blood rheology effect of Example 1 was better than that of Comparative Examples 1-5, and the differences in the data of each group in Example 1 were statistically significant (P<0.05), indicating that the fresh earthworm extract prepared by the present invention has the effect of reducing blood viscosity and improving blood rheology.

[0120] Table 2 Blood rheology test data

[0121]

[0122] Note: Compared with the blank group, * indicates P < 0.05, ** Indicates P<0.01.

[0123] Finally, it should be noted that the above content is only used to illustrate the technical solution of the present invention, rather than to limit the scope of protection of the present invention. Simple modifications or equivalent substitutions of the technical solution of the present invention by ordinary technicians in this field do not deviate from the essence and scope of the technical solution of the present invention.

Claims

1. A method for extracting fresh earthworm extract, characterized in that: The following steps are involved: (1) First, soak fresh earthworms in a buffer solution, homogenize, and filter to obtain a crude enzyme solution; (2) Mixing the crude enzyme solution with ammonium sulfate, taking the precipitate, dialyzing, centrifuging, and collecting the supernatant; (3) Finally, the supernatant is purified by anion exchange column and gel column in sequence, and the eluate is collected and dried to obtain: Wherein, the buffer solution in step (1) is a mixture of Tris hydrochloride and trehalose solution in a volume ratio of 5-8:1-2.

2. The extraction method according to claim 1, characterized in that The pH of the Tris hydrochloride is 7.4-7.8, the mass concentration of the trehalose solution is 10-15%, and the mass volume ratio of the fresh earthworm to the buffer solution is 1kg:3-7L.

3. The extraction method according to claim 1, characterized in that In step (1), the immersion temperature is 20-35° C., the immersion time is 30-60 min, the homogenization speed is 8000-10000 rpm, the homogenization time is 20-30 min, and the filtration uses an ultrafiltration membrane with a molecular weight cutoff of 30k-50k Da.

4. The extraction method according to claim 1, characterized in that In step (2), the mass concentration of ammonium sulfate in the crude enzyme solution is 30-40%, the mixing temperature is 2-5° C., the mixing time is 4-6 h, and after the mixing is completed, centrifugation is required at 5000-8000 rpm for 15-25 min. The dialysis process includes adding a phosphate buffer having a pH value of 7.6-7.8 to the precipitate to obtain a mixture, and then dialyzing in a phosphate buffer having a pH value of 7.6-7.

8.

5. The extraction method according to claim 1, characterized in that The model of the anion exchange column in step (3) is a 330 weakly alkaline epoxy anion exchange column, and the gel column includes a dextran gel column and a Q gel column.

6. The extraction method according to claim 5, characterized in that The model of the dextran gel column is Sephadex G-25 or Sephadex G-75, and the model of the Q gel column is Q-Sepharose 6FF or Q-Beads 6FF.

7. The extraction method according to claim 1, characterized in that The purification process in step (3) includes first filtering the supernatant through a 0.45 μm filter head to remove impurities, then loading 5-10 times the column volume of the supernatant on an anion exchange column and a gel column in sequence, and eluting with a 0.3-0.9 M sodium chloride solution, and the eluate needs to be concentrated by an ultrafiltration membrane with a molecular weight cutoff of 2k-3k Da, and the drying is freeze-drying or vacuum drying.

8. A fresh earthworm extract obtained by the extraction method according to any one of claims 1 to 7.

9. A fresh earthworm extract preparation, characterized in that: The invention comprises a fresh earthworm extract obtained by the extraction method according to any one of claims 1 to 7 or a fresh earthworm extract according to claim 8.

10. Use of the fresh earthworm extract obtained by the extraction method according to any one of claims 1 to 7 or the fresh earthworm extract according to claim 8 in the preparation of a medicine containing the fresh earthworm extract.

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