Method for rapidly extracting protamine sulfate

Abstract

The invention discloses a method for rapidly extracting protamine sulfate, which comprises the following steps: S1, mixing testis tissue and a sodium chloride solution, pulping, standing, centrifuging, and filtering to obtain a precipitate; s2, mixing the precipitate with a sulfuric acid solution, carrying out ultrasonic extraction by using at least two ultrasonic waves with different frequencies at the same time, and centrifuging and filtering to obtain an extracting solution; and S3, carrying out alcohol precipitation on the extracted mixed solution, and drying to obtain protamine sulfate. In the process of extracting protamine by using sulfuric acid, a plurality of ultrasonic waves with different frequencies are adopted to carry out ultrasonic treatment at the same time to generate more cavitation effects and mechanical effects, so that more protamine is dissolved out, and the extraction rate of protamine sulfate is improved.

Description

Technical Field

[0001] The present invention relates to the technical field of protamine sulfate extraction, and more specifically, to a method for rapidly extracting protamine sulfate. Background Art

[0002] As the largest regional high-quality species of Xinjiang's characteristic cold-water fish, white salmon has an annual output of up to 1,500 tons, of which 50% is used for processing. The testicular tissue produced during the processing is usually discarded as waste, but the protamine extracted from the testicular tissue of salmon, which forms protamine sulfate with sulfuric acid, is an alkaline protein sulfate rich in arginine. It has a strong alkaline group and can combine with strongly acidic heparin in the body to form a stable complex, thereby making heparin lose its anticoagulant ability. It also has a mild antithrombin prokinase effect. It is generally used clinically to counteract the anticoagulant effect caused by excessive heparin or after heart surgery. In addition, protamine sulfate can also be combined with insulin to prepare long-acting insulin. Protamine sulfate is also a natural and safe food preservative.

[0003] At present, there are many methods for extracting protamine sulfate, such as sulfuric acid method, hydrochloric acid method, enzymatic method, etc. These methods have been widely used, but there are still some problems such as low extraction rate of protamine sulfate and long extraction time. Therefore, a method for quickly extracting protamine sulfate is urgently needed. Summary of the invention

[0004] An object of the present invention is to solve at least the above problems and to provide at least the advantages which will be described hereinafter.

[0005] In order to achieve these purposes and other advantages according to the present invention, a method for rapidly extracting protamine sulfate is provided, comprising the following steps: S1, mixing the testicular tissue and the sodium chloride solution, beating, standing, centrifuging, and filtering to obtain a precipitate; S2, mixing the precipitate and the sulfuric acid solution, and simultaneously performing ultrasonic extraction using at least two ultrasonic waves of different frequencies, and obtaining an extract after centrifugation and filtration; S3. The extracted mixed solution is precipitated with alcohol and dried to obtain protamine sulfate.

[0006] Preferably, in step S1, the mass ratio of the testicular tissue to the sodium chloride solution is 1:5-10, and the concentration of the sodium chloride solution is 0.14-0.3 mol / L.

[0007] Preferably, silicate sol is added to the slurry obtained by beating, and stirred at 300-500 rpm for 20-30 min, and the mass ratio of the slurry to the silicate sol is 1:2-5.

[0008] Preferably, the method for preparing the silicate sol comprises the following steps: Sodium silicate solution and hydrochloric acid are mixed in a volume ratio of 4:1, wherein the mass fraction of the sodium silicate solution is 61% and the mass fraction of the hydrochloric acid is 10.5%.

[0009] Preferably, the beating temperature in step S1 is not higher than 4°C, the beating speed is 400-600 r / min, and the beating time is 1-2 min.

[0010] Preferably, in step S2, the precipitate and the sulfuric acid solution are mixed at a ratio of 1:1-5 g / mL, and the concentration of the sulfuric acid solution is 0.05-0.25 mol / L.

[0011] Preferably, the ultrasonic extraction operation uses three ultrasonic waves of different frequencies to perform ultrasonic extraction simultaneously, and the three different frequencies are 22-25 kHz, 28-30 kHz, and 40-50 kHz respectively; The ultrasonic power is 120-600 W, the ultrasonic time is 10-60 min, and the ultrasonic temperature is 10-50° C.

[0012] Preferably, the alcohol precipitation operation comprises the following steps: A1, the extraction mixture and ethanol are mixed in a volume ratio of 1:3-5, and after standing for 12-24 hours, centrifuged to obtain a first precipitate; A2. Add 6 to 10 times the mass of water to the first precipitate, stir to dissolve, let stand at 60 to 65°C for 30 min, filter to obtain a first filtrate, add 3 to 5 times the volume of ethanol to the first filtrate, stir, separate into layers, and discard the upper layer of ethanol; A3, add 6 to 10 times the weight of water, stir to dissolve, adjust the pH to 11.0 with 8 mol / L sodium hydroxide solution, let stand at 60 to 65°C for 30 min, and filter to obtain a second filtrate; A4. The second filtrate was allowed to stand at 4°C for 10-12 h, and the supernatant was discarded to obtain the second precipitate; A5. Add 6 to 10 times the weight of water to the second precipitate, stir to dissolve, adjust the pH to 3.0 with 2.5 mol / L sulfuric acid solution, then add 3 to 5 times the volume of ethanol, centrifuge to obtain the third precipitate.

[0013] Preferably, the centrifugal speed in step S1, step S2 and the alcohol precipitation process is 8000 r / min.

[0014] Preferably, the ultrasonic extraction operation uses three ultrasonic waves of different frequencies for simultaneous ultrasonication, and the three different frequencies are 22kHz, 28kHz, and 40kHz respectively; The ultrasonic power was 300 W, the ultrasonic time was 15 min, and the ultrasonic temperature was 10°C.

[0015] The present invention has at least the following beneficial effects: First, the present invention uses three ultrasonic waves of different frequencies to simultaneously perform ultrasonic treatment during the process of extracting protamine using sulfuric acid, thereby generating more cavitation effects and mechanical effects, thereby dissolving more protamine and improving its extraction rate.

[0016] Second, the present invention can accelerate the breaking of the DNA-protamine bond, reduce the amount of sulfuric acid used, and shorten the extraction time by simultaneously ultrasonicating with three ultrasonic waves of different frequencies, thereby improving the extraction rate and reducing the probability of protamine denaturation.

[0017] Third, the present invention adds silica sol to the slurry obtained by beating the testis tissue and the sodium chloride solution and stirs it, and uses the negatively charged silica sol to electrically neutralize the arginine (positively charged) in the protamine, promotes the aggregation of arginine, and thus produces adsorption and cross-linking between them, ultimately causing more protamine to precipitate and improve its extraction rate.

[0018] Other advantages, objectives and features of the present invention will be embodied in part through the following description, and in part will be understood by those skilled in the art through study and practice of the present invention. DETAILED DESCRIPTION

[0019] The present invention is further described in detail below in conjunction with embodiments so that those skilled in the art can implement the invention with reference to the description.

[0020] It should be noted that the experimental methods described in the following embodiments are conventional methods unless otherwise specified, and the reagents and materials can be obtained from commercial channels unless otherwise specified.

[0021] The equipment for ultrasonic extraction in the present invention is a three-frequency ultrasonic reactor purchased from Jiangsu Jiangda Wukesong Biotechnology Co., Ltd., and its model is Wks900B / 3S. The three-frequency ultrasonic reactor adopts a three-frequency (22kHz, 28 kHz, 40 kHz) combination, with adjustable power, and can realize free setting of different combinations of single frequency, dual frequency, and triple frequency.

[0022] The testicular tissues used in the following examples and comparative examples and the blank group are all from whitefish. The conventional nutritional components of the testicular tissues of whitefish are shown in Table 1 below: Table 1 General nutritional composition of testicular tissue of whitefish <Example 1> The method for rapidly extracting protamine sulfate comprises the following steps: S1. Mix 100 g of testicular tissue of whitefish and 500 g of 0.14 mol / L sodium chloride solution, beat at 600 r / min for 2 min at 4°C, let stand for 20 min, centrifuge at 8000 r / min for 10 min, and filter to obtain the precipitate; S2, the precipitate and 0.25 mol / L sulfuric acid solution were mixed at a ratio of 1:1 g / mL, and ultrasonic extraction was performed simultaneously in a triple-frequency ultrasonic reactor with three different frequencies of ultrasound for 60 min, centrifuged at a speed of 8000 r / min for 10 min, filtered, and an extract was obtained. The obtained precipitate was repeatedly extracted with sulfuric acid solution for 3 times, wherein the frequencies of the three different ultrasounds were 22 kHz, 28 kHz, and 40 kHz, respectively, and the ultrasonic power was 360 W; S3. The extracted mixed solution is subjected to alcohol precipitation operation and freeze-dried in a freeze dryer to obtain protamine sulfate.

[0023] The alcohol precipitation operation comprises the following steps: A1, the extraction mixture and ethanol were mixed at a volume ratio of 1:3, allowed to stand for 24 hours, and then centrifuged to obtain a first precipitate; A2. Add 6 times the mass of water to the first precipitate, stir to dissolve, let stand at 65°C for 30 min, filter to obtain the first filtrate, add 3 times the volume of ethanol to the first filtrate, stir, separate into layers, and discard the upper ethanol layer the next day; A3, add 6 times the weight of water, stir to dissolve, adjust the pH to 11.0 with 8 mol / L sodium hydroxide solution, let stand at 65°C for 30 min, and filter to obtain a second filtrate; A4, the second filtrate was allowed to stand at 4°C for 10 h, the supernatant was discarded, and the second precipitate was obtained; A5. Add 6 times the weight of water to the second precipitate, stir to dissolve, adjust the pH to 3.0 with 2.5 mol / L sulfuric acid solution, then add 3 times the volume of ethanol, centrifuge to obtain the third precipitate.

[0024] <Example 2> The method for rapidly extracting protamine sulfate comprises the following steps: S1. Mix 100 g of testicular tissue of whitefish and 800 g of 0.14 mol / L sodium chloride solution, beat at 600 r / min for 2 min at 4°C, let stand for 20 min, centrifuge at 8000 r / min for 10 min, and filter to obtain the precipitate; S2, the precipitate and 0.25 mol / L sulfuric acid solution were mixed at a ratio of 1:3 g / mL, and ultrasonic extraction was performed simultaneously in a triple-frequency ultrasonic reactor with three different frequencies of ultrasound for 60 min, centrifuged at a speed of 8000 r / min for 10 min, filtered, and an extract was obtained. The obtained precipitate was repeatedly extracted with sulfuric acid solution for 3 times, wherein the frequencies of the three different ultrasounds were 22 kHz, 28 kHz, and 40 kHz, respectively, and the ultrasonic power was 360 W; S3. The extracted mixed solution is subjected to alcohol precipitation operation and freeze-dried in a freeze dryer to obtain protamine sulfate.

[0025] The alcohol precipitation operation comprises the following steps: A1, the extraction mixture and ethanol were mixed at a volume ratio of 1:3, allowed to stand for 24 hours, and then centrifuged to obtain a first precipitate; A2. Add 6 times the mass of water to the first precipitate, stir to dissolve, let stand at 65°C for 30 min, filter to obtain the first filtrate, add 3 times the volume of ethanol to the first filtrate, stir, separate into layers, and discard the upper ethanol layer the next day; A3, add 6 times the weight of water, stir to dissolve, adjust the pH to 11.0 with 8 mol / L sodium hydroxide solution, let stand at 65°C for 30 min, and filter to obtain a second filtrate; A4, the second filtrate was allowed to stand at 4°C for 10 h, the supernatant was discarded, and the second precipitate was obtained; A5. Add 6 times the weight of water to the second precipitate, stir to dissolve, adjust the pH to 3.0 with 2.5 mol / L sulfuric acid solution, then add 3 times the volume of ethanol, centrifuge to obtain the third precipitate.

[0026] <Example 3> The method for rapidly extracting protamine sulfate comprises the following steps: S1. Mix 100 g of testicular tissue of whitefish and 1 kg of 0.14 mol / L sodium chloride solution, beat at 600 r / min for 2 min at 4°C, let stand for 20 min, centrifuge at 8000 r / min for 10 min, and filter to obtain the precipitate; S2, the precipitate and 0.25 mol / L sulfuric acid solution were mixed at a ratio of 1:5 g / mL, and ultrasonic extraction was performed simultaneously in a triple-frequency ultrasonic reactor with three different frequencies of ultrasound for 60 min, centrifuged at a speed of 8000 r / min for 10 min, filtered, and an extract was obtained. The obtained precipitate was repeatedly extracted with sulfuric acid solution for 3 times, wherein the frequencies of the three different ultrasounds were 22 kHz, 28 kHz, and 40 kHz, respectively, and the ultrasonic power was 360 W; S3. The extracted mixed solution is subjected to alcohol precipitation operation and freeze-dried in a freeze dryer to obtain protamine sulfate.

[0027] The alcohol precipitation operation comprises the following steps: A1, the extraction mixture and ethanol were mixed at a volume ratio of 1:3, allowed to stand for 24 hours, and then centrifuged to obtain a first precipitate; A2. Add 6 times the mass of water to the first precipitate, stir to dissolve, let stand at 65°C for 30 min, filter to obtain the first filtrate, add 3 times the volume of ethanol to the first filtrate, stir, separate into layers, and discard the upper ethanol layer the next day; A3, add 6 times the weight of water, stir to dissolve, adjust the pH to 11.0 with 8 mol / L sodium hydroxide solution, let stand at 65°C for 30 min, and filter to obtain a second filtrate; A4, the second filtrate was allowed to stand at 4°C for 10 h, the supernatant was discarded, and the second precipitate was obtained; A5. Add 6 times the weight of water to the second precipitate, stir to dissolve, adjust the pH to 3.0 with 2.5 mol / L sulfuric acid solution, then add 3 times the volume of ethanol, centrifuge to obtain the third precipitate.

[0028] <Example 4> The method for rapidly extracting protamine sulfate comprises the following steps: S1. Mix 100 g of testicular tissue of whitefish and 500 g of 0.14 mol / L sodium chloride solution, beat at 600 r / min for 2 min at 4°C, add 2 kg of silicate sol and stir at 300 rpm for 20 min, let stand for 20 min, centrifuge at 8000 r / min for 10 min, and filter to obtain the precipitate; S2, the precipitate and 0.25 mol / L sulfuric acid solution were mixed at a ratio of 1:1 g / mL, and ultrasonic extraction was performed simultaneously in a triple-frequency ultrasonic reactor with three different frequencies of ultrasound for 60 min, centrifuged at a speed of 8000 r / min for 10 min, filtered, and an extract was obtained. The obtained precipitate was repeatedly extracted with sulfuric acid solution for 3 times, wherein the frequencies of the three different ultrasounds were 22 kHz, 28 kHz, and 40 kHz, respectively, and the ultrasonic power was 360 W; S3. The extracted mixed solution is subjected to alcohol precipitation operation and freeze-dried in a freeze dryer to obtain protamine sulfate.

[0029] The preparation method of the silicate sol comprises the following steps: Sodium silicate solution and hydrochloric acid are mixed in a volume ratio of 4:1, wherein the mass fraction of the sodium silicate solution is 61% and the mass fraction of the hydrochloric acid is 10.5%. The prepared silicate sol has good stability and applicability.

[0030] The alcohol precipitation operation comprises the following steps: A1, the extraction mixture and ethanol were mixed at a volume ratio of 1:3, allowed to stand for 24 hours, and then centrifuged to obtain a first precipitate; A2. Add 6 times the mass of water to the first precipitate, stir to dissolve, let stand at 65°C for 30 min, filter to obtain the first filtrate, add 3 times the volume of ethanol to the first filtrate, stir, separate into layers, and discard the upper ethanol layer the next day; A3, add 6 times the weight of water, stir to dissolve, adjust the pH to 11.0 with 8 mol / L sodium hydroxide solution, let stand at 65°C for 30 min, and filter to obtain a second filtrate; A4, the second filtrate was allowed to stand at 4°C for 10 h, the supernatant was discarded, and the second precipitate was obtained; A5. Add 6 times the weight of water to the second precipitate, stir to dissolve, adjust the pH to 3.0 with 2.5 mol / L sulfuric acid solution, then add 3 times the volume of ethanol, centrifuge to obtain the third precipitate.

[0031] <Example 5> The method for rapidly extracting protamine sulfate comprises the following steps: S1. Mix 100 g of testicular tissue of whitefish and 800 g of 0.14 mol / L sodium chloride solution, beat at 600 r / min for 2 min at 4°C, add 4 kg of silicate sol and stir at 400 rpm for 25 min, let stand for 20 min, centrifuge at 8000 r / min for 10 min, and filter to obtain the precipitate; S2, the precipitate and 0.25 mol / L sulfuric acid solution were mixed at a ratio of 1:3 g / mL, and ultrasonic extraction was performed simultaneously in a triple-frequency ultrasonic reactor with three different frequencies of ultrasound for 60 min, centrifuged at a speed of 8000 r / min for 10 min, filtered, and an extract was obtained. The obtained precipitate was repeatedly extracted with sulfuric acid solution for 3 times, wherein the frequencies of the three different ultrasounds were 22 kHz, 28 kHz, and 40 kHz, respectively, and the ultrasonic power was 360 W; S3. The extracted mixed solution is subjected to alcohol precipitation operation and freeze-dried in a freeze dryer to obtain protamine sulfate.

[0032] The preparation method of the silicate sol comprises the following steps: Sodium silicate solution and hydrochloric acid are mixed in a volume ratio of 4:1, wherein the mass fraction of the sodium silicate solution is 61% and the mass fraction of the hydrochloric acid is 10.5%. The prepared silicate sol has good stability and applicability.

[0033] The alcohol precipitation operation comprises the following steps: A1, the extraction mixture and ethanol were mixed at a volume ratio of 1:3, allowed to stand for 24 hours, and then centrifuged to obtain a first precipitate; A2. Add 6 times the mass of water to the first precipitate, stir to dissolve, let stand at 65°C for 30 min, filter to obtain the first filtrate, add 3 times the volume of ethanol to the first filtrate, stir, separate into layers, and discard the upper ethanol layer the next day; A3, add 6 times the weight of water, stir to dissolve, adjust the pH to 11.0 with 8 mol / L sodium hydroxide solution, let stand at 65°C for 30 min, and filter to obtain a second filtrate; A4, the second filtrate was allowed to stand at 4°C for 10 h, the supernatant was discarded, and the second precipitate was obtained; A5. Add 6 times the weight of water to the second precipitate, stir to dissolve, adjust the pH to 3.0 with 2.5 mol / L sulfuric acid solution, then add 3 times the volume of ethanol, centrifuge to obtain the third precipitate.

[0034] <Example 6> The method for rapidly extracting protamine sulfate comprises the following steps: S1. Mix 100 g of testicular tissue of whitefish and 1 kg of 0.14 mol / L sodium chloride solution, beat at 600 r / min for 2 min at 4°C, add 5 kg of silicate sol and stir at 500 rpm for 30 min, let stand for 20 min, centrifuge at 8000 r / min for 10 min, and filter to obtain the precipitate; S2, the precipitate and 0.25 mol / L sulfuric acid solution were mixed at a ratio of 1:5 g / mL, and ultrasonic extraction was performed simultaneously in a triple-frequency ultrasonic reactor with three different frequencies of ultrasound for 60 min, centrifuged at a speed of 8000 r / min for 10 min, filtered, and an extract was obtained. The obtained precipitate was repeatedly extracted with sulfuric acid solution for 3 times, wherein the frequencies of the three ultrasounds were 22 kHz, 28 kHz, and 40 kHz, respectively, and the ultrasonic power was 360 W; S3. The extracted mixed solution is subjected to alcohol precipitation operation and freeze-dried in a freeze dryer to obtain protamine sulfate.

[0035] The preparation method of the silicate sol comprises the following steps: Sodium silicate solution and hydrochloric acid are mixed in a volume ratio of 4:1, wherein the mass fraction of the sodium silicate solution is 61% and the mass fraction of the hydrochloric acid is 10.5%. The prepared silicate sol has good stability and applicability.

[0036] The alcohol precipitation operation comprises the following steps: A1, the extraction mixture and ethanol were mixed at a volume ratio of 1:3, allowed to stand for 24 hours, and then centrifuged to obtain a first precipitate; A2. Add 6 times the mass of water to the first precipitate, stir to dissolve, let stand at 65°C for 30 min, filter to obtain the first filtrate, add 3 times the volume of ethanol to the first filtrate, stir, separate into layers, and discard the upper ethanol layer the next day; A3, add 6 times the weight of water, stir to dissolve, adjust the pH to 11.0 with 8 mol / L sodium hydroxide solution, let stand at 65°C for 30 min, and filter to obtain a second filtrate; A4, the second filtrate was allowed to stand at 4°C for 10 h, the supernatant was discarded, and the second precipitate was obtained; A5. Add 6 times the weight of water to the second precipitate, stir to dissolve, adjust the pH to 3.0 with 2.5 mol / L sulfuric acid solution, then add 3 times the volume of ethanol, centrifuge to obtain the third precipitate.

[0037] <Blank Group> The method for rapidly extracting protamine sulfate comprises the following steps: S1. Mix 100 g of testicular tissue of whitefish and 1 kg of 0.14 mol / L sodium chloride solution, beat at 600 r / min for 2 min at 4°C, let stand for 20 min, centrifuge at 8000 r / min for 10 min, and filter to obtain the precipitate; S2, the precipitate and 0.25 mol / L sulfuric acid solution were mixed at a ratio of 1:5 g / mL, extracted for 60 min, centrifuged at a speed of 8000 r / min for 10 min, filtered, and an extract was obtained. The obtained precipitate was repeatedly extracted with sulfuric acid solution for 3 times; S3. The extracted mixed solution is subjected to alcohol precipitation operation and freeze-dried in a freeze dryer to obtain protamine sulfate.

[0038] The alcohol precipitation operation comprises the following steps: A1, the extraction mixture and ethanol were mixed at a volume ratio of 1:3, allowed to stand for 24 hours, and then centrifuged to obtain a first precipitate; A2. Add 6 times the mass of water to the first precipitate, stir to dissolve, let stand at 65°C for 30 min, filter to obtain the first filtrate, add 3 times the volume of ethanol to the first filtrate, stir, separate into layers, and discard the upper ethanol layer the next day; A3, add 6 times the weight of water, stir to dissolve, adjust the pH to 11.0 with 8 mol / L sodium hydroxide solution, let stand at 65°C for 30 min, and filter to obtain a second filtrate; A4, the second filtrate was allowed to stand at 4°C for 10 h, the supernatant was discarded, and the second precipitate was obtained; A5. Add 6 times the weight of water to the second precipitate, stir to dissolve, adjust the pH to 3.0 with 2.5 mol / L sulfuric acid solution, then add 3 times the volume of ethanol, centrifuge to obtain the third precipitate.

[0039] <Test 1> The protamine sulfate extracted from Examples 1 to 6 and the blank group was weighed, and then the extraction rate was calculated, wherein the extraction rate is the percentage of protamine sulfate in the testis tissue of whitefish. The test results are shown in Table 2 below: Table 2 Extraction rate of protamine sulfate in each group According to the data in Table 2, compared with the blank group, the extraction rate of protamine sulfate in Examples 1 to 3 is significantly improved, indicating that the three-frequency simultaneous ultrasonic treatment can significantly improve the extraction of protamine in testicular tissue by sulfuric acid. The reason is that the simultaneous ultrasonic treatment of three different frequencies of ultrasound can produce more cavitation effect and mechanical effect, thereby dissolving more protamine and improving its extraction rate; and compared with Examples 1 to 3, the extraction rate of protamine sulfate in Examples 4 to 6 is significantly improved, indicating that the silica sol can further improve the extraction rate of protamine in testicular tissue by sulfuric acid on the basis of the simultaneous ultrasonic treatment of three different frequencies of ultrasound. The reason is that the negatively charged silica sol electrically neutralizes the arginine (positively charged) in the protamine, promotes the aggregation of arginine, thereby generating adsorption and cross-linking between each other, and finally precipitates more protamine, improving its extraction rate.

[0040] <Comparative Example 1> The method for rapidly extracting protamine sulfate comprises the following steps: S1. Mix 100 g of testicular tissue of whitefish and 1 kg of 0.14 mol / L sodium chloride solution, beat at 600 r / min for 2 min at 4°C, let stand for 20 min, centrifuge at 8000 r / min for 10 min, and filter to obtain the precipitate; S2, the precipitate and 0.25 mol / L sulfuric acid solution were mixed at a ratio of 1:5 g / mL, and extracted with 360W 22 kHz single-frequency ultrasound in a triple-frequency ultrasonic reactor for 60 min, centrifuged at 8000 r / min for 10 min, filtered, and the obtained precipitate was repeatedly extracted with sulfuric acid solution for 3 times; S3. The extracted mixed solution is subjected to alcohol precipitation operation and freeze-dried in a freeze dryer to obtain protamine sulfate.

[0041] The alcohol precipitation operation comprises the following steps: A1, the extraction mixture and ethanol were mixed at a volume ratio of 1:3, allowed to stand for 24 hours, and then centrifuged to obtain a first precipitate; A2. Add 6 times the mass of water to the first precipitate, stir to dissolve, let stand at 65°C for 30 min, filter to obtain the first filtrate, add 3 times the volume of ethanol to the first filtrate, stir, separate into layers, and discard the upper ethanol layer the next day; A3, add 6 times the weight of water, stir to dissolve, adjust the pH to 11.0 with 8 mol / L sodium hydroxide solution, let stand at 65°C for 30 min, and filter to obtain a second filtrate; A4, the second filtrate was allowed to stand at 4°C for 10 h, the supernatant was discarded, and the second precipitate was obtained; A5. Add 6 times the weight of water to the second precipitate, stir to dissolve, adjust the pH to 3.0 with 2.5 mol / L sulfuric acid solution, then add 3 times the volume of ethanol, centrifuge to obtain the third precipitate.

[0042] <Comparative Example 2> The method for rapidly extracting protamine sulfate comprises the following steps: S1. Mix 100 g of testicular tissue of whitefish and 1 kg of 0.14 mol / L sodium chloride solution, beat at 600 r / min for 2 min at 4°C, let stand for 20 min, centrifuge at 8000 r / min for 10 min, and filter to obtain the precipitate; S2, the precipitate and 0.25 mol / L sulfuric acid solution were mixed at a ratio of 1:5 g / mL, and extracted with 360W 28 kHz single-frequency ultrasound in a triple-frequency ultrasonic reactor for 60 min, centrifuged at 8000 r / min for 10 min, filtered, and the obtained precipitate was repeatedly extracted with sulfuric acid solution for 3 times; S3. The extracted mixed solution is subjected to alcohol precipitation operation and freeze-dried in a freeze dryer to obtain protamine sulfate.

[0043] The alcohol precipitation operation comprises the following steps: A1, the extraction mixture and ethanol were mixed at a volume ratio of 1:3, allowed to stand for 24 hours, and then centrifuged to obtain a first precipitate; A2. Add 6 times the mass of water to the first precipitate, stir to dissolve, let stand at 65°C for 30 min, filter to obtain the first filtrate, add 3 times the volume of ethanol to the first filtrate, stir, separate into layers, and discard the upper ethanol layer the next day; A3, add 6 times the weight of water, stir to dissolve, adjust the pH to 11.0 with 8 mol / L sodium hydroxide solution, let stand at 65°C for 30 min, and filter to obtain a second filtrate; A4, the second filtrate was allowed to stand at 4°C for 10 h, the supernatant was discarded, and the second precipitate was obtained; A5. Add 6 times the weight of water to the second precipitate, stir to dissolve, adjust the pH to 3.0 with 2.5 mol / L sulfuric acid solution, then add 3 times the volume of ethanol, centrifuge to obtain the third precipitate.

[0044] <Comparative Example 3> The method for rapidly extracting protamine sulfate comprises the following steps: S1. Mix 100 g of testicular tissue of whitefish and 1 kg of 0.14 mol / L sodium chloride solution, beat at 600 r / min for 2 min at 4°C, let stand for 20 min, centrifuge at 8000 r / min for 10 min, and filter to obtain the precipitate; S2, the precipitate and 0.25 mol / L sulfuric acid solution were mixed at a ratio of 1:5 g / mL, and extracted with 360W 40 kHz single-frequency ultrasound in a triple-frequency ultrasonic reactor for 60 min, centrifuged at 8000 r / min for 10 min, filtered, and the obtained precipitate was repeatedly extracted with sulfuric acid solution for 3 times; S3. The extracted mixed solution is subjected to alcohol precipitation operation and freeze-dried in a freeze dryer to obtain protamine sulfate.

[0045] The alcohol precipitation operation comprises the following steps: A1, the extraction mixture and ethanol were mixed at a volume ratio of 1:3, allowed to stand for 24 hours, and then centrifuged to obtain a first precipitate; A2. Add 6 times the mass of water to the first precipitate, stir to dissolve, let stand at 65°C for 30 min, filter to obtain the first filtrate, add 3 times the volume of ethanol to the first filtrate, stir, separate into layers, and discard the upper ethanol layer the next day; A3, add 6 times the weight of water, stir to dissolve, adjust the pH to 11.0 with 8 mol / L sodium hydroxide solution, let stand at 65°C for 30 min, and filter to obtain a second filtrate; A4, the second filtrate was allowed to stand at 4°C for 10 h, the supernatant was discarded, and the second precipitate was obtained; A5. Add 6 times the weight of water to the second precipitate, stir to dissolve, adjust the pH to 3.0 with 2.5 mol / L sulfuric acid solution, then add 3 times the volume of ethanol, centrifuge to obtain the third precipitate.

[0046] <Comparative Example 4> The method for rapidly extracting protamine sulfate comprises the following steps: S1. Mix 100 g of testicular tissue of whitefish and 1 kg of 0.14 mol / L sodium chloride solution, beat at 600 r / min for 2 min at 4°C, let stand for 20 min, centrifuge at 8000 r / min for 10 min, and filter to obtain the precipitate; S2, the precipitate and 0.25 mol / L sulfuric acid solution were mixed at a ratio of 1:5 g / mL, and ultrasonic extraction was performed simultaneously in a triple-frequency ultrasonic reactor with two ultrasonic waves of different frequencies for 60 min, centrifuged at a speed of 8000 r / min for 10 min, filtered, and an extract was obtained. The obtained precipitate was repeatedly extracted with sulfuric acid solution for 3 times, wherein the frequencies of the two different ultrasonic waves were 28 kHz and 40 kHz, respectively, and the ultrasonic power was 360 W; S3. The extracted mixed solution is subjected to alcohol precipitation operation and freeze-dried in a freeze dryer to obtain protamine sulfate.

[0047] The alcohol precipitation operation comprises the following steps: A1, the extraction mixture and ethanol were mixed at a volume ratio of 1:3, allowed to stand for 24 hours, and then centrifuged to obtain a first precipitate; A2. Add 6 times the mass of water to the first precipitate, stir to dissolve, let stand at 65°C for 30 min, filter to obtain the first filtrate, add 3 times the volume of ethanol to the first filtrate, stir, separate into layers, and discard the upper ethanol layer the next day; A3, add 6 times the weight of water, stir to dissolve, adjust the pH to 11.0 with 8 mol / L sodium hydroxide solution, let stand at 65°C for 30 min, and filter to obtain a second filtrate; A4, the second filtrate was allowed to stand at 4°C for 10 h, the supernatant was discarded, and the second precipitate was obtained; A5. Add 6 times the weight of water to the second precipitate, stir to dissolve, adjust the pH to 3.0 with 2.5 mol / L sulfuric acid solution, then add 3 times the volume of ethanol, centrifuge to obtain the third precipitate.

[0048] <Comparative Example 5> The method for rapidly extracting protamine sulfate comprises the following steps: S1. Mix 100 g of testicular tissue of whitefish and 1 kg of 0.14 mol / L sodium chloride solution, beat at 600 r / min for 2 min at 4°C, let stand for 20 min, centrifuge at 8000 r / min for 10 min, and filter to obtain the precipitate; S2, the precipitate and 0.25 mol / L sulfuric acid solution were mixed at a ratio of 1:5 g / mL, and a sequential multi-frequency (triple-frequency) method was used in a triple-frequency ultrasonic reactor, the frequencies of the three ultrasonic waves were 22 kHz, 28 kHz, and 40 kHz, and the ultrasonic power was 360 W. Each ultrasonic wave was performed for 20 min (a total of 60 min of ultrasonic extraction), and the mixture was centrifuged at a speed of 8000 r / min for 10 min and filtered to obtain an extract. The precipitate was repeatedly extracted with a sulfuric acid solution for 3 times; S3. The extracted mixed solution is subjected to alcohol precipitation operation and freeze-dried in a freeze dryer to obtain protamine sulfate.

[0049] The alcohol precipitation operation comprises the following steps: A1, the extraction mixture and ethanol were mixed at a volume ratio of 1:3, allowed to stand for 24 hours, and then centrifuged to obtain a first precipitate; A2. Add 6 times the mass of water to the first precipitate, stir to dissolve, let stand at 65°C for 30 min, filter to obtain the first filtrate, add 3 times the volume of ethanol to the first filtrate, stir, separate into layers, and discard the upper ethanol layer the next day; A3, add 6 times the weight of water, stir to dissolve, adjust the pH to 11.0 with 8 mol / L sodium hydroxide solution, let stand at 65°C for 30 min, and filter to obtain a second filtrate; A4, the second filtrate was allowed to stand at 4°C for 10 h, the supernatant was discarded, and the second precipitate was obtained; A5. Add 6 times the weight of water to the second precipitate, stir to dissolve, adjust the pH to 3.0 with 2.5 mol / L sulfuric acid solution, then add 3 times the volume of ethanol, centrifuge to obtain the third precipitate.

[0050] <Comparative Example 6> The method for rapidly extracting protamine sulfate comprises the following steps: S1. Mix 100 g of testicular tissue of whitefish and 1 kg of 0.14 mol / L sodium chloride solution, beat at 600 r / min for 2 min at 4°C, add 5 kg of water and stir at 500 rpm for 30 min, let stand for 20 min, centrifuge at 8000 r / min for 10 min, and filter to obtain the precipitate; S2, the precipitate and 0.25 mol / L sulfuric acid solution were mixed at a ratio of 1:5 g / mL, and ultrasonic extraction was performed simultaneously in a triple-frequency ultrasonic reactor with three different frequencies of ultrasound for 60 min, centrifuged at a speed of 8000 r / min for 10 min, filtered, and an extract was obtained. The obtained precipitate was repeatedly extracted with sulfuric acid solution for 3 times, wherein the frequencies of the three different ultrasounds were 22 kHz, 28kHz, and 40 kHz, respectively, and the ultrasonic power was 360 W; S3. The extracted mixed solution is subjected to alcohol precipitation operation and freeze-dried in a freeze dryer to obtain protamine sulfate.

[0051] The alcohol precipitation operation comprises the following steps: A1, the extraction mixture and ethanol were mixed at a volume ratio of 1:3, allowed to stand for 24 hours, and then centrifuged to obtain a first precipitate; A2. Add 6 times the mass of water to the first precipitate, stir to dissolve, let stand at 65°C for 30 min, filter to obtain the first filtrate, add 3 times the volume of ethanol to the first filtrate, stir, separate into layers, and discard the upper ethanol layer the next day; A3, add 6 times the weight of water, stir to dissolve, adjust the pH to 11.0 with 8 mol / L sodium hydroxide solution, let stand at 65°C for 30 min, and filter to obtain a second filtrate; A4, the second filtrate was allowed to stand at 4°C for 10 h, the supernatant was discarded, and the second precipitate was obtained; A5. Add 6 times the weight of water to the second precipitate, stir to dissolve, adjust the pH to 3.0 with 2.5 mol / L sulfuric acid solution, then add 3 times the volume of ethanol, centrifuge to obtain the third precipitate.

[0052] <Test 2> The protamine sulfate extracted from Comparative Examples 1 to 6 was weighed, and the extraction rate was calculated, and compared with Example 3, Example 6, and the blank group. The test results are shown in Table 3 below: Table 3 Extraction rate of protamine sulfate in each group According to the data in Table 3, the extraction rate of protamine sulfate in Comparative Examples 1 to 5 is significantly improved compared with the blank group, but the extraction rate is significantly lower than that in Example 3, indicating that single-frequency ultrasonic treatment, dual-frequency simultaneous ultrasonic treatment, and triple-frequency sequential ultrasonic treatment can all improve the extraction of protamine sulfate from testicular tissue by sulfuric acid, but the magnitude of the improvement in the extraction rate of protamine sulfate is not as good as that of simultaneous ultrasonic treatment with three different frequencies of ultrasound. The reason for this is that simultaneous ultrasonic treatment with three different frequencies of ultrasound can produce more cavitation effect and mechanical effect than single-frequency ultrasonic treatment, dual-frequency simultaneous ultrasonic treatment, and triple-frequency sequential ultrasonic treatment, thereby dissolving more protamine and improving its extraction rate; the extraction rate of protamine sulfate in Comparative Example 6 is not much different from that in Example 3, indicating that adding water and stirring for 30 more times can improve the extraction rate of protamine sulfate. min, and cannot significantly improve the extraction rate of protamine sulfate; compared with Comparative Example 6, the extraction rate of protamine sulfate in Example 6 is significantly improved, indicating that stirring the silica sol with the slurry can effectively promote the precipitation of protamine. The reason is that the negatively charged silica sol electrically neutralizes the arginine (positively charged) in the protamine, promotes the aggregation of arginine, thereby generating adsorption and cross-linking between each other, and finally precipitating more protamine, thereby improving its extraction rate.

[0053] <Determination of the biological potency of protamine sulfate> 1. Preparation of protamine sulfate solution Weigh 100.02 mg of the protamine sulfate extracted in Example 6, add 0.9% sodium chloride solution to prepare a 1 mg / mL protamine sulfate solution.

[0054] 2. Preparation of Heparin Standard Solution Weigh 150.05 mg of heparin standard with a unit potency of 180 IU / mg, add 0.9% sodium chloride solution to dissolve, and dilute to obtain 6 heparin standard solutions with different potency concentrations of 130 IU / mg, 135 IU / mg, 140 IU / mg, 145 IU / mg, 150 IU / mg, and 155 IU / mg.

[0055] 3. Verification method Take 6 test tubes, add 5 mL of protamine sulfate solution to each test tube, then add 5 mL of heparin standard solution with concentrations of 130 IU / mg, 135 IU / mg, 140 IU / mg, 145 IU / mg, 150 IU / mg, and 155 IU / mg to each test tube respectively. Let stand, take the supernatant, and measure the transmittance of the mixed solution in each test tube at a wavelength of 450 nm. Repeat 3 times.

[0056] Through the transmittance test, it was found that the heparin standard solution with a potency concentration of 145 IU / mg had the highest transmittance, which means that the biological potency of protamine sulfate is 145 IU / mg.

[0057] Although the embodiments of the present invention have been disclosed as above, they are not limited to the applications listed in the specification and the implementation modes. They can be fully applied to various fields suitable for the present invention. For those familiar with the art, additional modifications can be easily implemented. Therefore, without departing from the general concept defined by the claims and the scope of equivalents, the present invention is not limited to the specific details and the embodiments shown and described herein.

Claims

1. A method for rapidly extracting protamine sulfate, characterized in that: The following steps are involved: S1, mixing the testicular tissue and the sodium chloride solution, beating, standing, centrifuging, and filtering to obtain a precipitate; S2, mixing the precipitate and the sulfuric acid solution, and simultaneously performing ultrasonic extraction using at least two ultrasonic waves of different frequencies, and obtaining an extract after centrifugation and filtration; S3. The extracted mixed solution is precipitated with alcohol and dried to obtain protamine sulfate.

2. The method for rapidly extracting protamine sulfate according to claim 1, characterized in that: In step S1, the mass ratio of the testicular tissue to the sodium chloride solution is 1:5-10, and the concentration of the sodium chloride solution is 0.14-0.3 mol / L.

3. The method for rapidly extracting protamine sulfate according to claim 2, characterized in that: Add silicate sol to the slurry obtained by beating, and stir at 300-500 rpm for 20-30 min. The mass ratio of the slurry to the silicate sol is 1:2-5.

4. The method for rapidly extracting protamine sulfate according to claim 3, characterized in that: The preparation method of the silicate sol comprises the following steps: Sodium silicate solution and hydrochloric acid are mixed in a volume ratio of 4:1, wherein the mass fraction of the sodium silicate solution is 61% and the mass fraction of the hydrochloric acid is 10.5%.

5. The method for rapidly extracting protamine sulfate according to claim 1, characterized in that: In step S1, the beating temperature is not higher than 4°C, the beating speed is 400-600 r / min, and the beating time is 1-2 min.

6. The method for rapidly extracting protamine sulfate according to claim 2, characterized in that: In step S2, the precipitate and the sulfuric acid solution are mixed at a ratio of 1:1-5 g / mL, and the concentration of the sulfuric acid solution is 0.05-0.25 mol / L.

7. The method for rapidly extracting protamine sulfate according to claim 1, characterized in that: The ultrasonic extraction operation uses three different frequencies of ultrasound to perform ultrasonic extraction simultaneously, and the three different frequencies are 22-25 kHz, 28-30 kHz, and 40-50 kHz respectively; The ultrasonic power is 120-600 W, the ultrasonic time is 10-60 min, and the ultrasonic temperature is 10-50° C.

8. The method for rapidly extracting protamine sulfate according to claim 6, characterized in that: The alcohol precipitation operation comprises the following steps: A1, the extraction mixture and ethanol are mixed in a volume ratio of 1:3-5, and after standing for 12-24 hours, centrifuged to obtain a first precipitate; A2. Add 6 to 10 times the mass of water to the first precipitate, stir to dissolve, let stand at 60 to 65°C for 30 min, filter to obtain a first filtrate, add 3 to 5 times the volume of ethanol to the first filtrate, stir, separate into layers, and discard the upper layer of ethanol; A3, add 6 to 10 times the weight of water, stir to dissolve, adjust the pH to 11.0 with 8 mol / L sodium hydroxide solution, let stand at 60 to 65°C for 30 min, filter to obtain a second filtrate; A4. The second filtrate was allowed to stand at 4°C for 10-12 h, and the supernatant was discarded to obtain the second precipitate; A5. Add 6 to 10 times the weight of water to the second precipitate, stir to dissolve, adjust the pH to 3.0 with 2.5 mol / L sulfuric acid solution, then add 3 to 5 times the volume of ethanol, centrifuge to obtain the third precipitate.

9. The method for rapidly extracting protamine sulfate according to claim 8, characterized in that: The centrifugal speed in step S1, step S2 and the alcohol precipitation process is 8000 r / min.

10. The method for rapidly extracting protamine sulfate according to claim 8, characterized in that: The ultrasonic extraction operation uses three different frequencies of ultrasound to perform ultrasonic extraction simultaneously, and the three different frequencies are 22kHz, 28kHz, and 40kHz respectively; The ultrasonic power was 300 W, the ultrasonic time was 15 min, and the ultrasonic temperature was 10°C.

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