蚯蚓纤溶酶（蚓激酶）是从蚯蚓中提取并纯化到的一类能溶解血纤维蛋白的酶类，它存在于蚯蚓的消化道内。蚯蚓纤溶酶具有双重功能，除了能直接水解纤维蛋白外，还能激活血纤维蛋白溶酶原成血纤维蛋白溶酶，从而具有间接水解纤维蛋白的作用。动物在服用蚯蚓纤溶酶之后，产生了明显的抗栓和溶栓作用。
蚯蚓纤溶酶（蚓激酶）通过激活 Sirt1 信号减轻心肌缺血再灌注 (I-R) 损伤，从而增强自噬通量并减少 I-R 诱导的氧化损伤、炎症和细胞凋亡。
性状
本品为淡黄色至黄褐色粉末；味腥，有引湿性。
鉴别
- 取本品适量，加水制成每 1ml 中含 0.5mg 的溶液，照分光光度法（中国药典 2000 年版二部附录 IVA）测定，在 278nm 的波长处有最大吸收。
- 取本品适量，加 0.9% 氯化钠溶液制成每 1ml 中含 10mg 的溶液，作为供试品溶液；用 6 号针头取动物血 1 滴于试管底部，30 分钟后，加供试品溶液 1ml 置试管中，轻轻摇动，血凝块应在 60 分钟内溶解。以 0.9% 氯化钠溶液 1ml 为空白，同法操作，血凝块应不溶解。
检查
- 酸碱度：取本品，加水制成每 1ml 中含 1mg 的溶液，依法测定（中国药典 2000 年版二部附录 VIH），pH 值应为 6.0~8.0。
- 溶液的澄清度与颜色：取本品适量，加水制成每 1ml 中含 1mg 的溶液，溶液应澄清；如显色，与黄色 2 号标准比色液（中国药典 2000 年版二部附录 IXA 第一法）比较，不得更深。
- 干燥失重：取本品适量，以五氧化二磷为干燥剂，在 60℃减压干燥至恒重，减失重量不得超过 5.0%（中国药典 2000 年版二部附录 VIL）。
比活力测定
效价测定
试剂
- 0.01mol 磷酸盐缓冲液（pH7.8）：取磷酸氢二钠 3.58g，加水使溶解并稀释至 1000ml 为 A 液；取磷酸二氢钠（NaH₂PO₄・2H₂O）0.78g，加水使溶解并稀释至 500ml 为 B 液；将 A、B 两液混合至 pH 值为 7.8。
- 工作溶液：取 0.01mol/L 磷酸盐缓冲液（pH7.8）与 0.9% 氯化钠溶液（1:17）混合。
- 1.5% 琼脂糖溶液：取琼脂糖 1.5g，加工作溶液 100ml 加热溶解。
- 纤维蛋白原溶液：取纤维蛋白原适量，加工作溶液制成每 1ml 中含 1.5mg 的可凝蛋白溶液。
- 凝血酶溶液：取凝血酶，加 0.9% 氯化钠溶液制成每 1ml 中含 1BP 单位的溶液。
- 标准品溶液的制备：取蚓激酶标准品，用 0.9% 氯化钠溶液制成浓度分别为每 1ml 中含 10000、8000、6000、4000、2000 蚓激酶单位的溶液。
- 供试品溶液的制备：取本品适量，加 0.9% 氯化钠溶液使溶解并稀释成标准曲线范围内的浓度。
- 测定法：取纤维蛋白原溶液 39ml，置烧杯中，边搅拌边加入 55℃琼脂糖溶液 39ml、凝血酶溶液 3.0ml，立即混匀，快速倒入直径 14cm 塑料培养皿中，室温水平放置 1 小时，打孔。精密量取不同浓度的蚓激酶标准品溶液及供试品溶液各 10μl，分别点在同一平皿中，加盖，置 37℃恒温箱中反应 18 小时。取出后用卡尺测量溶圈垂直两直径，以激酶标准品单位数的对数为横坐标，垂直两直径乘积的对数为纵坐标，计算回归方程，将供试品垂直两直径乘积的对数代入回归方程，计算供试品效价单位数。标准品与供试品应各做两点，以平均值计算。
蛋白含量
取本品约 20mg，精密称定，照氮测定法（中国药典 2000 年版二部附录 VII D 第二法）测定，将结果乘以 6.25，即为供试品中蛋白含量，并计算每 1mg 供试品中的蛋白毫克数。
比活力
按下式计算比活力：
Earthworm Fibrinolytic Enzyme (Lumbrokinase) is a type of enzyme extracted and purified from earthworms that can dissolve fibrin. It is present in the digestive tract of earthworms. Earthworm Fibrinolytic Enzyme has a dual function: in addition to directly hydrolyzing fibrin, it can also activate plasminogen into plasmin, thereby exerting an indirect fibrin-hydrolyzing effect. After animals take Earthworm Fibrinolytic Enzyme, obvious antithrombotic and thrombolytic effects are produced.
Lumbokinase attenuates myocardial ischemia-reperfusion (I-R) injury through the activation of Sirt1 signaling, and thus enhances autophagic flux and reduces I-R-induced oxidative damage, inflammation and apoptosis.
Characteristics
This product is a pale yellow to yellowish-brown powder; it has a fishy odor and is hygroscopic.
Identification
- Take an appropriate amount of this product, add water to prepare a solution containing 0.5mg per 1ml, and determine according to the spectrophotometry (Appendix IV A of the 2000 Edition of Pharmacopoeia of the People's Republic of China, Volume II); there shall be a maximum absorption at a wavelength of 278nm.
- Take an appropriate amount of this product, add 0.9% sodium chloride solution to prepare a solution containing 10mg per 1ml as the test solution; use a No. 6 needle to take 1 drop of animal blood and place it at the bottom of a test tube. After 30 minutes, add 1ml of the test solution to the test tube, shake gently, and the blood clot shall dissolve within 60 minutes. Use 1ml of 0.9% sodium chloride solution as the blank, and perform the same operation; the blood clot shall not dissolve.
Tests
- Acidity and Alkalinity: Take this product, add water to prepare a solution containing 1mg per 1ml, and determine according to the method (Appendix VI H of the 2000 Edition of Pharmacopoeia of the People's Republic of China, Volume II); the pH value shall be 6.0 - 8.0.
- Clarity and Color of Solution: Take an appropriate amount of this product, add water to prepare a solution containing 1mg per 1ml; the solution shall be clear. If colored, it shall not be darker than the Yellow No. 2 Standard Color Solution (Method 1 of Appendix IX A of the 2000 Edition of Pharmacopoeia of the People's Republic of China, Volume II).
- Loss on Drying: Take an appropriate amount of this product, use phosphorus pentoxide as a desiccant, dry it under reduced pressure at 60℃ to constant weight; the weight loss shall not exceed 5.0% (Appendix VIII L of the 2000 Edition of Pharmacopoeia of the People's Republic of China, Volume II).Determination of Specific Activity
Titer Determination
Reagents
- 0.01mol phosphate buffer (pH 7.8): Take 3.58g of disodium hydrogen phosphate, add water to dissolve and dilute to 1000ml as Solution A; take 0.78g of sodium dihydrogen phosphate (NaH₂PO₄·2H₂O), add water to dissolve and dilute to 500ml as Solution B; mix Solutions A and B until the pH value reaches 7.8.
- Working Solution: Mix 0.01mol/L phosphate buffer (pH 7.8) and 0.9% sodium chloride solution at a ratio of 1:17.
- 1.5% Agarose Solution: Take 1.5g of agarose, add 100ml of the working solution, and heat to dissolve.
- Fibrinogen Solution: Take an appropriate amount of fibrinogen, add the working solution to prepare a coagulable protein solution containing 1.5mg per 1ml.
- Thrombin Solution: Take thrombin, add 0.9% sodium chloride solution to prepare a solution containing 1 BP unit per 1ml.
- Preparation of Standard Solution: Take the lumbrokinase standard, add 0.9% sodium chloride solution to prepare solutions with concentrations of 10,000, 8,000, 6,000, 4,000, and 2,000 lumbrokinase units per 1ml respectively.
- Preparation of Test Solution: Take an appropriate amount of this product, add 0.9% sodium chloride solution to dissolve and dilute to a concentration within the range of the standard curve.
- Determination Method: Take 39ml of the fibrinogen solution, place it in a beaker, add 39ml of the 55℃ agarose solution and 3.0ml of the thrombin solution while stirring, mix immediately, and quickly pour into a plastic petri dish with a diameter of 14cm. Place it horizontally at room temperature for 1 hour, then punch holes. Precisely measure 10μl each of the lumbrokinase standard solutions of different concentrations and the test solution, spot them on the same petri dish respectively, cover the dish, and place it in a 37℃ incubator for 18 hours of reaction. After taking it out, use a caliper to measure the vertical diameters of the lysis zones. Take the logarithm of the number of units of the kinase standard as the abscissa and the logarithm of the product of the vertical diameters as the ordinate, calculate the regression equation, and substitute the logarithm of the product of the vertical diameters of the test sample into the regression equation to calculate the titer units of the test sample. Both the standard and the test sample shall be tested in duplicate, and the average value shall be used for calculation.
Protein Content
Take about 20mg of this product, weigh it accurately, determine according to the nitrogen determination method (Method 2 of Appendix VII D of the 2000 Edition of Pharmacopoeia of the People's Republic of China, Volume II), multiply the result by 6.25 to get the protein content in the test sample, and calculate the milligrams of protein per 1mg of the test sample.
Specific Activity
Calculate the specific activity according to the following formula:
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