阅读说明：本技术 一种实验室使用活体水蛭提取水蛭素的装置及其方法 (device and method for extracting hirudin from living leech in laboratory ) 是由 刘子超 仝向荣 毕晓旭 苏源 袁永生 祝平 于 2019-12-05 设计创作，主要内容包括：本发明公开了一种实验室使用活体水蛭提取水蛭素的装置及其方法,涉及水蛭素提取技术领域。所述装置包括顶部开口的箱体、中隔板、升降盖板和挤压板,中隔板设置于箱体中部,中隔板上均匀分布有条形凹槽,条形凹槽底部开设有若干出液孔；中隔板上方设置有升降盖板,升降盖板上设置有诱导液盛装槽,诱导液盛装槽与条形凹槽相适配；升降盖板上方设置有挤压板,挤压板底部设置有凸部,凸部与诱导液盛装槽相适配。条形凹槽两侧设置有第一电极,诱导液盛装槽两侧设置有第二电极,第一电极和第二电极接触后导通。条形凹槽和升降盖板可将水蛭活动范围进行限制,通过诱导液盛装槽有效对水蛭进行诱导,促使其分泌唾液；通过挤压、电刺激促使水蛭分泌腺液。(The invention discloses a device and a method for extracting hirudin from live leeches in laboratories, and relates to the technical field of hirudin extraction, wherein the device comprises a box body with an opening at the top, a middle partition plate, a lifting cover plate and a squeezing plate, the middle partition plate is arranged in the middle of the box body, strip-shaped grooves are uniformly distributed on the middle partition plate, a plurality of liquid outlet holes are formed in the bottoms of the strip-shaped grooves, the lifting cover plate is arranged above the middle partition plate, an inducing liquid containing groove is formed in the lifting cover plate and matched with the strip-shaped grooves, the squeezing plate is arranged above the lifting cover plate, a convex part is arranged at the bottom of the squeezing plate and matched with the inducing liquid containing groove, electrodes are arranged on two sides of each strip-shaped groove, a second electrode is arranged on two sides of each inducing liquid containing groove, the electrodes are communicated after being contacted with the second electrodes, the strip-shaped grooves and the lifting cover plate can limit the moving range of the leeches, effectively induce the leeches through the inducing.)

The device for extracting hirudin from living leeches in the laboratories is characterized by comprising a box body (1) with an opening at the top, a middle partition plate (2), a lifting cover plate (3) and an extrusion plate (4), wherein the middle partition plate (2) is arranged in the middle of the box body (1), strip-shaped grooves (21) are uniformly distributed in the middle partition plate (2), a plurality of liquid outlet holes are formed in the bottoms of the strip-shaped grooves (21), the lifting cover plate (3) is arranged above the middle partition plate (2), an inducing liquid containing groove (31) is formed in the lifting cover plate (3), the inducing liquid containing groove (31) is matched with the strip-shaped grooves (21), the extrusion plate (4) is arranged above the lifting cover plate (3), a convex part (41) is arranged at the bottom of the extrusion plate (4), and the convex part (41) is matched with the inducing liquid containing groove (31.

2. The device for extracting hirudin from living leeches in laboratory according to claim 1, wherein the bar-shaped groove (21) is provided with electrodes (22) at both sides, the inducing liquid containing groove (31) is provided with a second electrode (32) at both sides, and the electrode (22) and the second electrode (32) are conducted after contacting.

3. The device for extracting hirudin from living leeches in laboratory according to claim 1, wherein the inducing liquid holding tank (31) is made of bullfrog skin.

4. The device for extracting hirudin from living leeches in laboratories is characterized in that the side wall of the box body (1) is provided with a water inlet (11) and a water outlet (12), the water inlet (11) is positioned above the middle partition plate (2), and the water outlet (12) is positioned at the bottom of the side wall of the box body (1).

The method for extracting hirudin from live leeches in laboratories is characterized by comprising the following steps:

1) starvation treatment: cleaning Hirudo, sterilizing, placing into vitamin C solution, starving for 24 hr, cleaning again, and sterilizing;

2) and (3) induction treatment: the leech is placed in the strip-shaped groove (21), the lifting cover plate (3) is covered, and the inducing liquid containing groove (31) contains the sterilized inducing liquid;

3) crude extraction of hirudin: after the induced liquid is sucked cleanly, extruding the leech in the strip-shaped groove (21) by using an extrusion plate (4) to secrete the glandular liquid, and taking out the leech after repeating for several times; washing the lifting cover plate (3) and the middle partition plate (2) with purified water to obtain a leech saliva sample, and leaching and centrifuging to obtain a precipitate to obtain a hirudin crude product;

4) and (3) dialysis purification: dialyzing the hirudin crude product, and performing rotary evaporation on the ultrafiltered supernatant to obtain a concentrated solution;

5) and (3) freeze drying: freeze-drying the concentrated solution in a freeze dryer to obtain the high-purity hirudin product.

6. The method for extracting hirudin from live leeches in laboratory according to claim 5, wherein the step 3) is performed by simultaneously energizing the device and stimulating leeches with pulse current through the strip-shaped groove (21), wherein the peak current value is 4-8 mA and the frequency is 10-20 times/sec.

7. The method for extracting hirudin from kinds of laboratory living leeches according to claim 1, wherein the leech cleaning and sterilizing in step 1) comprises rinsing leeches with deionized water to remove impurities on the surface, soaking in normal saline mixed with a sterilizing agent for 2-5 minutes, cleaning again, and draining.

Technical Field

The invention relates to the technical field of hirudin extraction, in particular to a device and a method for extracting hirudin from live leeches in laboratories.

Background

Animal experiments and clinical studies show that the hirudin can efficiently anticoagulate and resist thrombus, can prevent the blood stasis phenomenon of steps such as thrombin-catalyzed activation of coagulation factors and platelet reaction, and the like.

Researches show that a large amount of hirudin can be extracted from leeches and salivary gland secretions thereof. At present, the industrial production method of hirudin mostly uses the powder of dried leech and then crushed as the raw material for extraction, or uses living leech for pulping as the raw material for extraction. The method has many defects, mainly including low effective component content of hirudin after extraction, low yield, high impurity content, and dependence on leech resource supply. The laboratory needs hirudin with high purity and high active ingredients for research, and the extraction method has high efficiency and low cost and cannot depend on the supply of leech resources too much.

Disclosure of Invention

The invention aims to provide a device and a method for extracting hirudin from live leeches in laboratories, and solves the problems that leech resources are excessively relied on, the extracted hirudin has low effective components, low yield and high impurity content, and the experimental requirements are difficult to meet in the prior art.

The device for extracting hirudin from live leeches in laboratories is characterized by comprising a box body with an opening at the top, a middle partition plate, a lifting cover plate and a squeezing plate, wherein the middle partition plate is arranged in the middle of the box body, strip-shaped grooves are uniformly distributed in the middle partition plate, a plurality of liquid outlet holes are formed in the bottoms of the strip-shaped grooves, the lifting cover plate is arranged above the middle partition plate, an inducing liquid containing groove is formed in the lifting cover plate and matched with the strip-shaped grooves, the squeezing plate is arranged above the lifting cover plate, a convex part is arranged at the bottom of the squeezing plate, and the convex part is matched with the inducing liquid containing groove.

The technical scheme of step is that both sides of the strip-shaped groove are provided with a electrode, both sides of the inducing liquid containing groove are provided with a second electrode, and the electrode is conducted after contacting the second electrode.

The technical proposal of step is that the inducing liquid holding tank is made of bullfrog skin.

The technical scheme of step is that the side wall of the box body is provided with a water inlet and a water outlet, the water inlet is positioned above the middle partition plate, and the water outlet is positioned at the bottom of the side wall of the box body.

The invention also relates to a method for extracting hirudin from laboratories by using live leeches, which is characterized by comprising the following steps:

1) starvation treatment: cleaning Hirudo, sterilizing, placing into vitamin C solution, starving for 24 hr, cleaning again, and sterilizing;

2) and (3) induction treatment: placing leeches in the strip-shaped grooves, covering the lifting cover plate, and placing the sterilized inducing liquid in the inducing liquid containing groove;

3) crude extraction of hirudin: after the induced liquid is sucked cleanly, extruding the leech in the strip-shaped groove by using an extrusion plate to enable the leech to secrete saliva, and taking out the leech after repeating for several times; washing the lifting cover plate and the middle partition plate with purified water to obtain a leech saliva sample, leaching and centrifuging, and taking a precipitate to obtain a hirudin crude product;

4) and (3) dialysis purification: dialyzing the hirudin crude product, and performing rotary evaporation on the ultrafiltered supernatant to obtain a concentrated solution;

5) and (3) freeze drying: freeze-drying the concentrated solution in a freeze dryer to obtain the high-purity hirudin product.

, electrifying the device when the extrusion operation in the step 3) is carried out, and carrying out pulse current stimulation on the leech through the strip-shaped groove, wherein the current peak value is 4-8 mA, and the frequency is 10-20 times/second.

, the technical scheme is that the leech cleaning and sterilizing in the step 1) is to rinse impurities on the surface of the leech by using deionized water, then put the leech into normal saline mixed with a sterilizing agent to be soaked for 2-5 minutes, then clean the leech again, and filter the water.

The working principle is as follows: and (3) placing the cleaned leeches into a vitamin C aqueous solution for hungry treatment, cleaning and sterilizing again, draining water, and laying the leeches in the strip-shaped grooves individually. And simultaneously lowering the lifting cover plate to enable the inducing liquid containing groove to be in contact with the leech, so that the leech in a hungry state eats the inducing liquid and secretes saliva. Impurities or germs on the surface of the washed and sterilized leech can not be brought into the hirudin.

After the induced liquid is sucked cleanly, the leech is slightly extruded through the extrusion plate to be secreted by the glandular liquid, and the process can be repeated for many times, the electrodes are conducted after being contacted with each other while the extrusion is carried out, the strip-shaped grooves are locally electrified to electrically stimulate the leech above, and steps are carried out to stimulate the leech to secrete the glandular liquid.

The method can repeatedly extract hirudin from living Hirudo, and has high yield, low impurity content, and high production efficiency.

Compared with the prior art, the invention has the beneficial effects that simple devices and methods for extracting hirudin through leech living bodies are provided, the moving range of leeches can be limited through the strip-shaped grooves and the lifting cover plate, the subsequent operation is convenient, the leeches are effectively induced through the inducing liquid containing grooves, the leeches are promoted to secrete saliva, the leeches can be promoted to secrete gland liquid through extrusion and electrical stimulation.

Drawings

FIG. 1 is a schematic view showing the internal structure of the hirudin extraction apparatus of the present invention.

FIG. 2 is a flow chart of the hirudin extraction method of the present invention.

In the figure, 1-a box body, 2-a middle partition board, 3-a lifting cover board, 4-a squeezing board, 11-a water inlet, 12-a water outlet, 21-a strip-shaped groove, 22-an th electrode, 31-an inducing liquid containing groove, 32-a second electrode and 41-a convex part.

Detailed Description

For purposes of making the objects, aspects and advantages of the present invention more apparent, the following detailed description proceeds with reference to the accompanying drawings and examples.