A placental lipopolysaccharide preparation and preparation method thereof

Abstract

The present invention relates to the technical field of lipopolysaccharide preparations, specifically, to a placental lipopolysaccharide preparation and a preparation method thereof, comprising the following components: 90-95 parts by weight of placental tissue, 1-5 parts by weight of sodium carbonate, 0.1-1 parts by weight of trypsin, 0.1-0.5 parts by weight of leupeptin, 1-2 parts by weight of chloroform, 0.1-1 parts by weight of chondroitin sulfate, 5-10 parts by weight of trichloroacetic acid, 70-80 parts by weight of methanol, 0.1-1 parts by weight of ascorbic acid and 0.5-1 parts by weight of sodium chloride. Since leupeptin can inhibit the activity of trypsin, it protects components such as proteins and peptides that have auxiliary effects on immunomodulation from being destroyed, thereby ensuring the quality and efficacy of the final preparation. Adding chondroitin sulfate can form a physical barrier or interact with lipopolysaccharide intermolecularly, thereby reducing the exposure of lipopolysaccharide in a complex chemical environment and preventing it from undergoing structural changes or loss of activity due to changes in the chemical environment.

Description

Technical Field

[0001] The present invention relates to the technical field of lipopolysaccharide preparations, in particular to a placenta lipopolysaccharide preparation and a preparation method thereof. Background Art

[0002] Placental tissue is a kind of tissue rich in a variety of bioactive substances, including proteins, peptides, lipids, carbohydrates, enzymes and cytokines. These components have potential application value in immunomodulation, anti-inflammation, anti-oxidation, and promotion of wound healing. Especially in traditional medicine, placental tissue has a long history of being used to treat various diseases; lipopolysaccharide (LPS), also known as endotoxin, is an important component of the outer membrane of Gram-negative bacteria. Although LPS is mainly derived from bacteria, similar structures from certain mammalian sources (such as lipopolysaccharide-like molecules in placental tissue) have also been found and show unique biological properties. Studies have shown that placental tissue lipopolysaccharide is involved in the maternal-fetal immune tolerance mechanism and has a special role in inflammatory response and immunomodulation.

[0003] The existing extraction methods of placental tissue on the market often have the following problems: it is difficult to effectively remove impurities during the extraction process, resulting in low concentrations of target components; the thermal stress or other adverse conditions introduced during the processing process affect the structural integrity and biological activity of lipopolysaccharide. In view of this, we propose a placental lipopolysaccharide preparation and a preparation method thereof. Summary of the invention

[0004] The purpose of the present invention is to provide a placental lipopolysaccharide preparation and a preparation method thereof, so as to solve the problem proposed in the above-mentioned background technology that it is difficult to effectively remove impurities in the placental tissue lipopolysaccharide extraction process, resulting in low concentration of target components; and the thermal stress or other adverse conditions introduced during the processing process affect the structural integrity and biological activity of lipopolysaccharide.

[0005] To achieve the above object, the present invention provides a placental lipopolysaccharide preparation, comprising the following raw materials: 90-95 parts by weight of placental tissue, 1-5 parts by weight of sodium carbonate, 0.1-1 parts by weight of trypsin, 0.1-0.5 parts by weight of leupeptin, 1-2 parts by weight of chloroform, 0.1-1 parts by weight of chondroitin sulfate, 5-10 parts by weight of trichloroacetic acid, 70-80 parts by weight of methanol, 0.1-1 parts by weight of ascorbic acid and 0.5-1 parts by weight of sodium chloride.

[0006] In another aspect, the present invention provides a method for preparing a placental lipopolysaccharide preparation, which is used to prepare the above-mentioned placental lipopolysaccharide preparation, comprising the following steps:

[0007] S1.1. Weigh the following raw materials in parts by weight respectively: 90-95 parts by weight of placental tissue, 1-5 parts by weight of sodium carbonate, 0.1-1 parts by weight of trypsin, 0.1-0.5 parts by weight of leupeptin, 1-2 parts by weight of chloroform, 0.1-1 parts by weight of chondroitin sulfate, 5-10 parts by weight of trichloroacetic acid, 70-80 parts by weight of methanol, 0.1-1 parts by weight of ascorbic acid and 0.5-1 parts by weight of sodium chloride;

[0008] S1.2. Thaw the placenta tissue in a thawing room at 20°C for no more than 24 hours. Remove the blood from the placenta tissue, pick out the blood clots and fascia, rinse with drinking water until it is close to flesh color, and drain for later use.

[0009] S1.3, mincing the drained placental tissue with a high-speed disperser at a speed of 5000-8000 rpm for 3-5 min and weighing it, and alkalizing the squeezed placental tissue;

[0010] S1.4. Add 10,000 units of trypsin per 1 kg of placental tissue for trypsin digestion and hydrolysis; when the pH value is maintained at 9.0±0.2, add 1% by volume chloroform and continue digestion for 24-28 hours. When the amino nitrogen accounts for 30% of the total nitrogen, add leupeptin solution to obtain a digestion solution;

[0011] Trypsin has non-specific enzymatic effects during the digestion process. When the digestion reaches a certain stage (such as amino nitrogen / total nitrogen reaches 30%), the addition of leupeptin solution can effectively inhibit the excessive activity of trypsin. Leupeptin is a specific protease inhibitor that can bind to the active site of trypsin and prevent it from continuing to hydrolyze other proteins or peptides. This inhibitory effect helps to accurately control the extent of the digestion reaction, thereby improving the extraction efficiency of target components (such as lipopolysaccharide), reducing the hydrolysis of non-essential components, and maintaining the stability of the extract components.

[0012] In addition to lipopolysaccharide, there are also some proteins and peptides that have auxiliary effects on immunoregulation in placental tissue. If trypsin continues to act, the integrity of these components will be destroyed. Therefore, the addition of leupeptin can protect these potential active ingredients from excessive enzymatic hydrolysis, thereby ensuring the quality and efficacy of the final placental lipopolysaccharide preparation. In addition, some small molecule peptides with immunoregulatory activity will be destroyed in excessive trypsin digestion. Leupeptin can preserve these small molecule peptides by inhibiting trypsin, thereby enhancing the comprehensive immunoregulatory ability of the preparation.

[0013] S1.5, stirring the digestion liquid at a speed of 25 r / min, heating at 60°C for more than 20 h for pasteurization; subjecting the pasteurized digestion liquid to precipitation treatment to obtain a supernatant;

[0014] S1.6, adding methanol to the supernatant for precipitation; collecting the precipitate and performing freeze-drying, and collecting under a sterile environment to obtain placental lipopolysaccharide raw material;

[0015] S1.7. Dilute the placental lipopolysaccharide raw material with water for injection to a concentration of 18-25 μg / mL, add ascorbic acid and sodium chloride to prepare a solution, sterilize and filter in a Class B environment and local Class A, divide into containers, seal, and obtain the placental lipopolysaccharide preparation.

[0016] Preferably, in S1.3, the placental tissue is alkalinized by adding a 1% sodium carbonate solution in a ratio of 1:1 to the weight of the placental tissue so that the pH value of the solution reaches 10, and the solution is mixed and allowed to stand for 12-24 hours.

[0017] Preferably, in S1.4, the temperature of trypsin digestion and hydrolysis is 37±2°C, and the pH value is adjusted every 15-30 minutes with a 2 mol / L sodium hydroxide solution within 2 hours to keep the pH value at 9.0±0.2.

[0018] Preferably, in S1.4, the concentration of the leupeptin solution is 1-10 mM.

[0019] Preferably, in S1.5, the precipitation treatment is performed by adding a 0.1-1% chondroitin sulfate solution to the digestive fluid, then slowly adding 5% by mass trichloroacetic acid, and standing at room temperature for 16-48 hours; taking out the supernatant, and centrifuging the lower suspension at a speed of 4000r / min for 5 minutes to separate the supernatant.

[0020] In placental lipopolysaccharide preparations, active ingredients such as lipopolysaccharide undergo structural changes or inactivation during the subsequent precipitation process due to changes in the chemical environment (such as changes in pH and ionic strength caused by the addition of trichloroacetic acid); chondroitin sulfate can stabilize the active ingredients by forming a relatively stable complex with lipopolysaccharide and other ingredients, thereby reducing the exposure and damage of the active ingredients during the precipitation process and maintaining their biological activity; lipopolysaccharide contains a polysaccharide part in its structure, and chondroitin sulfate also belongs to a polysaccharide substance, and there are interactions between them, such as hydrogen bonds, electrostatic interactions, etc.; this interaction can shield the influence of adverse external factors on lipopolysaccharide to a certain extent, just like a protective film that wraps around lipopolysaccharide to ensure its structural integrity in a complex chemical treatment environment.

[0021] Chondroitin sulfate has the characteristic of being negatively charged in solution, and its molecular structure contains a large number of carboxyl and sulfate groups, which enable it to bind to other substances such as metal ions. In the digestive juice, chondroitin sulfate can interact with incompletely digested protein fragments or other macromolecular impurities. When trichloroacetic acid is added, the complex formed by chondroitin sulfate and impurities is more easily precipitated due to its change in the pH value and ionic environment of the solution. This process helps to more effectively separate impurities and target components, thereby improving the purity of lipopolysaccharide in the supernatant. In addition, through the synergistic effect of trichloroacetic acid, impurities are precipitated to the bottom layer, making the supernatant obtained by subsequent centrifugation purer, reducing the interference of impurities on the quality of the final placental lipopolysaccharide preparation.

[0022] Preferably, the speed of slowly adding trichloroacetic acid is 5-10 mL / min.

[0023] Preferably, in S1.6, methanol is added for precipitation treatment by adding methanol to the supernatant to a final concentration of 70%, standing for 12-15 hours, and collecting the precipitate by centrifugation at 5000 r / min for 5 minutes; adding 5-10 times the concentration of 0.9% sodium chloride solution to the precipitate, heating to dissolve the precipitate, and collecting the supernatant by centrifugation at 5000 r / min for 5 minutes; adding methanol to the collected supernatant to a final concentration of 80%, standing for 15-20 hours, and collecting the precipitate by centrifugation at 4000 r / min for 5 minutes.

[0024] Preferably, the heating temperature for heating and dissolving the precipitate is 45-50°C.

[0025] Preferably, in S1.6, the freeze-drying treatment is to maintain the precipitate at -40°C for 1 hour, then heat it to 1°C within 3 hours and keep it for 3 hours, then heat it to 15°C and keep it for 2 hours, and finally heat it to 22°C and keep it for 2 hours.

[0026] Compared with the prior art, the present invention has the following beneficial effects:

[0027] 1. In the placental lipopolysaccharide preparation and its preparation method, leupeptin is added because it can specifically inhibit the activity of trypsin, thereby preventing the target component (such as lipopolysaccharide) from excessive enzymatic hydrolysis, making the extraction process more controllable; in addition, during the enzymatic hydrolysis process, leupeptin can protect components such as proteins and peptides that have an auxiliary effect on immunomodulation from being destroyed, thereby ensuring the quality and efficacy of the final preparation.

[0028] 2. In the placental lipopolysaccharide preparation and its preparation method, the addition of chondroitin sulfate can form a physical barrier or interact with lipopolysaccharide intermolecularly, thereby reducing the exposure of lipopolysaccharide to a complex chemical environment and preventing it from undergoing structural changes or loss of activity due to changes in the chemical environment; when trichloroacetic acid is added, the pH value and ion environment of the solution change, and chondroitin sulfate can combine with impurities and precipitate them to the bottom layer, thereby making the subsequent supernatant purer, which is beneficial to improving the quality of the final preparation. DETAILED DESCRIPTION

[0029] The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0030] The invention provides a placental lipopolysaccharide preparation, which comprises the following raw materials: 90-95 parts by weight of placental tissue, 1-5 parts by weight of sodium carbonate, 0.1-1 parts by weight of trypsin, 0.1-0.5 parts by weight of leupeptin, 1-2 parts by weight of chloroform, 0.1-1 parts by weight of chondroitin sulfate, 5-10 parts by weight of trichloroacetic acid, 70-80 parts by weight of methanol, 0.1-1 parts by weight of ascorbic acid and 0.5-1 parts by weight of sodium chloride.

[0031] When choosing placental tissue as raw material, it is necessary to ensure that it undergoes rigorous testing, including testing for infectious diseases such as hepatitis B surface antigen, hepatitis C virus, syphilis spirochete and HIV, to ensure that all indicators are negative to ensure safe use.

[0032] Example 1: A method for preparing a placental lipopolysaccharide preparation, comprising the following steps:

[0033] S1.1. Weigh the following raw materials in parts by weight respectively: 93 parts by weight of placental tissue, 3 parts by weight of sodium carbonate, 0.4 parts by weight of trypsin, 0.3 parts by weight of leupeptin, 1.5 parts by weight of chloroform, 0.7 parts by weight of chondroitin sulfate, 8 parts by weight of trichloroacetic acid, 75 parts by weight of methanol, 0.5 parts by weight of ascorbic acid and 0.9 parts by weight of sodium chloride;

[0034] S1.2. Thaw the placenta tissue in a thawing room at 20°C for no more than 24 hours. Remove the blood from the placenta tissue, pick out the blood clots and fascia, rinse with drinking water until it is close to flesh color, and drain for later use.

[0035] S1.3, mince the drained placenta tissue with a high-speed disperser at a speed of 5000 rpm for 5 minutes and weigh it, add 1% sodium carbonate solution to the squeezed placenta tissue at a weight ratio of 1:1 to make the solution pH value reach 10, mix well and let it stand for 24 hours;

[0036] S1.4. Add 10,000 units of trypsin per 1 kg of placental tissue for trypsin digestion and hydrolysis at 37°C. Adjust the pH value with 2 mol / L sodium hydroxide solution every 20 min within 2 hours to keep the pH value at 9.0. Add 1% by volume chloroform and continue digestion for 28 hours. When amino nitrogen accounts for 30% of the total nitrogen, add 5 mM leupeptin solution to obtain a digestion solution.

[0037] S1.5. Stir the digestion liquid at a speed of 25 r / min, and heat it at 60°C for more than 20 h for pasteurization; add a 0.5% chondroitin sulfate solution to the pasteurized digestion liquid, and then add 5% trichloroacetic acid by mass at a speed of 5 mL / min, and let it stand at room temperature for 48 h; take out the supernatant, and centrifuge the lower suspension at a speed of 4000 r / min for 5 min to separate the supernatant to obtain the supernatant;

[0038] S1.6, adding methanol to the supernatant to make the final concentration reach 70%, after standing for 15 hours, centrifuging at 5000r / min for 5 minutes to collect the precipitate; adding 5 times the concentration of 0.9% sodium chloride solution to the precipitate, heating to 45°C to dissolve the precipitate, centrifuging at 5000r / min for 5 minutes to collect the supernatant; adding methanol to the collected supernatant to make the final concentration reach 80%, after standing for 20 hours, centrifuging at 4000r / min for 5 minutes to collect the precipitate; after collecting the precipitate, freeze-drying the precipitate, first maintaining it at -40°C for 1 hour, heating it to 1°C within 3 hours and keeping it warm for 3 hours, then heating it to 15°C and keeping it warm for 2 hours, and finally heating it to 22°C and keeping it warm for 2 hours, collecting it under a sterile environment to obtain the placental lipopolysaccharide raw material;

[0039] S1.7. Dilute the placental lipopolysaccharide raw material with water for injection to a concentration of 20 μg / mL, add ascorbic acid and sodium chloride to prepare a solution, sterilize and filter in a Class B environment and local Class A, divide into containers, seal, and obtain the placental lipopolysaccharide preparation.

[0040] Example 2: A method for preparing a placental lipopolysaccharide preparation, comprising the following steps:

[0041] S1.1. Weigh the following raw materials in parts by weight respectively: 93 parts by weight of placental tissue, 3 parts by weight of sodium carbonate, 0.4 parts by weight of trypsin, 0.5 parts by weight of leupeptin, 1.5 parts by weight of chloroform, 0.7 parts by weight of chondroitin sulfate, 8 parts by weight of trichloroacetic acid, 75 parts by weight of methanol, 0.5 parts by weight of ascorbic acid and 0.9 parts by weight of sodium chloride;

[0042] S1.2. Thaw the placenta tissue in a thawing room at 20°C for no more than 24 hours. Remove the blood from the placenta tissue, pick out the blood clots and fascia, rinse with drinking water until it is close to flesh color, and drain for later use.

[0043] S1.3, mince the drained placenta tissue with a high-speed disperser at a speed of 5000 rpm for 5 minutes and weigh it, add 1% sodium carbonate solution to the squeezed placenta tissue at a weight ratio of 1:1 to make the solution pH value reach 10, mix well and let it stand for 24 hours;

[0044] S1.4. Add 10,000 units of trypsin per 1 kg of placental tissue for trypsin digestion and hydrolysis at 37°C. Adjust the pH value with 2 mol / L sodium hydroxide solution every 20 min within 2 hours to keep the pH value at 9.0. Add 1% by volume chloroform and continue digestion for 28 hours. When amino nitrogen accounts for 30% of the total nitrogen, add 5 mM leupeptin solution to obtain a digestion solution.

[0045] S1.5. Stir the digestion liquid at a speed of 25 r / min, and heat it at 60°C for more than 20 h for pasteurization; add a 0.5% chondroitin sulfate solution to the pasteurized digestion liquid, and then add 5% trichloroacetic acid by mass at a speed of 5 mL / min, and let it stand at room temperature for 48 h; take out the supernatant, and centrifuge the lower suspension at a speed of 4000 r / min for 5 min to separate the supernatant to obtain the supernatant;

[0046] S1.6, adding methanol to the supernatant to make the final concentration reach 70%, after standing for 15 hours, centrifuging at 5000r / min for 5 minutes to collect the precipitate; adding 5 times the concentration of 0.9% sodium chloride solution to the precipitate, heating to 45°C to dissolve the precipitate, centrifuging at 5000r / min for 5 minutes to collect the supernatant; adding methanol to the collected supernatant to make the final concentration reach 80%, after standing for 20 hours, centrifuging at 4000r / min for 5 minutes to collect the precipitate; after collecting the precipitate, freeze-drying the precipitate, first maintaining it at -40°C for 1 hour, heating it to 1°C within 3 hours and keeping it warm for 3 hours, then heating it to 15°C and keeping it warm for 2 hours, and finally heating it to 22°C and keeping it warm for 2 hours, collecting it under a sterile environment to obtain the placental lipopolysaccharide raw material;

[0047] S1.7. Dilute the placental lipopolysaccharide raw material with water for injection to a concentration of 20 μg / mL, add ascorbic acid and sodium chloride to prepare a solution, sterilize and filter in a Class B environment and local Class A, divide into containers, seal, and obtain the placental lipopolysaccharide preparation.

[0048] Example 3: A method for preparing a placental lipopolysaccharide preparation, comprising the following steps:

[0049] S1.1. Weigh the following raw materials in parts by weight respectively: 93 parts by weight of placental tissue, 3 parts by weight of sodium carbonate, 0.4 parts by weight of trypsin, 0.3 parts by weight of leupeptin, 1.5 parts by weight of chloroform, 0.1 parts by weight of chondroitin sulfate, 8 parts by weight of trichloroacetic acid, 75 parts by weight of methanol, 0.5 parts by weight of ascorbic acid and 0.9 parts by weight of sodium chloride;

[0050] S1.2. Thaw the placenta tissue in a thawing room at 20°C for no more than 24 hours. Remove the blood from the placenta tissue, pick out the blood clots and fascia, rinse with drinking water until it is close to flesh color, and drain for later use.

[0051] S1.3, mince the drained placenta tissue with a high-speed disperser at a speed of 5000 rpm for 5 minutes and weigh it, add 1% sodium carbonate solution to the squeezed placenta tissue at a weight ratio of 1:1 to make the solution pH value reach 10, mix well and let it stand for 24 hours;

[0052] S1.4. Add 10,000 units of trypsin per 1 kg of placental tissue for trypsin digestion and hydrolysis at 37°C. Adjust the pH value with 2 mol / L sodium hydroxide solution every 20 min within 2 hours to keep the pH value at 9.0. Add 1% by volume chloroform and continue digestion for 28 hours. When amino nitrogen accounts for 30% of the total nitrogen, add 5 mM leupeptin solution to obtain a digestion solution.

[0053] S1.5. Stir the digestion liquid at a speed of 25 r / min, and heat it at 60°C for more than 20 h for pasteurization; add a 0.5% chondroitin sulfate solution to the pasteurized digestion liquid, and then add 5% trichloroacetic acid by mass at a speed of 5 mL / min, and let it stand at room temperature for 48 h; take out the supernatant, and centrifuge the lower suspension at a speed of 4000 r / min for 5 min to separate the supernatant to obtain the supernatant;

[0054] S1.6, adding methanol to the supernatant to make the final concentration reach 70%, after standing for 15 hours, centrifuging at 5000r / min for 5 minutes to collect the precipitate; adding 5 times the concentration of 0.9% sodium chloride solution to the precipitate, heating to 45°C to dissolve the precipitate, centrifuging at 5000r / min for 5 minutes to collect the supernatant; adding methanol to the collected supernatant to make the final concentration reach 80%, after standing for 20 hours, centrifuging at 4000r / min for 5 minutes to collect the precipitate; after collecting the precipitate, freeze-drying the precipitate, first maintaining it at -40°C for 1 hour, heating it to 1°C within 3 hours and keeping it warm for 3 hours, then heating it to 15°C and keeping it warm for 2 hours, and finally heating it to 22°C and keeping it warm for 2 hours, collecting it under a sterile environment to obtain the placental lipopolysaccharide raw material;

[0055] S1.7. Dilute the placental lipopolysaccharide raw material with water for injection to a concentration of 20 μg / mL, add ascorbic acid and sodium chloride to prepare a solution, sterilize and filter in a Class B environment and local Class A, divide into containers, seal, and obtain the placental lipopolysaccharide preparation.

[0056] Example 4: A method for preparing a placental lipopolysaccharide preparation, comprising the following steps:

[0057] S1.1. Weigh the following raw materials in parts by weight respectively: 93 parts by weight of placental tissue, 3 parts by weight of sodium carbonate, 0.4 parts by weight of trypsin, 0.5 parts by weight of leupeptin, 1.5 parts by weight of chloroform, 0.9 parts by weight of chondroitin sulfate, 8 parts by weight of trichloroacetic acid, 75 parts by weight of methanol, 0.5 parts by weight of ascorbic acid and 0.9 parts by weight of sodium chloride;

[0058] S1.2. Thaw the placenta tissue in a thawing room at 20°C for no more than 24 hours. Remove the blood from the placenta tissue, pick out the blood clots and fascia, rinse with drinking water until it is close to flesh color, and drain for later use.

[0059] S1.3, mince the drained placenta tissue with a high-speed disperser at a speed of 5000 rpm for 5 minutes and weigh it, add 1% sodium carbonate solution to the squeezed placenta tissue at a weight ratio of 1:1 to make the solution pH value reach 10, mix well and let it stand for 24 hours;

[0060] S1.4. Add 10,000 units of trypsin per 1 kg of placental tissue for trypsin digestion and hydrolysis at 37°C. Adjust the pH value with 2 mol / L sodium hydroxide solution every 20 min within 2 hours to keep the pH value at 9.0. Add 1% by volume chloroform and continue digestion for 28 hours. When amino nitrogen accounts for 30% of the total nitrogen, add 5 mM leupeptin solution to obtain a digestion solution.

[0061] S1.5. Stir the digestion liquid at a speed of 25 r / min, and heat it at 60°C for more than 20 h for pasteurization; add a 0.5% chondroitin sulfate solution to the pasteurized digestion liquid, and then add 5% trichloroacetic acid by mass at a speed of 5 mL / min, and let it stand at room temperature for 48 h; take out the supernatant, and centrifuge the lower suspension at a speed of 4000 r / min for 5 min to separate the supernatant to obtain the supernatant;

[0062] S1.6, adding methanol to the supernatant to make the final concentration reach 70%, after standing for 15 hours, centrifuging at 5000r / min for 5 minutes to collect the precipitate; adding 5 times the concentration of 0.9% sodium chloride solution to the precipitate, heating to 45°C to dissolve the precipitate, centrifuging at 5000r / min for 5 minutes to collect the supernatant; adding methanol to the collected supernatant to make the final concentration reach 80%, after standing for 20 hours, centrifuging at 4000r / min for 5 minutes to collect the precipitate; after collecting the precipitate, freeze-drying the precipitate, first maintaining it at -40°C for 1 hour, heating it to 1°C within 3 hours and keeping it warm for 3 hours, then heating it to 15°C and keeping it warm for 2 hours, and finally heating it to 22°C and keeping it warm for 2 hours, collecting it under a sterile environment to obtain the placental lipopolysaccharide raw material;

[0063] S1.7. Dilute the placental lipopolysaccharide raw material with water for injection to a concentration of 20 μg / mL, add ascorbic acid and sodium chloride to prepare a solution, sterilize and filter in a Class B environment and local Class A, divide into containers, seal, and obtain the placental lipopolysaccharide preparation.

[0064] Example 5: A method for preparing a placental lipopolysaccharide preparation, comprising the following steps:

[0065] S1.1. Weigh the following raw materials in parts by weight respectively: 90 parts by weight of placental tissue, 1 part by weight of sodium carbonate, 0.1 part by weight of trypsin, 0.1 part by weight of leupeptin, 1 part by weight of chloroform, 0.1 part by weight of chondroitin sulfate, 5 parts by weight of trichloroacetic acid, 70 parts by weight of methanol, 0.1 part by weight of ascorbic acid and 0.5 part by weight of sodium chloride;

[0066] S1.2. Thaw the placenta tissue in a thawing room at 20°C for no more than 24 hours. Remove the blood from the placenta tissue, pick out the blood clots and fascia, rinse with drinking water until it is close to flesh color, and drain for later use.

[0067] S1.3, mince the drained placenta tissue with a high-speed disperser at a speed of 5000 rpm for 5 minutes and weigh it, add 1% sodium carbonate solution to the squeezed placenta tissue at a weight ratio of 1:1 to make the solution pH value reach 10, mix well and let it stand for 24 hours;

[0068] S1.4. Add 10,000 units of trypsin per 1 kg of placental tissue for trypsin digestion and hydrolysis at 37°C. Adjust the pH value with 2 mol / L sodium hydroxide solution every 20 min within 2 hours to keep the pH value at 9.0. Add 1% by volume chloroform and continue digestion for 28 hours. When amino nitrogen accounts for 30% of the total nitrogen, add 5 mM leupeptin solution to obtain a digestion solution.

[0069] S1.5. Stir the digestion liquid at a speed of 25 r / min, and heat it at 60°C for more than 20 h for pasteurization; add a 0.5% chondroitin sulfate solution to the pasteurized digestion liquid, and then add 5% trichloroacetic acid by mass at a speed of 5 mL / min, and let it stand at room temperature for 48 h; take out the supernatant, and centrifuge the lower suspension at a speed of 4000 r / min for 5 min to separate the supernatant to obtain the supernatant;

[0070] S1.6, adding methanol to the supernatant to make the final concentration reach 70%, after standing for 15 hours, centrifuging at 5000r / min for 5 minutes to collect the precipitate; adding 5 times the concentration of 0.9% sodium chloride solution to the precipitate, heating to 45°C to dissolve the precipitate, centrifuging at 5000r / min for 5 minutes to collect the supernatant; adding methanol to the collected supernatant to make the final concentration reach 80%, after standing for 20 hours, centrifuging at 4000r / min for 5 minutes to collect the precipitate; after collecting the precipitate, freeze-drying the precipitate, first maintaining it at -40°C for 1 hour, heating it to 1°C within 3 hours and keeping it warm for 3 hours, then heating it to 15°C and keeping it warm for 2 hours, and finally heating it to 22°C and keeping it warm for 2 hours, collecting it under a sterile environment to obtain the placental lipopolysaccharide raw material;

[0071] S1.7. Dilute the placental lipopolysaccharide raw material with water for injection to a concentration of 20 μg / mL, add ascorbic acid and sodium chloride to prepare a solution, sterilize and filter in a Class B environment and local Class A, divide into containers, seal, and obtain the placental lipopolysaccharide preparation.

[0072] Comparative Example 1: The method of Example 4 was adopted to remove leupeptin in the preparation method of placental lipopolysaccharide preparation.

[0073] Comparative Example 2: The method of Example 4 was adopted to remove chondroitin sulfate in the preparation method of placental lipopolysaccharide preparation.

[0074] The present invention provides a placental lipopolysaccharide preparation prepared by adding leupeptin and chondroitin sulfate, wherein the performance index test items and test standards of the placental lipopolysaccharide preparation are as follows:

[0075] According to GB / T 26794-2011, add equal amounts of LAL reagent and diluted sample or standard endotoxin solution to a microtiter plate or test tube. The total volume of each well / tube is usually 0.1 mL. Mix gently and immediately place in a 37±1°C constant temperature incubator or incubator for 60 minutes. Use the standard endotoxin concentration as the horizontal axis and the corresponding OD value as the vertical axis to calculate the lipopolysaccharide content.

[0076] According to GB / T 14233.2-2005 standard, the purity of lipopolysaccharide was determined by high performance liquid chromatography.

[0077] The placental lipopolysaccharide preparations prepared in the above Examples 1-5 and Comparative Examples 1-2 were tested according to the above standards, and the obtained data are shown in the table:

[0078] Performance data of placental lipopolysaccharide preparations of Examples 1-5 and Comparative Examples 1-2

[0079]

[0080] From the above data, it can be seen that the placental lipopolysaccharide preparations in Examples 1-5 show significantly high purity and high content; Taking Example 4 as the optimal example, combined with Comparative Example 1, it can be seen that in the preparation method of the placental lipopolysaccharide preparation, when leupeptin is removed, the purity and content of the placental lipopolysaccharide preparation are significantly reduced;

[0081] Leuprorelin plays a key role in the preparation process, as it can inhibit the excessive enzymatic reaction of trypsin. When leuprorelin is removed, the activity of trypsin is out of control, resulting in excessive enzymatic reaction. In this case, trypsin will decompose lipopolysaccharide in the placental tissue and other components that protect or synergize lipopolysaccharide. In the absence of leuprorelin, excessive hydrolysis of these bound components by trypsin will damage the lipopolysaccharide structure, release it from its original bound state, and make it more easily lost in subsequent separation and purification steps, thereby reducing the lipopolysaccharide content. In addition, without the inhibition of leuprorelin, trypsin will continue to hydrolyze various proteins in the placental tissue, producing a large amount of impurities such as protein fragments. In subsequent separation and purification steps such as precipitation and centrifugation, excessive impurities will interfere with the separation of lipopolysaccharide. In the trichloroacetic acid precipitation treatment stage, a large amount of protein impurities are precipitated together with lipopolysaccharide or form a complex co-precipitation system, making it difficult to effectively separate lipopolysaccharide from impurities, which will result in a decrease in the purity of lipopolysaccharide in the final preparation because the mixing of impurities increases the proportion of non-lipopolysaccharide components in the preparation.

[0082] The presence of leupeptin helps to maintain a relatively stable reaction system, in which there is a synergistic effect between lipopolysaccharide, other active ingredients and various additives; when leupeptin is removed, this synergistic stabilization system is destroyed, and ingredients such as chondroitin sulfate added during the preparation process have a synergistic relationship with lipopolysaccharide in stabilizing the structure and function; however, excessive enzymatic hydrolysis destroys this synergistic relationship, resulting in changes in the structure and functional properties of lipopolysaccharide, making it more vulnerable to damage during subsequent processing, thereby reducing the purity and content of lipopolysaccharide in the preparation.

[0083] Example 4 is the best example, and combined with Comparative Example 2, it can be seen that in the preparation method of the placental lipopolysaccharide preparation, when chondroitin sulfate is removed, the purity and content of the placental lipopolysaccharide preparation are significantly reduced;

[0084] Chondroitin sulfate can combine with impurities in the digestive juice, and when trichloroacetic acid changes the pH and ionic environment of the solution, it makes these impurities easier to precipitate; when chondroitin sulfate is removed, the precipitation effect of impurities becomes worse, and a large number of impurities (such as incompletely digested protein fragments, other macromolecular impurities, etc.) cannot be effectively separated from the solution, and will remain in the supernatant with lipopolysaccharide or interfere with each other in the subsequent processing process; in the final preparation, the presence of these impurities leads to a decrease in the purity of lipopolysaccharide because the proportion of non-lipopolysaccharide components is relatively increased; chondroitin sulfate can interact with lipopolysaccharide and provide protection for lipopolysaccharide. It can maintain the stability of lipopolysaccharide by forming a physical barrier or interacting with lipopolysaccharide intermolecularly (such as hydrogen bonds, electrostatic effects, etc.); without the protective effect of chondroitin sulfate, lipopolysaccharide is more susceptible to changes in the chemical environment (such as changes in pH, temperature, etc.) in subsequent processing steps (such as adding methanol precipitation treatment, heating to dissolve precipitation, etc.), resulting in structural changes or loss of activity, thereby reducing the content of lipopolysaccharide.

[0085] The above shows and describes the basic principles, main features and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited by the above embodiments. The above embodiments and descriptions are only preferred examples of the present invention and are not intended to limit the present invention. Without departing from the spirit and scope of the present invention, the present invention may have various changes and improvements, which fall within the scope of the present invention. The scope of protection of the present invention is defined by the attached claims and their equivalents.

Claims

1. A placental lipopolysaccharide preparation, characterized in that: The invention comprises the following raw materials: 90-95 parts by weight of placental tissue, 1-5 parts by weight of sodium carbonate, 0.1-1 parts by weight of trypsin, 0.1-0.5 parts by weight of leupeptin, 1-2 parts by weight of chloroform, 0.1-1 parts by weight of chondroitin sulfate, 5-10 parts by weight of trichloroacetic acid, 70-80 parts by weight of methanol, 0.1-1 parts by weight of ascorbic acid and 0.5-1 parts by weight of sodium chloride; The preparation method of placental lipopolysaccharide preparation is as follows: S1.

1. Weigh the following raw materials in parts by weight respectively: 90-95 parts by weight of placental tissue, 1-5 parts by weight of sodium carbonate, 0.1-1 parts by weight of trypsin, 0.1-0.5 parts by weight of leupeptin, 1-2 parts by weight of chloroform, 0.1-1 parts by weight of chondroitin sulfate, 5-10 parts by weight of trichloroacetic acid, 70-80 parts by weight of methanol, 0.1-1 parts by weight of ascorbic acid and 0.5-1 parts by weight of sodium chloride; S1.

2. Thaw the placenta tissue in a thawing room at 20°C for no more than 24 hours. Remove the blood from the placenta tissue, pick out the blood clots and fascia, rinse with drinking water until it is close to flesh color, and drain for later use. S1.3, mincing the drained placental tissue with a high-speed disperser at a speed of 5000-8000 rpm for 3-5 min and weighing it, and alkalizing the squeezed placental tissue; S1.

4. Add 10,000 units of trypsin per 1 kg of placental tissue for trypsin digestion and hydrolysis; when the pH value is maintained at 9.0±0.2, add 1% by volume chloroform and continue digestion for 24-28 hours. When the amino nitrogen accounts for 30% of the total nitrogen, add leupeptin solution to obtain a digestion solution; S1.5, stirring the digestion liquid at a speed of 25 r / min, heating at 60°C for more than 20 h for pasteurization; subjecting the pasteurized digestion liquid to precipitation treatment to obtain a supernatant; S1.6, adding methanol to the supernatant for precipitation treatment; The precipitate is collected and freeze-dried, and then collected under a sterile environment to obtain the placental lipopolysaccharide raw material; S1.7, diluting the placental lipopolysaccharide raw material with water for injection to a concentration of 18-25 μg / mL, adding ascorbic acid and sodium chloride to prepare a solution, sterilizing and filtering in a Class B environment and a local Class A environment, dispensing into containers, and sealing to obtain a placental lipopolysaccharide preparation; In S1.4, the concentration of the leupeptin solution is 1-10 mM; In S1.5, the precipitation treatment is performed by adding a 0.1-1% chondroitin sulfate solution to the digestive fluid, then slowly adding 5% trichloroacetic acid by mass, and standing at room temperature for 16-48 hours; taking out the supernatant, and centrifuging the lower suspension at a speed of 4000r / min for 5 minutes to separate the supernatant.

2. The method for preparing the placental lipopolysaccharide preparation according to claim 1, characterized in that: In S1.3, the placental tissue is alkalinized by adding a 1% sodium carbonate solution in a ratio of 1:1 to the weight of the placental tissue to adjust the pH value of the solution to 10, and then the solution is mixed and left for 12-24 hours.

3. The method for preparing the placental lipopolysaccharide preparation according to claim 1, characterized in that: In S1.4, the temperature of trypsin digestion and hydrolysis is 37±2°C, and the pH value is adjusted every 15-30 minutes with a 2 mol / L sodium hydroxide solution within 2 hours to keep the pH value at 9.0±0.

2.

4. The method for preparing the placental lipopolysaccharide preparation according to claim 1, characterized in that: Slowly add trichloroacetic acid at a rate of 5-10 mL / min.

5. The method for preparing the placental lipopolysaccharide preparation according to claim 1, characterized in that: In the S1.6, methanol is added for precipitation treatment by adding methanol to the supernatant to a final concentration of 70%, standing for 12-15 hours, and collecting the precipitate by centrifugation at 5000 r / min for 5 minutes; adding 5-10 times the concentration of 0.9% sodium chloride solution to the precipitate, heating to dissolve the precipitate, and collecting the supernatant by centrifugation at 5000 r / min for 5 minutes; adding methanol to the collected supernatant to a final concentration of 80%, standing for 15-20 hours, and collecting the precipitate by centrifugation at 4000 r / min for 5 minutes.

6. The method for preparing the placental lipopolysaccharide preparation according to claim 5, characterized in that: The heating temperature for heating and dissolving the precipitate is 45-50°C.

7. The method for preparing the placental lipopolysaccharide preparation according to claim 1, characterized in that: In the S1.6, the freeze-drying treatment is to maintain the precipitate at -40°C for 1 hour, then raise the temperature to 1°C within 3 hours and keep it for 3 hours, then raise the temperature to 15°C and keep it for 2 hours, and finally raise the temperature to 22°C and keep it for 2 hours.

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