Sodium chondroitin sulfate with high cartilage repair effect and preparation method thereof

Abstract

The invention relates to the technical field of sodium chondroitin sulfate, in particular to sodium chondroitin sulfate with a high cartilage repair effect and a preparation method thereof.The sodium chondroitin sulfate initial feed liquid is provided, the pH value of the sodium chondroitin sulfate initial feed liquid is adjusted to a preset acidic pH value, and at the preset reaction temperature, the pH value and the reaction temperature are maintained for reaction time, so that the chondroitin sulfate with the high cartilage repair effect is obtained. The method comprises the following steps: adding a sulfated disaccharide unit CS-0 into a feed liquid to precipitate out a non-sulfated disaccharide unit CS-0 in the feed liquid, carrying out solid-liquid separation on the feed liquid, and removing the CS-0 precipitate to obtain a supernatant; pumping the supernatant into a gradient membrane separation system under controlled transmembrane pressure difference and feed liquid temperature, and separating the supernatant into three component flows with different molecular weight intervals; the concentration of the three component flows is monitored, the volume flow of each component flow is dynamically adjusted according to the preset molecular weight and mass distribution proportion, the chondroitin sulfate sodium product with the fixed molecular weight distribution is obtained, and the biological activity and the application value of the chondroitin sulfate sodium are improved.

Description

Technical Field

[0001] This invention relates to the technical field of chondroitin sulfate sodium, specifically to a chondroitin sulfate sodium with high cartilage repair activity and its preparation method. Background Technology

[0002] Chondroitin sulfate sodium is a naturally occurring glycosaminoglycan, primarily found in animal cartilage tissue. It possesses bioactivities such as promoting cartilage repair, alleviating arthritis symptoms, and improving joint lubrication, and is widely used in pharmaceuticals, health products, and biomaterials. Traditionally, chondroitin sulfate sodium is extracted from cartilage raw materials such as nasal bones, trachea, or laryngeal bones of animals like pigs and cattle, involving processes such as alkaline extraction, enzymatic hydrolysis, and precipitation purification. These methods aim to separate and purify CS, but often face problems such as imprecise molecular weight control, high residual levels of non-sulfated disaccharide units (CS-O), and uneven product distribution, resulting in limited product bioavailability and repair efficacy.

[0003] Several methods for preparing chondroitin sulfate sodium have been disclosed in the prior art. For example, CN103214595A discloses a method for preparing chondroitin sulfate sodium, which includes steps such as enzymatic hydrolysis of cartilage raw materials, resin adsorption, salt solution elution, oxidative decolorization, ultrafiltration, and ethanol precipitation. This method achieves the extraction and purification of CS through the combined hydrolysis of trypsin and trypsin enzymes and the adsorption of strongly basic anion exchange resin. However, the problem with this technology is that the existing alkaline-enzymatic precipitation method easily leads to low chondroitin sulfate sodium content, excessive heavy metal and nitrogen content, and non-compliant ignition residue in the product. At the same time, the dry matter yield and content of the product are low, resulting in limited economic value and failure to meet export standards.

[0004] For example, announcement number CN112724279A discloses a method for preparing chondroitin sulfate sodium, which involves boiling and pulverizing cartilage raw materials, extracting by mixing and grinding sodium hydroxide and silica, alkaline extraction, alkaline protease hydrolysis, adsorption filtration, and fractional ethanol precipitation. This method emphasizes promoting the separation of glycosaminoglycans from the core protein through grinding and using low-concentration ethanol to precipitate impurities before purification with high-concentration ethanol to improve yield and purity. However, analysis of this technology shows that traditional alkaline extraction methods consume large amounts of sodium hydroxide solution, leading to increased wastewater discharge, high costs, and environmental pollution; the extraction time is long, which is not conducive to industrial-scale production; the crude product has a high protein impurity content (>5%), requiring complex purification steps, which easily causes CS degradation and reduced yield.

[0005] For example, CN118702841B discloses a process for preparing high-purity, low-molecular-weight chondroitin sulfate sodium, which combines microwave radiation and ultrasonic treatment with an enzymatic purification method. This method includes steps such as raw material immersion in liquid nitrogen and pulverization, intermittent microwave and ultrasonic synergistic treatment under alkaline conditions, hydrolysis with alkaline protease and papain, resin adsorption, alcohol precipitation, and ultrafiltration. This process improves degradation efficiency through intermittent radiation, achieving high-purity extraction of low-molecular-weight CS. However, the common use of chemicals such as benzyl chloride and benzyl chloride introduces impurities, affecting product usability; balancing purity and yield is difficult; and chemical degradation or continuous radiation methods are inefficient, resulting in limited bioavailability.

[0006] Announcement No. CN102190740B discloses a method for preparing chondroitin sulfate, proposing a process involving alkaline enzymatic hydrolysis, alcohol precipitation, oxidation, and emulsification dehydration. This includes heating hydrolysis under alkaline conditions, treatment with 2709 alkaline protease, oxidation and decolorization with hydroxide or potassium permanganate, and emulsification-assisted dehydration. This method produces a product with a loose texture, fine particles, low organic solvent residue (<0.2%), and high content (>98%). However, traditional alcohol precipitation methods tend to form thick, lumpy precipitates, leading to difficult dehydration, high organic solvent residue (≥2%), complex operation, high energy consumption, long production cycles, and affecting product quality stability.

[0007] In summary, while existing chondroitin sulfate sodium preparation technologies can achieve basic extraction, they generally suffer from problems such as incomplete removal of non-sulfated impurities like CS-0, uncontrollable molecular weight degradation, uneven distribution, difficulty in balancing purity and yield, and heavy environmental burden. These defects lead to unstable cartilage repair effects in the product, failing to meet the needs of high-quality pharmaceutical applications. Summary of the Invention

[0008] Therefore, the purpose of this invention is to provide sodium chondroitin sulfate with high cartilage repair function and its preparation method, which can simultaneously achieve CS-O precipitation and controllable molecular weight reduction during acid treatment, and formulate a product with a fixed molecular weight distribution through gradient membrane separation and dynamic flow control, so as to improve the bioactivity and application value of sodium chondroitin sulfate.

[0009] To achieve the above objectives, the present invention provides the following technical solution:

[0010] A method for preparing sodium chondroitin sulfate with high cartilage repair activity includes the following steps:

[0011] S1. Acid treatment and sedimentation steps: Provide an initial solution of sodium chondroitin sulfate and adjust its pH value to a preset acidic pH value. Maintain this pH value and reaction temperature at a preset reaction temperature for a preset reaction time so that the non-sulfated disaccharide unit CS-0 in the solution precipitates out, and at the same time achieves controllable acid hydrolysis of sodium chondroitin sulfate to reduce its molecular weight.

[0012] S2, Solid-liquid separation step: The liquid after step S1 is subjected to solid-liquid separation to remove CS-0 precipitate and obtain supernatant;

[0013] S3, Gradient Separation Step: The supernatant is pumped into the gradient membrane separation system under controlled transmembrane pressure difference and feed temperature. The supernatant is separated into three component streams with different molecular weight cutoffs by passing through three stages of ultrafiltration membranes with different molecular weight cutoffs.

[0014] S4. Dynamic preparation step: The concentration of the three component streams is monitored in real time, and the volumetric flow rate of each of the three component streams is adjusted in real time and dynamically according to the preset molecular weight mass distribution ratio. The component streams are mixed in proportion to obtain a sodium chondroitin sulfate product with a fixed molecular weight distribution.

[0015] The present invention is further configured such that: in step S1, the initial concentration of the feed solution is 5-15% w / v;

[0016] pH adjustment is achieved by slowly adding 0.1-1M hydrochloric acid solution dropwise while stirring at 100-300 rpm under pH electrode monitoring until the pH stabilizes at the preset value. The maintenance process involves monitoring the pH value with a pH electrode every 10-20 minutes and fine-tuning the pH value by adding hydrochloric acid solution.

[0017] The present invention is further configured as follows: in step S1, the preset acidic pH value is pH 3.8-4.2, the adjustment process is continuously monitored using a glass pH electrode, and the pH change curve is recorded; the preset reaction temperature is 35°C to 45°C, the temperature is maintained by a PID temperature controller and a platinum resistance temperature sensor, and the feed solution is preheated to the target temperature before the reaction begins; the preset reaction time is 1.5 to 2.5 hours, and the feed solution is cooled to 10-20°C at the end of the reaction.

[0018] The present invention is further configured as follows: In step S2, the solid-liquid separation is first performed by allowing the precipitate to settle for 10-30 minutes, and then by centrifuging at 4000-6000 rpm for 15-25 minutes; after separation, the precipitate is rinsed once with deionized water and the supernatant is combined.

[0019] The present invention is further configured such that: in step S3, a peristaltic pump is used to control the flow rate of 0.2-1.5 L / min during the pumping process; the transmembrane pressure difference is maintained at 0.05-0.4 MPa, and the pressure difference value is monitored every 5 minutes during the separation process; the feed liquid temperature is controlled at 15-35°C.

[0020] The present invention is further configured such that: in step S3, the ultrafiltration membrane module is a spiral wound type, and the gradient membrane separation system includes ultrafiltration membrane modules with molecular weight cutoffs of 5000 Da, 10000 Da, and 20000 Da, and a pre-filter with a pore size of 50-100 μm is set before each membrane stage; the permeate of each membrane stage is used as the feed for the next stage, and the retentate of each stage is collected as the component stream.

[0021] The present invention is further configured such that: in step S4, concentration data is collected every 3-10 seconds using an online refractometer and transmitted to the controller via a data cable in real time;

[0022] The preset ratio is 20:50:30 for low molecular weight components: medium molecular weight components: high molecular weight components.

[0023] The present invention is further configured such that: the control algorithm in step S4 is a mass flow ratio control algorithm, which specifically includes:

[0024] S41. Calculate the target mass flow rate of each component stream based on the preset molecular weight mass distribution ratio and the target total mass flow rate. The calculation formula is:

[0025] ;

[0026] S42. Obtain the actual concentration of each component stream in real time using online concentration meters and online flow meters. and actual volumetric flow rate And calculate the actual mass flow rate:

[0027] Data collection frequency is every 2-5 seconds;

[0028] S43. Comparison via PID controller and It also outputs control signals to adjust the actual volumetric flow rate. , making Dynamically approaching and maintaining The parameters of the PID controller are set as follows: proportional coefficient Kp is 0.5-2.0, integral time Ti is 10-30 seconds, and derivative time Td is 1-5 seconds, and are set before startup through the controller interface.

[0029] The present invention is further configured such that the control strategy in step S43 specifically includes the following steps:

[0030] Multidimensional rheological parameter acquisition: in monitoring actual concentration Simultaneously, the fluid temperature of each component stream is monitored in real time. ;

[0031] Based on the Arrhenius-type rheological model, the dynamic viscosity coefficients of each component stream are calculated in real time. The calculation formula is:

[0032]

[0033] Where A is the preset consistency coefficient and B is the concentration index. R is the activation energy for the flow, and R is the gas constant.

[0034] According to the target quality flow and the dynamic viscosity coefficient The valve opening degree is calculated using an inverse model of valve flow characteristics. The calculation formula is:

[0035]

[0036] in, It is the inverse function of the inherent flow characteristics of the control valve. For fluid density, This is the valve flow coefficient after viscosity correction;

[0037] The feedback control quantity calculated by the PID controller With the opening degree of the basic valve The signals are superimposed to obtain the control signals for the final actuator. :

[0038] .

[0039] A sodium chondroitin sulfate product with high cartilage repair activity obtained by a method.

[0040] Compared with the shortcomings of the prior art, the beneficial effects of the present invention are as follows:

[0041] By utilizing the synergistic effect of specific pH and temperature, the specific precipitation and removal of the low-activity component (non-sulfated disaccharide unit CS-0) was achieved, reducing its content from over 8% to below 4%. This significantly increased the relative proportion of highly active CS-4 and CS-6, increasing the total charge density of the product, thereby promoting chondrocyte proliferation and anti-inflammatory activity.

[0042] By introducing a PID control algorithm based on mass flow ratio, the technical problems of wide molecular weight distribution and batch-to-batch quality instability in traditional process products are solved, resulting in products with high uniformity and repeatability. Attached Figure Description

[0043] Figure 1 This is a flowchart illustrating the preparation process of the present invention. Detailed Implementation

[0044] Reference Figure 1 To further illustrate the embodiments of the present invention, the present invention provides a sodium chondroitin sulfate with high cartilage repair activity, the chemical nature of which is a glycosaminoglycan. This sodium chondroitin sulfate product is mainly composed of repeating sulfated disaccharide units, each disaccharide unit containing one D-glucuronic acid (GlcA) and one N-acetyl-D-galactosamine (GalNAc). Regarding the chemical linkage:

[0045] Inside the disaccharide unit: the C1 position of D-glucuronic acid is linked to the C3 position of N-acetyl-D-galactosamine via a β-1,3-glycosidic bond;

[0046] Between disaccharide units: The C1 position of N-acetyl-D-galactosamine is linked to the C4 position of D-glucuronic acid in the next disaccharide unit via a β-1,4-glycosidic bond.

[0047] The sulfate group (-SO_3Na) is mainly attached to the C4 position (CS-4) or C6 position (CS-6) of N-acetyl-D-galactosamine.

[0048] The product of this invention is in the form of a disodium salt, and the theoretical chemical formula of its monosulfated disaccharide unit is: .

[0049] Compared to commercially available products, this product reduces the content of non-sulfated disaccharide units (CS-0) (to below 4.0%), thereby increasing the relative proportion of highly active sulfated units such as CS-4 and CS-6, and enhancing the overall charge density and bioactivity of the product.

[0050] The preparation method provided by this invention specifically includes the following steps:

[0051] The first step, acid treatment and precipitation: taking advantage of the reduced solubility of CS-0 at a specific acidic pH value, it is precipitated out. At the same time, the polysaccharide chain is hydrolyzed (acid hydrolysis) under acidic conditions to initially reduce the molecular weight.

[0052] First, an initial solution of chondroitin sulfate sodium is provided. This solution is preferably prepared by dissolving crude chondroitin sulfate sodium in deionized water, and its initial concentration is preferably 5-15% (w / v).

[0053] Subsequently, under stirring at 100-300 rpm, 0.1-1M hydrochloric acid solution is slowly added dropwise to the feed solution through an automatic pH control system (including a glass pH electrode and an acid metering pump) to adjust the pH value to a preset value, preferably pH 3.8-4.2. During the adjustment process, pH changes are continuously monitored using a glass pH electrode, and pH change curves can be recorded for process traceability.

[0054] Once the pH value stabilizes at the preset value, timing begins. The feed solution temperature is maintained at the preset reaction temperature (preferably 35°C to 45°C) via a PID temperature controller and a platinum resistance temperature sensor (through a heating jacket). The feed solution can be preheated to the target temperature before the reaction begins.

[0055] The reaction was maintained at this pH and temperature for 1.5 to 2.5 hours. During the maintenance process, to ensure a constant pH, the control system preferably monitored the pH every 10-20 minutes using a pH electrode and fine-tuned the pH by adding a very small amount of hydrochloric acid solution.

[0056] Once the reaction time is reached, the liquid is immediately cooled to 10-20°C using an ice-water bath or cooling jacket to terminate the acid hydrolysis reaction and lock in the molecular weight and CS-0 precipitation state.

[0057] The purpose of the second step, the solid-liquid separation step, is to separate the CS-0 (and other insoluble substances) precipitated in the first step from the dissolved chondroitin sulfate sodium, which is rich in CS-4 / CS-6.

[0058] After cooling the liquid in the first step, let it stand for 10-30 minutes to allow large particles to settle naturally. Then, pump the liquid into a high-speed centrifuge and centrifuge at 4000-6000 rpm for 15-25 minutes.

[0059] After centrifugation, collect the supernatant. To improve the yield, the precipitate can be rinsed once with a small amount of deionized water, centrifuged again, and the supernatant from this second centrifugation can be combined with the previous one to obtain a supernatant rich in CS-4 / CS-6 and with a significantly reduced CS-0 content.

[0060] The third step, gradient separation step, involves precisely fractionating the supernatant obtained in the second step according to molecular weight, providing raw materials for the dynamic formulation in the fourth step.

[0061] The combined supernatant from the second step is pumped into a gradient membrane separation system. This system preferably consists of three ultrafiltration membrane modules connected in series. The membrane modules are preferably spiral wound, with a pre-filter with a pore size of 50-100 μm placed before each membrane stage to remove any small particles that may remain from the second step and protect the ultrafiltration membrane.

[0062] The molecular weight cutoff (MWCO) of these three ultrafiltration membranes are 5000 Da, 10000 Da, and 20000 Da, respectively.

[0063] During the separation process, the feed liquid inflow rate is maintained at 0.2-1.5 L / min through precise pump (such as peristaltic pump) and valve control, and the transmembrane pressure difference (TMP) is maintained at 0.05-0.4 MPa through inlet pressure sensor and outlet valve. At the same time, the feed liquid temperature is controlled at 15-35°C through built-in circulating water bath system to ensure stable separation efficiency.

[0064] When the feed liquid flows through the system:

[0065] The permeate from the first stage (5000 Da) (<5000 Da) is discharged as a byproduct or waste.

[0066] The first stage (5000 Da) retentate (>5000 Da) enters the second stage (10000 Da).

[0067] The permeate (5000-10000 Da) from the second stage (10000 Da) was collected as a low molecular weight component stream.

[0068] The retentate from the second stage (10000Da) (>10000Da) enters the third stage (20000Da).

[0069] The permeate (10,000-20,000 Da) from the third stage (20,000 Da) was collected as a medium molecular weight component stream.

[0070] The third stage (20000 Da) retentate (>20000 Da) was collected as the high molecular weight component stream. Thus, three component streams with different molecular weight ranges were obtained.

[0071] The fourth step, dynamic formulation step, involves precisely mixing the different molecular weight components obtained in the third step according to the specified proportions to obtain a final product with a constant molecular weight distribution and high consistency between batches.

[0072] This step is implemented using an algorithm based on mass flow rate ratio control.

[0073] In the third step, online concentration meters (preferably online refractometers) and online flow meters (preferably electromagnetic flow meters) are installed on the outlet pipelines of the three component streams (low, medium, and high). A precision proportional control valve (flow actuator) is also installed. The control system (PLC or DCS) collects real-time concentration data from the online refractometer every 3-10 seconds.

[0074] The control system calculates the target mass flow rate that each component stream (i=1,2,3) should achieve at any given moment based on a preset molecular weight mass distribution ratio (e.g., low:medium:high = 20:50:30 mass ratio) and a target total mass flow rate (e.g., 1-5 g / min). :

[0075]

[0076] : Target mass flow rate of component flow i (unit: g / min);

[0077] : The preset mass ratio of component stream i ( );

[0078] Total production rate set by the operator (unit: g / min)

[0079] The control system collects real-time data from the online concentration meter and online flow meter every 2-5 seconds and calculates the current actual mass flow rate of each component. ;

[0080]

[0081]

[0082] ;

[0083] Meanwhile, in order to eliminate the interference of viscosity fluctuations on flow control, the system uses a preset Arrhenius-type rheological model to calculate the dynamic viscosity coefficient of the fluid in real time. :

[0084]

[0085] Where A is the consistency coefficient and B is the concentration index. R is the flow activation energy, and R is the gas constant. These parameters are pre-stored in the controller.

[0086] Composite closed-loop control: The control system runs an independent control loop for each component flow and outputs a total control signal. This signal is supplied to the precision proportional control valve. and feedback items composition.

[0087] feedforward term Based on target flow rate and real-time viscosity Estimate the opening degree of the basic valve in advance to offset the resistance caused by viscosity changes:

[0088]

[0089] in, It is the inverse function of the inherent flow characteristics of the control valve. For fluid density, This is the valve flow coefficient after viscosity correction;

[0090] Feedback items PID control: The controller calculates the error between the target value and the actual value. The compensation opening degree is calculated using a PID algorithm:

[0091]

[0092] in:

[0093] These are the proportional, integral, and derivative gain coefficients of the PID controller, respectively.

[0094] In this embodiment, these parameters are preferably set as follows before startup: proportional coefficient The integral time is 0.5-2.0. The derivative time is 10-30 seconds. It takes 1-5 seconds.

[0095] Final execution: .

[0096] regulating valve according to Adjusting the opening degree changes the actual volumetric flow rate. .

[0097] Through this composite closed-loop control, even if the concentration or temperature of the upstream component streams fluctuates (leading to viscosity changes), the system can predict and adjust before significant deviations in flow rate occur, using a feedforward term and fine-tuning via PID control. This ensures that the actual mass flow rate is always locked at the target value. Ultimately, the three component streams are mixed in precise mass ratios to obtain a sodium chondroitin sulfate product with a low molecular weight distribution (low CV value).

[0098] Example 1:

[0099] S1. Take the initial chondroitin sulfate sodium solution with a concentration of 10% (w / v). While stirring at 100 rpm, slowly add 0.1M hydrochloric acid solution dropwise using the automatic pH control system to adjust the pH value to the preset value of 3.8. Maintain the reaction at this pH value and the preset reaction temperature of 35°C for 1.5 hours. During this period, monitor and fine-tune the pH value every 20 minutes. After the reaction time is reached, immediately cool the solution to 25°C.

[0100] S2. After cooling, allow the liquid to stand for 10 minutes, then pump it into a high-speed centrifuge and centrifuge at 4000 rpm for 15 minutes. Collect the supernatant and rinse the precipitate once with deionized water, then combine the supernatants.

[0101] S3. Pump the combined supernatant into the gradient membrane separation system, control the pumping flow rate to maintain the transmembrane pressure difference at 0.05 MPa, and control the feed temperature at 15°C. Collect the component streams in the low, medium and high molecular weight ranges respectively.

[0102] S4. Start the dynamic preparation system and preset the rheological model parameters of chondroitin sulfate sodium in the controller: consistency coefficient A = 0.05, concentration index B = 2.5, and activation energy of flow. =18,500 J / mol. The target mass ratio (low:medium:high) is set to 20:50:30. PID settings: Set the PID controller parameters as follows... The runtime system, based on data from an online refractometer and temperature sensor, automatically adjusts the flow actuators of each component stream using a feedforward-feedback composite algorithm, ultimately producing product 1.

[0103] Example 2:

[0104] S1. Take the initial chondroitin sulfate sodium solution with a concentration of 10% (w / v). While stirring at 200 rpm, slowly add 0.5 M hydrochloric acid solution dropwise using the automatic pH control system to adjust the pH to the preset value of 4.0. Maintain the reaction at this pH and the preset reaction temperature of 40°C for 2.0 hours. During this period, monitor and fine-tune the pH every 15 minutes. After the reaction time is reached, immediately cool the solution to 15°C.

[0105] S2. After cooling, allow the liquid to stand for 20 minutes, then pump it into a high-speed centrifuge and centrifuge at 5000 rpm for 20 minutes. Collect the supernatant and rinse the precipitate once with deionized water, then combine the supernatants.

[0106] S3. Pump the combined supernatant into the gradient membrane separation system (same as in Example 1), control the pumping flow rate to maintain the transmembrane pressure difference at 0.2 MPa, control the feed temperature at 25°C, and collect the three component streams separately.

[0107] S4. Start the dynamic preparation system and preset the rheological model parameters of chondroitin sulfate sodium in the controller: consistency coefficient A = 0.05, concentration index B = 2.5, and activation energy of flow. =18,500 J / mol. The target mass ratio (low:medium:high) is set to 20:50:30. PID settings: Set the PID controller parameters as follows... The runtime system uses data from an online refractometer and temperature sensor to automatically adjust the flow actuators of each component flow using a feedforward-feedback composite algorithm, ultimately producing product 2.

[0108] Example 3:

[0109] S1. Take the initial chondroitin sulfate sodium solution with a concentration of 15% (w / v). While stirring at 300 rpm, slowly add 1.0 M hydrochloric acid solution dropwise using the automatic pH control system to adjust the pH to the preset value of 4.2. Maintain the reaction at this pH and the preset reaction temperature of 45°C for 2.5 hours. During this period, monitor and fine-tune the pH every 10 minutes. After the reaction time is reached, immediately cool the solution to 20°C.

[0110] S2. After cooling, allow the liquid to stand for 30 minutes, then pump it into a high-speed centrifuge and centrifuge at 6000 rpm for 25 minutes. Collect the supernatant and rinse the precipitate once with deionized water, then combine the supernatants.

[0111] S3. Pump the combined supernatant into the gradient membrane separation system (same as in Example 1), control the pumping flow rate to maintain the transmembrane pressure difference at 0.4 MPa, control the feed temperature at 35°C, and collect the three component streams separately.

[0112] S4. Start the dynamic preparation system and preset the rheological model parameters of chondroitin sulfate sodium in the controller: consistency coefficient A = 0.05, concentration index B = 2.5, and activation energy of flow. =18,500 J / mol. The target mass ratio (low:medium:high) is set to 20:50:30. PID settings: Set the PID controller parameters as follows... The runtime system uses data from an online refractometer and temperature sensor to automatically adjust the flow actuators of each component flow using a feedforward-feedback composite algorithm, ultimately producing product 3.

[0113] Comparative Example 1: This comparative example uses the same crude raw material as the example, but step S1 (acid treatment and sedimentation) is omitted. The crude raw material is directly subjected to steps S2 (centrifugation parameters are the same as in Example 2), S3 (membrane separation parameters are the same as in Example 2), and S4 (dynamic formulation parameters are the same as in Example 2) to finally prepare product 4.

[0114] Comparative Example 2: This comparative example performed steps S1 (parameters same as in Example 2), S2 (parameters same as in Example 2), and S3 (parameters same as in Example 2), but omitted step S4 (dynamic formulation). Instead, the three component streams separated in S3 were simply mixed in a volume ratio of 1:1:1 (instead of the mass flow rate control of the present invention) to obtain product 5.

[0115] Performance testing methods:

[0116] In all examples and comparative examples, the same batch of crude chondroitin sulfate sodium was used (tested to have a CS-O content of 8.2% and a molecular weight distribution standard deviation of 15.3%). The performance of the final product was tested using the following methods:

[0117] CS-0 content was determined using high performance liquid chromatography.

[0118] The coefficient of variation (CV) was determined using gel permeation chromatography. CV is the ratio of the standard deviation (SD) of the molecular weight distribution to the weight-average molecular weight (Mw), expressed as a percentage (CV = SD / Mw × 100%). A smaller CV value indicates a narrower molecular weight distribution range and better product uniformity.

[0119] Chondrocyte proliferation rate: The MTT assay was used (the blank control group was 100%).

[0120] Anti-inflammatory activity (NO inhibition rate): determined using the Griess method.

[0121] The details are shown in Table 1 below:

[0122] Group CS-0 content (%) Molecular weight coefficient of variation (CV) (%) chondrocyte proliferation rate (%) Anti-inflammatory activity (NO inhibition rate, %) Product 1 3.9 7.6 122% 46% Product 2 3.6 7.2 128% 52% Product 3 3.8 7.4 125% 49% Product 4 8.1 11.5 103% 22% Product 5 3.7 15.1 126% 50% crude raw materials 8.2 15.3 100% 20%

[0123] Comparative Example 1, which did not undergo S1, had a CS-O content (8.1%) essentially the same as the crude raw material, and extremely low bioactivity (proliferation rate 103%, inhibition rate 22%). In contrast, Examples 1, 2, and 3 all showed significantly reduced CS-O content to below 4%, with a substantial increase in bioactivity. This demonstrates that the acid treatment and sedimentation steps in S1 are crucial for removing CS-O and improving bioactivity.

[0124] Comparative Example 2 performed S1 (CS-0 content 3.7%, activity 50%), but did not perform S4. The coefficient of variation (CV) was as high as 15.1%, consistent with the crude product, indicating that simple volume mixing cannot control the molecular weight distribution. In contrast, Example 2, through the dynamic formulation algorithm of S4, strictly controlled the CV at 7.2%. This proves that step S4 is the key to achieving uniform and controllable molecular weight of the product.

[0125] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any ordinary changes and substitutions made by those skilled in the art within the scope of the technical solution of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for preparing sodium chondroitin sulfate with high cartilage repair activity, characterized in that, Includes the following steps: S1. Acid treatment and sedimentation steps: Provide an initial solution of sodium chondroitin sulfate and adjust its pH value to a preset acidic pH value. Maintain this pH value and reaction temperature at a preset reaction temperature for a preset reaction time so that the non-sulfated disaccharide unit CS-0 in the solution precipitates out, and at the same time achieves controllable acid hydrolysis of sodium chondroitin sulfate to reduce its molecular weight. S2, Solid-liquid separation step: The liquid after step S1 is subjected to solid-liquid separation to remove CS-0 precipitate and obtain supernatant. S3, Gradient Separation Step: The supernatant is pumped into the gradient membrane separation system under controlled transmembrane pressure difference and feed temperature. The supernatant is separated into three component streams with different molecular weight cutoffs by passing through three stages of ultrafiltration membranes with different molecular weight cutoffs. S4. Dynamic preparation step: The concentration of the three component streams is monitored in real time, and the volumetric flow rate of each of the three component streams is adjusted in real time and dynamically according to the preset molecular weight mass distribution ratio. The component streams are mixed in proportion to obtain a sodium chondroitin sulfate product with a fixed molecular weight distribution.

2. The method for preparing sodium chondroitin sulfate with high cartilage repair activity according to claim 1, characterized in that, In step S1, the initial concentration of the solution is 5-15% (w / v); pH adjustment is achieved by slowly adding 0.1-1M hydrochloric acid solution while stirring at 100-300 rpm under pH electrode monitoring until the pH stabilizes at the preset value; the maintenance process includes monitoring the pH value every 10-20 minutes using a pH electrode and fine-tuning the pH value by adding hydrochloric acid solution.

3. The method for preparing sodium chondroitin sulfate with high cartilage repair activity according to claim 1, characterized in that, In step S1, the preset acidic pH value is pH 3.8-4.

2. The adjustment process is continuously monitored using a glass pH electrode, and the pH change curve is recorded. The preset reaction temperature is 35°C to 45°C. The temperature is maintained by a PID temperature controller and a platinum resistance temperature sensor. The feed solution is preheated to the target temperature before the reaction begins. The preset reaction time is 1.5 to 2.5 hours. The feed solution is cooled to 10-20°C at the end of the reaction.

4. The method for preparing sodium chondroitin sulfate with high cartilage repair activity according to claim 1, characterized in that, In step S2, the solid-liquid separation is first carried out by letting the precipitate settle for 10-30 minutes, and then centrifuging it at 4000-6000 rpm for 15-25 minutes. After separation, the precipitate is rinsed once with deionized water and the supernatant is combined.

5. The method for preparing sodium chondroitin sulfate with high cartilage repair activity according to claim 1, characterized in that, In step S3, a peristaltic pump is used to control the flow rate at 0.2-1.5 L / min during the pumping process; the transmembrane pressure difference is maintained at 0.05-0.4 MPa, and the pressure difference value is monitored every 5 minutes during the separation process; the feed temperature is controlled at 15-35°C.

6. The method for preparing sodium chondroitin sulfate with high cartilage repair activity according to claim 1, characterized in that, In step S3, the ultrafiltration membrane module is a spiral wound type, and the gradient membrane separation system includes ultrafiltration membrane modules with molecular weight cutoffs of 5000 Da, 10000 Da, and 20000 Da. A pre-filter with a pore size of 50-100 μm is set before each membrane stage. The permeate of each membrane stage is used as the feed for the next stage, and the retentate of each stage is collected as the component stream.

7. The method for preparing sodium chondroitin sulfate with high cartilage repair activity according to claim 1, characterized in that, In step S4, concentration data is collected every 3-10 seconds using an online refractometer and transmitted to the controller via a data cable in real time; the preset ratio is a mass ratio of low molecular weight component: medium molecular weight component: high molecular weight component of 20:50:

30.

8. The method for preparing sodium chondroitin sulfate with high cartilage repair activity according to claim 1, characterized in that, The control algorithm in step S4 is a mass flow ratio control algorithm, which specifically includes: Based on the preset molecular weight mass distribution ratio and the target total mass flow rate, the target mass flow rate of each component stream is calculated. The calculation formula is: ； S42. Obtain the actual concentration of each component stream in real time using online concentration meters and online flow meters. and actual volumetric flow rate And calculate the actual mass flow rate: Data collection frequency is every 2-5 seconds; S43. Comparison via PID controller and It also outputs control signals to adjust the actual volumetric flow rate. , making Dynamically approaching and maintaining The parameters of the PID controller are set as follows: proportional coefficient Kp is 0.5-2.0, integral time Ti is 10-30 seconds, and derivative time Td is 1-5 seconds, and are set before startup through the controller interface.

9. The method for preparing sodium chondroitin sulfate with high cartilage repair activity according to claim 8, characterized in that, The control strategy in step S43 specifically includes the following steps: Multidimensional rheological parameter acquisition: in monitoring actual concentration Simultaneously, the fluid temperature of each component stream is monitored in real time. ; Based on the Arrhenius-type rheological model, the dynamic viscosity coefficients of each component stream are calculated in real time. The calculation formula is: ； Where A is the preset consistency coefficient and B is the concentration index. R is the activation energy for the flow, and R is the gas constant. According to the target quality flow and the dynamic viscosity coefficient The valve opening degree is calculated using an inverse model of valve flow characteristics. The calculation formula is: ； in, It is the inverse function of the inherent flow characteristics of the control valve. For fluid density, This is the valve flow coefficient after viscosity correction; The feedback control quantity calculated by the PID controller ( ) and the opening degree of the basic valve The signals are superimposed to obtain the control signals for the final actuator. : 。 10. A sodium chondroitin sulfate product with high cartilage repair activity, characterized in that, The product is prepared by the method of any one of claims 1-9.

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