An antioxidant anti-aging pn complex and a method for preparing the same

Abstract

This invention belongs to the field of biopharmaceutical manufacturing technology and discloses an antioxidant and anti-aging PN complex and its preparation method, comprising: polydeoxyribonucleotide, epigallocatechin gallate, divalent magnesium ions, sodium ascorbate phosphate, citrate-sodium citrate buffer system, and water; the divalent magnesium ions form chelates with the phosphate groups on the polydeoxyribonucleotide molecular chain and the phenolic hydroxyl groups of epigallocatechin gallate, respectively, so that epigallocatechin gallate forms a continuously distributed molecular coating layer on the surface of the polydeoxyribonucleotide molecular chain; this invention solves the problem of heterogeneous gel aggregation caused by excessively fast reaction kinetics in the preparation of biopharmaceutical macromolecules by transforming the unavoidable disordered crosslinking between chains in conventional mixing processes into highly ordered intramolecular homogeneous encapsulation.

Description

Technical Field

[0001] This invention relates to an antioxidant and anti-aging PN complex and its preparation method, belonging to the field of biopharmaceutical manufacturing technology. Background Technology

[0002] Currently, polydeoxyribonucleic acid (PDA) is used to induce fibroblast proliferation and promote extracellular matrix regeneration. Formulations prepared using this active ingredient have significant biological effects in skin tissue repair and anti-aging applications. However, the phosphodiester backbone of PDA exhibits high chemical sensitivity under physiological conditions, making it highly susceptible to degradation by local tissue nucleases and prone to oxidative breakage under oxidative stress. Conventional methods involve physically mixing small-molecule antioxidants with PDA. In such mixtures, small-molecule antioxidants, due to their low molecular weight and high water solubility, rapidly diffuse outwards along the concentration gradient and participate in systemic circulation after entering the tissue. In contrast, large PDA molecules, due to their low diffusion coefficient, remain in the injection site. This difference in pharmacokinetic parameters between the components leads to a spatiotemporal dissociation between the antioxidant components and the biomolecules. Without the protection of the antioxidant layer, PDA is directly exposed to the oxidative stress microenvironment, causing structural degradation of the active ingredient before it can exert its function.

[0003] To inhibit the degradation of biomacromolecules, metal ion-mediated coordination reactions are introduced to achieve stable anchoring between components. However, in actual manufacturing processes, the coordination reaction kinetics of divalent metal ions are much higher than the conformational unfolding rate of polydeoxyribonucleotide macromolecules. This rate mismatch causes metal ions to initiate random and irreversible cross-linking between chains upon contact with the macromolecule, resulting in heterogeneous gel clumps. The heterogeneous phase leads to a large number of defensive vacuum regions in the polydeoxyribonucleotide chain backbone. Simply increasing the stirring intensity or changing the component concentration cannot overcome the aggregation phenomenon caused by the difference in kinetic rates, thus limiting the orderly assembly of antioxidant components along the topological direction of nucleic acid monomolecule chains.

[0004] Analysis revealed the following shortcomings of the existing technology: 1. Imbalance in the diffusion rates of components in the physical mixing system leads to the failure of the antioxidant protective layer; 2. The disordered aggregation of macromolecular chains induced by mismatch in coordination reaction kinetics limits the microscopic homogeneous encapsulation.

[0005] Therefore, how to overcome the kinetic barriers in the coordination process between polydeoxyribonucleotides and metal ions, and achieve the sequential assembly and homogeneous locking of antioxidant components on the biomolecular backbone, has become the technical problem to be solved by this invention. Summary of the Invention

[0006] To address the problems in the background art, the technical solution of the present invention is as follows: an antioxidant and anti-aging PN complex, comprising polydeoxyribonucleotides, epigallocatechin gallate, divalent magnesium ions, sodium ascorbate phosphate, a citrate-sodium citrate buffer system, and water: Polydeoxyribonucleic acid is a product obtained by enzymatic hydrolysis and purification of salmon sperm deoxyribonucleic acid. The purity of polydeoxyribonucleic acid is not less than 98.5 wt%, the weight average molecular weight is 300 kDa to 500 kDa, and the polydispersity index (PDI) is 1.2 to 1.5. Based on a total weight of 100 parts for the complex, the following components are present: polydeoxyribonucleotides: 0.5 to 2.0 parts; epigallocatechin gallate: 0.1 to 0.5 parts; sodium ascorbate phosphate: 0.05 to 0.12 parts; citrate-sodium citrate buffer system: 0.1 to 0.3 parts; and water: to make up the balance to 100 parts. The divalent magnesium ions are provided by magnesium chloride, and the molar ratio of the divalent magnesium ions to the phosphate groups on the polydeoxyribonucleotide molecular chain is 1:10 to 2:10; the polydeoxyribonucleotide, epigallocatechin gallate, and divalent magnesium ions have a coordination coating structure in an environment with a temperature of 2°C to 6°C and a pH of 5.5 to 6.2. Coordination coating structure refers to the chelation between divalent magnesium ions and the phosphate groups on the polydeoxyribonucleotide molecular chain and the phenolic hydroxyl groups of epigallocatechin gallate, so that epigallocatechin gallate forms a continuously distributed molecular coating layer on the surface of the polydeoxyribonucleotide molecular chain.

[0007] Preferably, the molecular coating layer formed by polydeoxyribonucleotide, epigallocatechin gallate, and divalent magnesium ions is in metastable equilibrium at a temperature of 2°C to 6°C. After mixing, the aqueous medium is heated to 37°C to 48°C at a heating rate of 1.0°C / min to 1.5°C / min and held for 30 min to 60 min to induce thermal desorption of the hydrated layer of divalent magnesium ions and drive the molecular coating layer from the metastable state to the equilibrium steady state. The physical shielding space formed by the molecular coating layer on the outside of the polydeoxyribonucleotide molecular chain has a thickness of 2 nm to 10 nm, and the molecular coating layer covers the phosphodiester bond sites on the polydeoxyribonucleotide molecular chain.

[0008] Preferably, the total content of divalent metal impurity ions in the polydeoxyribonucleotide is less than 5 ppm; the purity of epigallocatechin gallate is higher than 98.0 wt%, and the caffeine content in epigallocatechin gallate is less than 0.1 wt%.

[0009] Preferably, the purity of sodium ascorbate phosphate is higher than 99.0 wt%; the weight ratio of sodium ascorbate phosphate to epigallocatechin gallate is 1:2 to 1:5.

[0010] Preferably, the osmolar concentration of the complex is between 280 mOsm / kg and 320 mOsm / kg; the complex is electrically neutral in an environment with a pH of 5.8 to 6.0.

[0011] Preferably, the coordination coating structure has temperature-responsive characteristics; the coordination saturation of divalent magnesium ions on the surface of polydeoxyribonucleotide molecular chains increases with increasing ambient temperature in the range of 2°C to 37°C; the surface density of the molecular coating layer of the complex at 37°C is higher than that at 6°C.

[0012] Preferably, sodium ascorbate phosphate undergoes an oxidation reaction when the dissolved oxygen concentration in the complex system is higher than 0.1 mg / L, thereby inhibiting the oxidation reaction inside the complex by capturing and scavenging free radicals; epigallocatechin gallate forms a multi-site chelation network with divalent magnesium ions through phenolic hydroxyl sites.

[0013] Preferably, after the complex is left to stand at 25°C for 48 hours, the change rate of absorbance at 260 nm is less than 5%; after the complex is incubated in a solution containing 10 U / mL deoxynucleotidase I for 24 hours, the residual rate of polydeoxyribonucleotidyl is not less than 85 wt%.

[0014] A method for preparing an antioxidant and anti-aging PN complex includes the following steps: Step 1001: Dissolve polydeoxyribonucleotides, epigallocatechin gallate, sodium ascorbate phosphate, and citrate-sodium citrate buffer system in water, and adjust the pH of the resulting aqueous medium to 5.5 to 6.2. Step 1002: Turn on the cooling unit and control the temperature of the aqueous medium to 2°C to 6°C; Step 1003: Under conditions of 2°C to 6°C, a solution containing divalent magnesium ions is added dropwise to an aqueous medium at a uniform rate, and the mixture is mixed for 1 to 4 hours at a temperature of 2°C to 6°C and a pH of 5.5 to 6.2. After mixing is completed, the aqueous medium is programmed to be heated to 37°C to 48°C at a heating rate of 1.0°C / min to 1.5°C / min, and held at this temperature for 30 to 60 minutes to drive the thermal desorption of the hydrated layer of divalent magnesium ions, thereby inducing the formation of a continuously distributed molecular coating layer on the surface of polydeoxyribonucleotide monomolecules by epigallocatechin gallate.

[0015] Compared with the prior art, the beneficial effects of the present invention are: Firstly, in the antioxidant and anti-aging PN complex, by using a constant temperature of 2℃ to 6℃ combined with high-speed shearing, the steric hindrance of the magnesium ion hydration layer is used to suppress instantaneous random coordination. The programmed temperature rise of 1.0℃ / min to 1.5℃ / min drives the orderly desorption of the hydration layer, inducing the sequential coordination of divalent magnesium ions along the molecular chain. This process transforms disordered cross-linking into homogeneous encapsulation within a single molecule, solving the common problem of heterogeneous gel aggregation in macromolecular formulations.

[0016] Secondly, this invention relies on the high-density anchoring of EGCG and PN molecular chains to construct a physical defense layer, directly blocking the contact of nucleases with cleavage sites in tissues. The dynamic coordination network locks the antioxidant components around the nucleic acid, avoiding the rapid diffusion and dissipation of small molecules, realizing strong spatiotemporal coupling between defense and regeneration components, prolonging the structural survival of polydeoxyribonucleotides in vivo, and ensuring stable regeneration signal stimulation.

[0017] Third, this invention utilizes sodium ascorbate phosphate to capture and scavenge free radicals, acting as a sacrificial layer to offset oxidative losses during preparation and storage. Combined with precise control of osmotic pressure and electroneutrality, it ensures the chemical structural integrity of the ternary coordination network before delivery to the clinical end. This mechanism enhances the physicochemical stability of the formulation under complex biochemical environments and ensures quality consistency in large-scale industrial production. Attached Figure Description

[0018] Figure 1 This is a schematic diagram illustrating the formation principle of the molecular-level coordination coating structure of the PN complex involved in this invention. Figure 2 This is a diagram of the PN composite production process monitoring and closed-loop temperature control system involved in this invention. Detailed Implementation

[0019] The technical solutions of the embodiments of this application will be clearly described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of this application, not all embodiments. All other embodiments obtained by those skilled in the art based on the embodiments of this application are within the scope of protection of this application.

[0020] Example 1: This example relates to an antioxidant and anti-aging PN complex, comprising polydeoxyribonucleotides, epigallocatechin gallate, divalent magnesium ions, sodium ascorbate phosphate, a citrate-sodium citrate buffer system, and water. Polydeoxyribonucleic acid is a product obtained by enzymatic hydrolysis and purification of salmon sperm deoxyribonucleic acid. The purity of polydeoxyribonucleic acid is not less than 98.5 wt%, the weight average molecular weight is 300 kDa to 500 kDa, and the polydispersity index (PDI) is 1.2 to 1.5. Based on a total weight of 100 parts for the complex, the following components are present: polydeoxyribonucleotides: 0.5 to 2.0 parts; epigallocatechin gallate: 0.1 to 0.5 parts; sodium ascorbate phosphate: 0.05 to 0.12 parts; citrate-sodium citrate buffer system: 0.1 to 0.3 parts; and water: to make up the balance to 100 parts. The divalent magnesium ions are provided by magnesium chloride, and the molar ratio of the divalent magnesium ions to the phosphate groups on the polydeoxyribonucleotide molecular chain is 1:10 to 2:10; the polydeoxyribonucleotide, epigallocatechin gallate, and divalent magnesium ions have a coordination coating structure in an environment with a temperature of 2°C to 6°C and a pH of 5.5 to 6.2. Coordination coating structure refers to the chelation between divalent magnesium ions and the phosphate groups on the polydeoxyribonucleotide molecular chain and the phenolic hydroxyl groups of epigallocatechin gallate, so that epigallocatechin gallate forms a continuously distributed molecular coating layer on the surface of the polydeoxyribonucleotide molecular chain.

[0021] In this embodiment, the molecular coating layer formed by polydeoxyribonucleotides, epigallocatechin gallate, and divalent magnesium ions is in a metastable equilibrium at a temperature of 2°C to 6°C. After mixing, the aqueous medium is heated to 37°C to 48°C at a heating rate of 1.0°C / min to 1.5°C / min and held for 30 min to 60 min to induce thermal desorption of the hydrated layer of divalent magnesium ions and drive the molecular coating layer from a metastable state to an equilibrium steady state. The physical shielding space formed by the molecular coating layer on the outside of the polydeoxyribonucleotide molecular chain has a thickness of 2 nm to 10 nm, and the molecular coating layer covers the phosphodiester bond sites on the polydeoxyribonucleotide molecular chain.

[0022] The total content of divalent metal impurity ions in the polydeoxyribonucleotides described in this embodiment is less than 5 ppm; the purity of epigallocatechin gallate is higher than 98.0 wt%, and the caffeine content in epigallocatechin gallate is less than 0.1 wt%.

[0023] The sodium ascorbate phosphate described in this embodiment has a purity higher than 99.0 wt%; the weight ratio of sodium ascorbate phosphate to epigallocatechin gallate is 1:2 to 1:5.

[0024] The osmolar concentration of the complex described in this embodiment is 280 mOsm / kg to 320 mOsm / kg; the complex is electrically neutral in an environment with a pH of 5.8 to 6.0.

[0025] The coordination coating structure described in this embodiment has temperature-responsive characteristics; the coordination saturation of divalent magnesium ions on the surface of polydeoxyribonucleotide molecular chains increases with the increase of ambient temperature in the range of 2℃ to 37℃; the surface density of the molecular coating layer of the complex at a temperature of 37℃ is higher than that at a temperature of 6℃.

[0026] In this embodiment, sodium ascorbate phosphate undergoes an oxidation reaction when the dissolved oxygen concentration in the complex system is higher than 0.1 mg / L, thereby inhibiting the oxidation reaction inside the complex by capturing and scavenging free radicals; epigallocatechin gallate forms a multi-site chelation network with divalent magnesium ions through phenolic hydroxyl sites.

[0027] The complex described in this embodiment, after standing at 25°C for 48 hours, showed an absorbance change rate of less than 5% at 260 nm; after incubation in a solution containing 10 U / mL deoxynucleotidase I for 24 hours, the residual rate of polydeoxyribonucleotidyl is not less than 85 wt%.

[0028] This embodiment relates to a method for preparing an antioxidant and anti-aging PN complex, comprising the following steps: Step 1001: Dissolve polydeoxyribonucleotides, epigallocatechin gallate, sodium ascorbate phosphate, and citrate-sodium citrate buffer system in water, and adjust the pH of the resulting aqueous medium to 5.5 to 6.2. Step 1002: Turn on the cooling unit and control the temperature of the aqueous medium to 2°C to 6°C; Step 1003: Under conditions of 2°C to 6°C, a solution containing divalent magnesium ions is added dropwise to an aqueous medium at a uniform rate, and the mixture is mixed for 1 to 4 hours at a temperature of 2°C to 6°C and a pH of 5.5 to 6.2. After mixing is completed, the aqueous medium is programmed to be heated to 37°C to 48°C at a heating rate of 1.0°C / min to 1.5°C / min, and held at this temperature for 30 to 60 minutes to drive the thermal desorption of the hydrated layer of divalent magnesium ions, thereby inducing the formation of a continuously distributed molecular coating layer on the surface of polydeoxyribonucleotide monomolecules by epigallocatechin gallate.

[0029] Example 2: In the manufacturing process of biopharmaceuticals involving skin tissue repair, this example addresses the situation where heterogeneous gel clumps are induced in an aqueous medium due to mismatched coordination reaction rates between polydeoxyribonucleic acid (PDA) with a weight-average molecular weight of 300kDa to 500kDa, epigallocatechin gallate, and divalent magnesium ions. The preparation method of the antioxidant and anti-aging PN complex provided by this invention is as follows: PDA with a purity of not less than 98.5wt% and a polydispersity index (PDI) of 1.2 to 1.5, obtained by enzymatic hydrolysis and purification of salmon sperm DNA, is selected. This PDA is dissolved in an aqueous medium along with epigallocatechin gallate with a purity higher than 98.0wt% and a caffeine content lower than 0.1wt%. The pH of the aqueous medium is adjusted to the range of 5.5 to 6.2 using a citrate-sodium citrate buffer system, and the initial temperature of the aqueous medium is controlled at 2°C to 6°C by starting a circulating cooling unit.

[0030] Under a constant temperature environment of 2℃ to 6℃, a magnesium chloride solution containing divalent magnesium ions was added dropwise to an aqueous medium according to a molar ratio of phosphate groups of polydeoxyribonucleotides, epigallocatechin gallate, and divalent magnesium ions of 10:3:1.5. Simultaneously, a high-speed dispersion device was activated to apply a constant shear force of 3200 rpm to the aqueous medium. Due to the low temperature environment of 2℃ to 6℃, a dense hydration layer formed on the surface of the divalent magnesium ions due to the directional arrangement of water molecules. This process facilitated the reaction between the divalent magnesium ions and the polydeoxyribonucleotides... A physical barrier is established between the phosphate group of the polyoxyribonucleotide and the phenolic hydroxyl group of epigallocatechin gallate, maintaining a homogeneous metastable equilibrium in the ternary system under high-speed shear. After the addition is complete, the aqueous medium is heated to 42°C at a rate of 1.2°C / min using a circulating cooling unit, and then stirred at low speed for 40 minutes at this temperature. As the temperature rises, the hydrated layer of divalent magnesium ions undergoes thermal desorption, and the coordination binding sites are exposed in an orderly manner along the topological structure of the polydeoxyribonucleotide molecular chain, driving the molecule to desorb. Epigallocatechin gallate forms a 5 nm thick, continuously distributed molecular coating layer on the surface of polydeoxyribonucleotide (PDNU) molecules via charge bridging with divalent magnesium ions. During this thermoinduced desorption process, the phosphate groups of the PDNU and the phenolic hydroxyl groups of epigallocatechin gallate provide a synergistic thermodynamic driving force through a chelation effect, lowering the apparent activation energy during magnesium ion desolvation. The upper temperature limit of 42 °C ensures that the incremental heat energy provided by the system precisely matches the ternary coordination network. The activation barrier, while disrupting the ion hydration layer, prevents the thermal degradation of polydeoxyribonucleotide macromolecules, thereby achieving precise exposure and orderly anchoring of coordination sites at the surface scale. At this time, the transmittance of the aqueous medium at a wavelength of 600 nm increased from 35% in the initial mixed state to 94%, confirming that epigallocatechin gallate has physically shielded the phosphodiester bond sites on the polydeoxyribonucleotide chain, thereby blocking the attack of nucleases on the nucleic acid backbone and providing antioxidant protection in the physiological environment.

[0031] Example 3: This example, in verifying the degradation pattern of the antioxidant and anti-aging PN complex in a biomimetic skin interstitial fluid environment containing nucleases and reactive oxygen species, confirms the physical shielding strength of the coordination coating structure for the biological activity of polydeoxyribonucleotides. The experiment employed an evaluation system consisting of a dynamic light scattering instrument with 0.1 nm resolution and an electrochemical oxygen reduction monitoring module. This system had a temperature control accuracy better than ±0.05℃. Oxidative stress conditions in damaged skin tissue were simulated by adding 15.0 U / L nuclease I and 50.0 μmol / L hydrogen peroxide to a phosphate buffer solution with a pH of 7.40. The amount of divalent magnesium ions added was considered. The decision-making process balances the coverage density of coordination sites on the phosphate backbone with the colloidal stability of the system. If the molar ratio of divalent magnesium ions to phosphate groups is less than 1:10, the distribution density of coordination bridging sites is insufficient to support the formation of a continuously distributed coating layer on the surface of polydeoxyribonucleotides by epigallocatechin gallate, resulting in gaps in the physical shielding space. If the molar ratio is greater than 2:10, it will induce excessive bridging between multiple molecular chains and produce heterogeneous precipitation. Based on this decision-making rule, a gradient verification was carried out by selecting a control group with a molar ratio of divalent magnesium ions to phosphate groups of 0.5:10, an experimental group with a molar ratio of 1.5:10, and an out-of-range group with a molar ratio of 2.5:10.

[0032] The evolution of physicochemical indicators of each sample group under simulated physiological stress was monitored. For the physical mixed control group without added divalent magnesium ions, after 4.0 hours of nuclease I treatment, the hydrodynamic radius of polydeoxyribonucleotides shrank from the initial 82.4 nm to 15.8 nm, and the content of free nucleotides in the system increased from the initial 1.2 wt% to 68.5 wt%, indicating that the nucleic acid backbone lacking coordination structure protection had undergone deep degradation. For the experimental group prepared using the process in Example 2, after continuous treatment under the same oxidative stress conditions for 24.0 hours, its weight-average molecular weight retention rate was 92.3%, and the transmittance of the system remained between 93.5% and 94.2%, confirming that the coordination network mediated by divalent magnesium ions constructed a deterministic physical barrier against nuclease attack on the surface of polydeoxyribonucleotide single molecular chains. This barrier blocked the effective contact between enzyme molecules and nucleic acid backbone by occupying and spatially isolating phosphodiester bond sites.

[0033] The influence of the core variable, oxidative stress intensity, on the stability of the complex was analyzed. When the hydrogen peroxide concentration increased linearly from 50.0 μmol / L to 200.0 μmol / L, the weight-average molecular weight retention rate of the experimental group fluctuated by less than 1.8%, demonstrating tolerance to oxidative damage. In contrast, the retention rate of the 0.5:10 control group, due to the discontinuous molecular coating layer, rapidly decreased from 41.6% to 22.4% with increasing oxidative intensity. The data showed that only when the component ratio was within the optimal window of 10:2:1 to 10:4:2 for the molar ratio of phosphate groups, epigallocatechin gallate, and divalent magnesium ions in the polydeoxyribonucleotide, and combined with a molecular sequential coordination process involving a linear temperature increase from 2℃ to 42℃, could the transmittance of the complex at 600 nm wavelength be increased from below 40.0% to above 92.0%, forming a stable molecular coating structure. This confirms the technical value of the method of this invention in maintaining the structural integrity of polydeoxyribonucleotides under complex biochemical environments. Furthermore, to confirm the physicochemical stability and anti-enzymatic degradation ability of the complex, standardized performance verification was performed on multiple batches of complex samples prepared according to the optimal window (molar ratio 10:2:1 to 10:4:2). First, the samples were placed in a constant temperature environment of 25°C for 48 hours, and the absorbance value at 260 nm was monitored in real time using a UV spectrophotometer. The experimental results showed that the absorbance fluctuation deviation of each sample was between 2.8% and 4.5%, all lower than the preset change rate threshold of 5%, confirming that the coordination coating structure effectively anchors the biochemical structure of polydeoxyribonucleotides at room temperature. Subsequently, the complex was incubated in a simulated physiological environment containing 10 U / mL deoxyribonuclease I for 24 hours. High performance liquid chromatography (HPLC) detection showed that the residual rate of polydeoxyribonucleotides was stable in the range of 87.2 wt% to 93.5 wt%, not lower than the performance benchmark of 85 wt%, proving the high structural stability of the complex of the present invention under physiological stress and storage conditions.

[0034] Example 4: In this example, when dealing with a 10L batch production scale biopharmaceutical manufacturing process, and facing the situation where the radial temperature gradient caused by thermal inertia in a large-capacity system leads to uneven coordination layer thickness, the implementation details of the antioxidant and anti-aging PN complex and its preparation method are as follows: A 10L stainless steel reactor with a Teflon-coated impeller and a jacketed cooling circulation system is used. Polydeoxyribonucleotides with a purity of not less than 98.5 wt% and a polydispersity index (PDI) of 1.35 and epigallocatechin gallate with a purity of not less than 98.0 wt% are dissolved in water for injection. The content of sodium ascorbate phosphate is 0.1 parts per 100 parts of the total weight of the complex. The pH of the resulting aqueous medium is adjusted to 5.8 using a citric acid-sodium citrate buffer system. After starting the jacketed circulation system to lower the initial temperature of the material to 4°C, the stirring speed inside the reactor is maintained at 450 rpm. Under rpm conditions, a 0.5 mol / L magnesium chloride solution was uniformly added dropwise at a flow rate of 15.0 ml / min using a peristaltic pump, ensuring a molar ratio of divalent magnesium ions to polydeoxyribonucleotide phosphate groups of 1.5:10. Simultaneously, the redox potential ΔE, regulated by sodium ascorbate phosphate, was monitored in real time by an electrochemical oxygen reduction monitoring module, stabilizing it between -150 mV and -100 mV to suppress component auto-oxidation. The electrochemical monitoring module employed pulse potential detection technology, with the detection pulse width controlled at the microsecond level. By shortening the polarization time, ion migration and deposition on the electrode surface were effectively suppressed. Furthermore, the electrolytic cell measurement point was located downstream of the optical path detection point of the transmission spectrometer. Combined with the diffusion and dilution effect of the high-speed shear flow field inside the vessel, this ensured that any transient minor disturbances that might occur near the electrode would not flow back to the optical detection window, thereby eliminating physical cross-interference between different monitoring methods.

[0035] After the dripping is completed, the reaction system activates an adaptive heating mode, adjusting the flow rate of the heat transfer medium in the jacket based on real-time readings from multiple temperature sensors within the reactor. This allows the material to heat up to 42°C at a linear rate of 1.2°C / min. During this heating process, material samples are periodically extracted and fed into an online dynamic light scattering detection module. The module monitors the evolution of the molecular coating thickness H in real time using the correlation between particle Brownian motion velocity and hydrodynamic radius. The online detection module incorporates a microfluidic pressure-reducing and flow-stabilizing unit along the sampling path, instantly introducing the extracted material samples into a resting measurement cell. At the measurement moment, the sample circulation feed is shut off, allowing the fluid to remain in the detection optical path area for 3 seconds to restore pure Brownian motion. The built-in high-pass filter of the detection system filters out overall mechanical vibration interference with frequencies below 10Hz, extracting only the coherent light intensity fluctuation signal driven by molecular thermal motion. The incremental information of the hydrodynamic radius is calculated using an autocorrelation function, thus avoiding convection noise generated by stirring and shearing. The formula for calculating the thickness H is as follows: H=R clad -Rcore Where H is the thickness of the molecular coating layer, and R is... clad R is the hydrodynamic radius of the complex measured after the coordination reaction is complete. core The initial hydrodynamic radius of the polydeoxyribonucleotide monomolecule was measured before adding magnesium chloride solution. When the H value output by the detection module stabilized at 5.5 nm and the transmittance of the system at a wavelength of 600 nm increased from 42.0% to 93.8%, heating was stopped and the heat exchanger was quickly started to cool the material to 25°C. After the prepared complex was placed at 25°C for 48 hours, the fluctuation deviation of its hydrodynamic radius was less than 0.5 nm, and the transmittance remained above 93.0%.

[0036] In the context of biopharmaceutical mass production under varying environmental temperature fluctuations, the system maintains the antioxidant kinetics stability of the complex by adjusting the addition ratio of sodium ascorbate phosphate. With a total complex weight of 100 parts, when the sodium ascorbate phosphate content is at the lower limit of 0.1 parts, the redox potential ΔE of the system shows an increasing trend during storage, leading to oxidative cross-linking of the phenolic hydroxyl groups of epigallocatechin gallate, which in turn causes a positive deviation in the molecular coating thickness H and induces the formation of fine clusters. When the sodium ascorbate phosphate content increases to the upper limit of 2.0 parts, the increased sodium ion concentration in the system generates an electrostatic shielding effect, weakening the coordination binding energy between the polydeoxyribonucleotide phosphate groups and divalent magnesium ions, resulting in a decrease in the density of the molecular coating. By determining the amount of sodium ascorbate phosphate within the range of 0.05 to 0.12 parts, the redox potential fluctuation rate δ in the aqueous medium remains stable within 48 hours, ensuring the structural survival of the complex in the physiological microenvironment.

[0037] Example 5: In this example, when the system faces the situation where the uneven initial charge distribution of polydeoxyribonucleotides from different extraction batches affects the consistency of molecular coating growth, the preparation method performs the following pre-calibration before adding magnesium chloride solution: By detecting the basic conductivity σ0 of the aqueous medium and calculating the ionization equilibrium deviation, the magnesium ion concentration compensation coefficient λ for this batch of raw materials is determined according to a preset linear correspondence to ensure that the effective charge density of divalent magnesium ions in the ternary coordination network is maintained at a constant baseline. The online particle size analysis unit is then activated to perform a baseline scan of the initial mixed solution, and the obtained initial hydrodynamic radius R is used to determine the baseline. core This serves as a reference input for subsequent adaptive heating rate calculations, enabling polydeoxyribonucleotide molecular chains under different physical states to complete conformational remodeling within the same dynamic response time.

[0038] In a biopharmaceutical manufacturing scenario involving continuous stirring, the system executes the following judgment logic regarding the stability boundary during the molecular coating formation process; the real-time monitoring device extracts the transmittance T of the aqueous medium.p The first derivative of the redox potential changes with time t, and combined with the redox potential fluctuation rate δ fed back by the electrochemical monitoring module, establishes a real-time response operator S, where the calculation formula for the real-time response operator S is as follows: S=(dT) p ) / dt×δ, where S is the real-time response operator; T p δ represents the transmittance percentage; t represents the heating time; and δ represents the redox potential fluctuation rate. When the heating rate is between 1.0℃ / min and 1.5℃ / min and the value of operator S remains below the threshold of 0.02 for five consecutive sampling cycles, it is determined that the molecular coating layer on the surface of the polydeoxyribonucleotide has reached an equilibrium structure. The control unit instructs the heat exchange system to reduce the material temperature to below 25℃ at a cooling rate of not less than 5.0℃ / min. This procedure controls the thickness fluctuation of the molecular coating layer of the complex within an error range of 0.5nm.

[0039] When the system faces deviations in the molecular weight distribution of polydeoxyribonucleic acid (PDA) due to the heterogeneity of raw materials, the preparation method follows a standardized verification procedure based on the source attributes. PDAs obtained from salmon sperm DNA through enzymatic hydrolysis and purification, with a purity of not less than 98.5 wt% and a polydispersity index (PDI) between 1.2 and 1.5, are selected. The total content of residual divalent metal impurities in the material is detected using atomic absorption spectrometry and locked to a baseline below 5 ppm. The buffer salt concentration of the citrate-sodium citrate buffer system is dynamically adjusted based on the measured impurity content to eliminate the competitive occupation of non-target metal ions in the coordination process between divalent magnesium ions and phosphate groups. This procedure, combined with online feedback control of the real-time response operator S, compresses the transmittance deviation of the complexes prepared from different batches of materials at 600 nm wavelength to within 1.5%, achieving a high degree of uniformity in the physical morphology and biochemical activity of the coordination coating structure.

[0040] Example 6: In this example, during the pre-deployment calibration of a biopharmaceutical manufacturing system targeting different production environment background noise benchmarks, the control threshold of the real-time response operator S is determined by executing the following sensitivity optimization procedure: In a Class 10,000 cleanroom environment equipped with a constant-temperature circulation unit, simulated mechanical stirring at 450 rpm is applied to solute-free water for injection. Background transmittance fluctuation data is recorded using a high-sensitivity online transmission spectrometer, and the mean fluctuation value of the basic response operator under no-load conditions is calculated. The mean fluctuation of the basic response operator Defined as the statistical average of the absolute value of the first derivative of the rate of change of background transmittance of water for injection with time at a stirring speed of 450 rpm, during calibration, the spectrometer continuously sampled for 120 seconds at a frequency of 100 Hz. By performing discrete difference calculation on the obtained transmittance curve, the intensity of background fluctuation caused by mechanical vibration, light source drift and solvent microbubbles was extracted and used as the zero-point benchmark for determining whether the reaction has reached physical equilibrium. Polydeoxyribonucleotide standards with a weight-average molecular weight gradient of 300 kDa to 500 kDa were selected, and coordination reactions were performed at a pH of 5.8 and a temperature of 4 ℃. The first derivative characteristics of the molecular coating thickness H when it reaches the steady-state range of 2 nm to 10 nm were statistically analyzed, and the correlation between the measured value of the real-time response operator S and the physical stability index was established. The control threshold Γ was determined according to the following formula. ,in, To control the threshold, ζ is the mean fluctuation of the basic response operator caused by environmental background noise, and ζ is the safety margin factor, which is set to 0.15 under static stirring environment. The control threshold Γ determined under this procedure is 0.02. The selection of this threshold enables the system to distinguish between transient transmittance noise caused by local flow field disturbance in the reactor and equilibrium state formed by molecular self-assembly, thus avoiding the risk of premature process shutdown caused by environmental signal glitches.

[0041] When the system encounters a situation where the molecular weight polydispersity index (PDI) of the raw material polydeoxyribonucleotides fluctuates within the range of 1.2 to 1.5, leading to inconsistent chain segment conformation adjustment rates, the system determines the dropping flow rate v using the following adaptive parameter matrix calibration procedure. f Before the material enters the reactor, the system extracts the characterization parameters of the batch of material and calculates the flow rate compensation coefficient based on the correspondence between the polydispersity index (PDI) and the dynamic response time of macromolecular chain unfolding. The dropping flow rate v f The calculation formula is as follows; v f =v0×(1-α×(PDI-1.2)), where v f To adapt the dripping flow rate, v0 is the baseline flow rate, set to 15.0 ml / min. α is the flow channel characteristic constant, calibrated to 0.45 based on the ratio of the reactor inner diameter to the agitator diameter. PDI is the polydispersity index of the molecular weight of the polydeoxyribonucleotides in the batch being treated. The derivation logic of this formula is based on the conformational relaxation kinetics of polydeoxyribonucleotide segments in a shear flow field. The PDI value reflects the heterogeneity of polymer molecular chain size, and materials with higher PDI have a wider conformational relaxation time distribution in the aqueous phase. The overall circulating shear rate is correlated with the surface segment relaxation rate through the flow channel characteristic constant α. When PDI increases, the dripping flow rate v0 is reduced. fTo prolong the local residence time of divalent magnesium ions at the dropping interface, ensuring that nucleotide molecular chains of different lengths can complete sequential coordination with magnesium ions within their specific conformational response time, thereby compensating for the surface coating heterogeneity caused by the heterogeneity of raw materials, when using materials with a molecular weight polydispersity index (PDI) of 1.5 for preparation, the system instructs the peristaltic pump to reduce the dropping flow rate to approximately 12.98 ml / min. This action prolongs the contact time between divalent magnesium ions and heterogeneous nucleotide segments, reducing the uniformity deviation of the final molecular coating layer on the surface of polydeoxyribonucleotide molecular chains from 15.0% to below 3.0%.

[0042] It will be apparent to those skilled in the art that the present invention is not limited to the details of the exemplary embodiments described above, and that the present invention can be implemented in other specific forms without departing from the spirit or essential characteristics of the present invention.

[0043] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention.

Claims

1. An antioxidant and anti-aging PN complex, characterized in that, Includes polydeoxyribonucleotides, epigallocatechin gallate, divalent magnesium ions, sodium ascorbate phosphate, citrate-sodium citrate buffer system, and water: Polydeoxyribonucleic acid is a product obtained by enzymatic hydrolysis and purification of salmon sperm deoxyribonucleic acid. The purity of polydeoxyribonucleic acid is not less than 98.5 wt%, the weight average molecular weight is 300 kDa to 500 kDa, and the polydispersity index (PDI) is 1.2 to 1.

5. Based on a total weight of 100 parts for the complex, the following components are present: polydeoxyribonucleotides: 0.5 to 2.0 parts; epigallocatechin gallate: 0.1 to 0.5 parts; sodium ascorbate phosphate: 0.05 to 0.12 parts; citrate-sodium citrate buffer system: 0.1 to 0.3 parts; and water: to make up the balance to 100 parts. The divalent magnesium ions are provided by magnesium chloride, and the molar ratio of the divalent magnesium ions to the phosphate groups on the polydeoxyribonucleotide molecular chain is 1:10 to 2:10; the polydeoxyribonucleotide, epigallocatechin gallate, and divalent magnesium ions have a coordination coating structure in an environment with a temperature of 2°C to 6°C and a pH of 5.5 to 6.

2. Coordination coating structure refers to the chelation between divalent magnesium ions and the phosphate groups on the polydeoxyribonucleotide molecular chain and the phenolic hydroxyl groups of epigallocatechin gallate, so that epigallocatechin gallate forms a continuously distributed molecular coating layer on the surface of the polydeoxyribonucleotide molecular chain.

2. The antioxidant and anti-aging PN complex according to claim 1, characterized in that, The molecular coating layer formed by polydeoxyribonucleotides, epigallocatechin gallate, and divalent magnesium ions is in metastable equilibrium at a temperature of 2°C to 6°C. After mixing, the aqueous medium is heated to 37°C to 48°C at a heating rate of 1.0°C / min to 1.5°C / min and held for 30 min to 60 min to induce thermal desorption of the hydrated layer of divalent magnesium ions and drive the molecular coating layer from the metastable state to the equilibrium steady state. The physical shielding space formed by the molecular coating layer on the outside of the polydeoxyribonucleotide molecular chain has a thickness of 2 nm to 10 nm, and the molecular coating layer covers the phosphodiester bond sites on the polydeoxyribonucleotide molecular chain.

3. The antioxidant and anti-aging PN complex according to claim 1, characterized in that, The total content of divalent metal impurity ions in polydeoxyribonucleotides is less than 5 ppm; the purity of epigallocatechin gallate is higher than 98.0 wt%, and the caffeine content in epigallocatechin gallate is less than 0.1 wt%.

4. The antioxidant and anti-aging PN complex according to claim 1, characterized in that, The purity of sodium ascorbate phosphate is higher than 99.0 wt%; the weight ratio of sodium ascorbate phosphate to epigallocatechin gallate is 1:2 to 1:

5.

5. The antioxidant and anti-aging PN complex according to claim 1, characterized in that, The osmolar concentration of the complex ranges from 280 mOsm / kg to 320 mOsm / kg; the complex is electrically neutral in an environment with a pH range of 5.8 to 6.

0.

6. The antioxidant and anti-aging PN complex according to claim 1, characterized in that, The coordination coating structure exhibits temperature-responsive characteristics; the coordination saturation of divalent magnesium ions on the surface of polydeoxyribonucleotide molecular chains increases with increasing ambient temperature in the range of 2℃ to 37℃; the surface density of the molecular coating layer of the complex at 37℃ is higher than that at 6℃.

7. The antioxidant and anti-aging PN complex according to claim 1, characterized in that, Sodium ascorbate phosphate undergoes oxidation when the dissolved oxygen concentration in the complex system is higher than 0.1 mg / L, thereby inhibiting the oxidation reaction inside the complex by capturing and scavenging free radicals; epigallocatechin gallate forms a multi-site chelation network with divalent magnesium ions through phenolic hydroxyl sites.

8. The antioxidant and anti-aging PN complex according to claim 1, characterized in that, After the complex was left to stand at 25°C for 48 h, the absorbance change rate at 260 nm was less than 5%; after the complex was incubated in a solution containing 10 U / mL deoxynucleotidase I for 24 h, the residual rate of polydeoxyribonucleotidyl is not less than 85 wt%.

9. A method for preparing an antioxidant and anti-aging PN complex, used to prepare the antioxidant and anti-aging PN complex according to claim 1, characterized in that, Includes the following steps: Step 1001: Dissolve polydeoxyribonucleotides, epigallocatechin gallate, sodium ascorbate phosphate, and citrate-sodium citrate buffer system in water, and adjust the pH of the resulting aqueous medium to 5.5 to 6.

2. Step 1002: Turn on the cooling unit and control the temperature of the aqueous medium to 2°C to 6°C; Step 1003: Under conditions of 2°C to 6°C, a solution containing divalent magnesium ions is added dropwise to an aqueous medium at a uniform rate, and the mixture is mixed for 1 to 4 hours at a temperature of 2°C to 6°C and a pH of 5.5 to 6.

2. After mixing is completed, the aqueous medium is programmed to be heated to 37°C to 48°C at a heating rate of 1.0°C / min to 1.5°C / min, and held at this temperature for 30 to 60 minutes to drive the thermal desorption of the hydrated layer of divalent magnesium ions, thereby inducing the formation of a continuously distributed molecular coating layer on the surface of polydeoxyribonucleotide monomolecules by epigallocatechin gallate.

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