Preparation And In Vitro Pharmacodynamic Investigation Of NMN-PNS Complex Slow-Release Formulations

The NMN-PNS compound hydrogel patch is developed by combining Panax notoginseng saponin and nicotinamide mononucleotide.Such a novel formulation combines Panax ginseng saponin’s effects in improving blood microcirculation and inhibiting platelet aggregation with the effect of nicotinamide mononucleotide in reducing vascular collagen deposition and obstruction.The novel formulation is a synergistic,accurately dosed,less toxic topical formulation for the treatment of ocular blood stasis（hereafter referred to as NMN-PNS compounded hydrogel patch）.The paper is divided into the following sections:Part I:Research on the preparation process of NMN-PNS complex slow-release formulation（1）Examination of the synergistic effect of Panax notoginseng saponin with nicotinamide mononucleotide.In vitro anticoagulation experiment was used to investigate the efficacy of total saponins of Panax notoginseng combined with niacinamide mononucleotide,taking the prolongation of coagulation time as the evaluation index.By comparing the compound drug with the single dose sample group,the preliminary test results of the bulk drug showed that,while reducing the total amount of Panax notoginseng saponins,the compound drug extended the coagulation time about1.5 times that of the single dose of Panax notoginseng saponins group and 1.77 times that of the niacinamide mononucleotide group.It was found that Panax notoginseng saponins combined with niacinamide mononucleotides had a synergistic anticoagulant effect of reducing medication dose.（2）Process study of total saponin liposomes from Panax ginseng.The liposomes of Panax ginseng total saponin were prepared using the film hydration method with soy lecithin and cholesterol as the lipid material.The liposome’s encapsulation rate and drug loading capacity were determined as the evaluation indexes.The optimal preparation of Panax ginseng saponin liposomes was screened by single-factor and response surface optimization experiments:drug-lipid ratio of 1:4,phospholipid ratio of 1:8,hydration temperature of 25℃,and hydration time of 60 min.The average encapsulation rate of the obtained Panax ginseng total saponin liposomes was 80.95%with an RSD of 1.07%,and the average drug loading was 15.21%with an RSD of 1.91%.The appearance of the lipid vesicles was monolayer and sphere-like,and the particle size was（183.8±9.32）nm.The particle size and properties were used as the evaluation indexes of the freeze-drying process.The prepared liposomes of Panax notoginseng were freeze-dried,and the preparation process and prescription were determined by single-factor and orthogonal experiments as follows:mannitol as the freeze-drying protective agent,the concentration of 2.5%,pre-freezing temperature-80℃and pre-freezing time 16 h.The FTIR and DSC characterization of the prepared liposomal lyophilized formulations showed that only hydrogen bonding and van der Waals forces were present between the drug and the lipid material,and no changes in the chemical properties of the drug were observed.（3）Compound hydrogel patch preparation and matrix prescription screening.Sodium polyacrylate and propanetriol were mixed in a mass ratio of 1:4 to form phase I;0.3%carbomer solution was added to the prescribed amount of panaxoside liposomes and nicotinamide mononucleotide to form phase II;phase I and phase II were mixed in equal amounts and added to phase III cross-linking agent（5%aluminum chloride and EDTA-2Na）solution to form a compound hydrogel agent.The above-prepared drug-containing hydrogel was evenly coated on the non-woven fabric,rolled and flattened by a pressure roller,and left for 24 h to obtain the compounded hydrogel patch.The optimum preparation process and prescription conditions for the compound hydrogel patch were determined using single-factor and response surface optimization experiments with the following quality factors as quality scoring criteria:0.3%Carbomer solution concentration of 43.75%,Sodium polyacrylate concentration of4.69%,Propanetriol concentration of 18.75%and Aluminum chloride concentration of1.56%.（4）In vitro drug release and transdermal permeation performance study of compounded hydrogel patches.The in vitro release of the PNS-NMN compound gel patch and PNS_Lip-NMN compound hydrogel patch was investigated by in vitro dialysis method.The results showed that the release of panaxosides from the non-liposome-forming panaxoside complex gel was under the primary release pattern with the equation:Q_t=72.642(1-e^-0.077t),while the release of panaxosides from the liposome-forming panaxoside complex gel was by the Higuchi model with the equation:Q_t=10.055t^1/2+0.521,and nicotinamide,The mononucleotides showed a one-stage release pattern due to their water solubility.The percutaneous permeability of the PNS-NMN prodrug compounded gel patch and the PNS_Lip-NMN compounded hydrogel patch were investigated separately using a Franz diffusion cell.The results showed that 3%nitrogen ketone was the best permeation promoter for this formulation,and the transdermal permeation of the PNS_Lip-NMN compounded hydrogel patch was approximately 4 times more effective than that of the PNS-NMN prodrug compounded hydrogel patch.Part II:Establishment of quality standards and stability study of compounded hydrogel patches（1）Development of a method for determining compounded formulations’content.In this study,it was found that the UV absorption peaks of PNS and NMN were similar,and their absorption wavelengths were close to each other,so it was difficult to determine their contents simultaneously by UV absorption methods without pre-separation.Therefore,the UV-second-order derivative method was chosen.The data obtained from the UV absorption spectra were smoothed using the Savitzky-Golay method in Origin software to remove other irrelevant interferences.The second-order derivatives were derived to establish the method for the determination of the compound content.The linear regressions were performed for the concentration of PNS and NMN and the second-order derivative values,respectively.The linear regression equations were C=-14017D-5.4056,R²=0.9988（PNS）,C=-24254D-3.3501,and R²=0.9892（NMN）,respectively,with good linearity.The method was applied to the determination of the active ingredients in six groups of samples.The RSD of PNS was about 0.23%,and the spiked recovery was about 99.05%,while the RSD of NMN was about 0.18%,and the spiked recovery was about 99.86%.（2）The establishment of quality standards for NMN-PNS compound slow-release formulations.A complete quality evaluation system was established for the compounded hydrogel patches in terms of appearance,paste content,fugacity,and adhesion,respectively,concerning the 2020 edition of the Chinese Pharmacopoeia.The prepared compounded hydrogel patches appeared uniformly coated with light yellow gel,and the initial adhesive force was determined by the rolling ball ramp stop method to be in the range of 24.6-25.6 gauge steel balls.The paste content per patch was 20.72±0.38g/100cm².（3）The stability of the compounded hydrogel patch was investigated:the high temperature,high humidity,and 4500lx intense light irradiation experiments were conducted on the compounded hydrogel patch,respectively.It was found that high humidity and bright light irradiation did not affect the quality of the patches,while the drug content decreased at a high temperature of 60°C.The patches were stable at 4°C,25°C,and 45°C.The storage conditions for this compounded hydrogel formulation were determined to be sealed and stored at room temperature.Part III:Safety and in vitro pharmacodynamic studies of the NMN-PNS combination sustained release formulation（1）In vivo skin irritation tests were conducted using New Zealand White rabbits to assess the safety performance of the formulation.Their experimental results showed no skin irritation,such as redness,swelling,and plaques,in both intact and broken skin groups of the compound hydrogel patch.（2）The in vitro efficacy study of compound hydrogel patch showed that:the thrombolysis efficiency and the thrombolysis efficiency of PNS and NMN alone and in combination were compared by in vitro anticoagulant test and in vitro thrombolysis test,and the combined dosage of medicinal drugs was determined.The experimental results showed that under the exact dosage,the prolongation of coagulation time of ATPP as the evaluation index,the prolongation of coagulation time of combined administration was about 1.2 times that of PNS liposome single-dose group and 1.7 times that of NMN single-dose group.In combination,the dosage of PNS was only 50%of that of PNS alone.The combination of PNS and NMN showed a tendency for synergistic effect,and compared with the single drug,it had a significant advantage of less dosage and enhanced efficacy.Through the in vitro thrombolytic experiment of the compound drug,the thrombolysis efficiency of the prepared compound drug was 1.6 times higher than that of the total saponins of Panax notoginseng single dose,and the effect was not noticeable compared with the single dose of niacinamide single nucleotide.In vitro efficacy studies of NMN-PNS compound hydrogel patch found that the concentration ratio of PNS to NMN 3:1 had the best thrombolysis and anticoagulation effect.The NMN-PNS compound slow-release formulation developed in this project is simple and easy to prepare.The drug release in vitro shows regularity,and the in vitro efficacy study found that its efficacy has a synergistic effect and the dose is reduced,which provides a reliable and detailed theoretical basis for the modernization of Panax notoginseng and the application of nicotinamide mononucleotide in topical formulations.