Research On The Stabilization And Protective Effect Of Tea Saponin On Hesperidin Nanocrystals And Their Anti-inflammatory Activities

Inflammation plays an important part in the pathogenesis of many diseases,such as cancer,arthritis,allergies,neurodegenerative diseases,and so on.Hesperidin（HDN）,a flavonoid compound,is rich in citrus plants.A large number of studies have shown that HDN is beneficial for a variety of diseases,mainly including inflammation,diabetes,Alzheimer’s disease and cancers.HDN has a strong inhibitory effect on arthritis,acute and chronic inflammation,colitis,respiratory tract inflammation,and other inflammation.It was found that HDN had significant anti-inflammatory activities in ancient China.Whereas,due to the lack of ionized groups,HDN showed poorly water-soluble,which affected its clinical curative effect.Nanosuspensions is a kind of drug delivery system which used surfactant as stabilizers aid to help the drug evenly disperse in the state of suspension liquid.It is one of the key strategies in the development of nanosuspensions preparation that adding stabilizers can make nanosuspensions maintain good physical stability during the preparation.At present,the commonly used stabilizers of nanosuspensions are mostly from chemical synthesis,such as Sodium Dodecyl Sulfate（SDS）,Hydroxypropyl Methyl Cellulose（HPMC）,Povidone K30（PVP K30）,Tween 80 and so on.They are used as stabilizers in nanosuspensions with large dosage,poor biocompatibility and certain toxicity.Due to the potential safety risks caused by long-term use of chemical synthetic stabilizers,the exploration of natural stabilizers has attracted extensive attention from researchers.In recent years,the researches on natural stabilizers are increasing day by day.Tea saponins（TS）,a pentacyclic triterpene compound extracted from tea trees,is a kind of natural nonionic surfactant with good surface activity.Based on the fact that TS has amphiphilic structure and surface activity,we hypothesized that TS could maintain a high degree of dispersion of drugs in suspensions and prevent irreversible agglomeration of nanosuspensions through a combination of saccharide during freeze-drying.Therefore,the study focused on the development and utilization of natural stabilizers.In this study,Hesperidin nanosuspensions（HDN-NS）was prepared by using TS as the natural stabilizer of nanosuspensions,instead of the traditional stabilizer,to improve the solubility of HDN.The ability of TS to form and stabilize HDN-NS,and the influence of TS on the redispersion of hesperidin solid nanocrystals（HDN-SN）were evaluated.Finally,the anti-inflammatory activity of HDN-SN was further studied.Using TS as a stabilizer to prepare HDN green nano-preparations can reduce the dosage of synthetic excipients,improve the safety of preparations,broaden the selection range of excipients used in nanosuspensions,and expand the application of traditional Chinese medicine.Meanwhile,the solubility,medicinal properties and bioavailability of HDN were improved by preparing nanosuspensions,which provide a reference for the development of HDN preparations and clinical research,as well as a new idea for the treatment of inflammation.The main contents and results of the study are as follows.（1）In vitro analysis method of HDN was established.HPLC method was used to determine the content of HDN in HDN-SN and analyze the dissolution of HDN in vitro.（2）To study the preparation process of HDN-NS.HDN-NS was prepared by high-speed shearing combined with high-pressure homogenization.The stability ability of TS was compared with that of five kinds of traditional stabilizers.In comparison with the commonly used stabilizers,such as SDS,HPMC,PVP K30,Polyethylene Glycol400（PEG 400）,Poloxamer 188（P 188）and glycyrrhizic acid（GA）,the mean particle size（261.0±2.3 nm）and PI（0.037±0.032）of HDN-NS stabilized by TS were the smallest at the same concentration（0.10%,w/v）,which proved the superior stabilizing efficiency of TS.The single factor method was used to investigate the dosage of stabilizer,drug concentration,shearing velocity,shearing time,homogeneous pressure,homogeneous cycles.According to the single-factor experiment,four preparation processes with significant influence were selected,and Box-Behnken response surface design with four factors and three levels was used to optimize the formulation and preparation process of HDN-NS.The optimal preparation process of HDN was as follows:the concentration of HDN was 1.0 mg·m L^-1,the concentration of TS was0.09%,high-speed shearing speed was 16000 rpm,shearing time was 2 min,the homogeneous cycles were 6 cycles at 35 MPa and 12 cycles at 98 MPa.HDN-NS was milky suspensions with mean particle size of 258.8±2.1 nm and PI of 0.070±0.024.At4℃and 25℃,the particle size and PI of HDN-NS have no significant change,so it can maintain good stability.（3）To study the stabilization mechanism of TS.By investigating the influence of critical micelle concentration（CMC）of TS,pH,ionic strength,zeta potential on HDN-NS,the result showed that TS appears to stabilize the HDN-NS by electrostatic effect.The zeta potential of HDN-NS was（-23.16±1.12）m V,The CMC of TS was 2.5mmol·L^-1.（4）The freeze-drying method was used to convert HDN-NS to HDN-SN,and the effect of TS on the redispersibility of HDN-SN was investigated.The optimal cryoprotectant was lactose and TS with a total concentration of 5%（w/v）,and the concentration ratio of TS and lactose was 1:1.The mean particle size of HDN-SN was（266.5±9.0）nm and PI of HDN-SN was 0.092±0.055 after reconstitution,indicating the best redispersibility.A combination of TS and lactose can effectively prevent the irreversible aggregation and Crystal growth of HDN-SN during freeze-drying.（5）The physicochemical properties of HDN-SN were investigated.The drug content in HDN-SN was（12.43±0.15）%.The cumulative dissolution rate of HDN-SN reached 37.19%within 120 minutes,increased by 126%over the coarse drug.Transmission electron microscope（TEM）and scanning electron microscope（SEM）results show that the HDN nanocrystals are irregular lumps of crystals.Fourier transform infrared（FT-IR）and X-Ray powder diffraction（XRD）results showed that the chemical structure and crystal form of HDN did not change during the preparation process,but the crystallinity decreased.The results of powder properties investigation showed that the heap density of HDN-SN was 0.263 g·cm^-3;the vibrational density of HDN-SN was 0.335 g·cm^-3;the Angle of repose of HDN-SN was 54.9°,indicating that the powder fluidity of HDN-SN was poor,and glidants should be added into HDN-SN for subsequent production and processing.The properties of HDN-SN were stable under high temperature and strong light irradiation.However,at 75%humidity,HDN-SN was slightly hygroscopic and slightly darker in color,suggesting that HDN-SN should be stored under dry conditions.（6）To study the in vivo anti-inflammatory activity of HDN-SN.The inhibitory effect of HDN-SN on acute inflammation was preliminarily evaluated by xylene-induced ear swelling in mice.The inhibition rate of HDN-SN was 47.7%,which was2.17 times higher than that of the coarse drug.The inhibitory effect of HDN-SN on acute inflammation was stronger than that of TS HDN and physical mixture.The above results indicated that the anti-inflammatory activity of HDN can be improved by preparing HDN as nanosuspension.