Study On The Identification Of Self-assembled Nanoparticles From Shaoyao Gancao Decoction And Its Effect On Compatibility

Shaoyao Gancao Decoction(SGD)is derived from“Treatise on Febrile Diseases”edited by Zhang Zhongjing in the Han Dynasty and is composed of Paeoniae Radix Alba(Baishao)and licorice(Gancao)with traditional Chinese medicine(TCM)effect of reconciling the liver and spleen,softening the tendons and relieving pain.SGD is one of the most commonly used classic prescriptions in TCM in the past dynasties and has a rich clinical application basis.It is also one of the 100classic prescriptions in the"Ancient Classics List(First Batch)"issued by the State Administration of TCM in 2018.However,paeoniflorin(as main active component of Shaoyao)and glycyrrhizic acid(as main active component of Gancao)have the problems of high hydrophilicity or low solubility,leading poor oral absorption.The compatibility of SGD could promote the absorption of paeoniflorin and enhance the anti-inflammatory and analgesic effects.The mechanism of compatibility was probable that the glycyrrhetinic acid,which was metabolized from glycyrrhizic acid,could inhibit the activity of efflux transporters,and thereby promoting the absorption of paeoniflorin.However,the absorption of the main active components from SGD was better than mixed suspension of Shaoyao and Gancao single decoction(MSSGD),and there was no significant difference in the content of main active components between SGD and MSSGD.Therefore,it was not sufficient to explain the compatibility mechanism from the effect of the active components on the efflux transporter.Inspired by the molecular recognition and self-assembly phenomenon of the chemical components of TCM,this study attempted to explain the SGD compatibility mechanism from the self-assembly behavior of the chemical components of SGD.Firstly,paeoniflorin loaded glycyrrhizic acid self-assembled micelles(Pae-GL SAMs)was constructed and the influence of SAMs on the absorption of paeoniflorin was evaluated.Secondly,aggregation caused quench(ACQ)probe was applied to identify and monitor the self-assembled nanoparticles(SAN)from SGD(SGD-SAN)in vitro and in vivo,and study the in vivo process of SGD-SAN,with a view clarify the in vivo behavior of SGD-SAN.Thirdly,under the premise of proving the existence and absorption of SGD-SAN,the function of SAN in compatibility was further studied.Finally,the SGD-SAN was isolated from decoction and its material basis and activity were preliminary identified and evaluated.The main study contents were as follows:1.Preparation and absorption evaluation of Pae-GL SAMsThe Pae-GL SAMs were prepared by ultrasonic dispersion method.The particle size,PDI and zeta potential of Pae-GL SAMs were(58.89±4.24)nm,0.194±0.010and-(24.40±1.90)m V,respectively.The entrapment efficiency(EE)and drug loading(DL)were(42.21±0.89)%and(8.66±0.25)%,respectively.The TEM image of Pae-GL SAMs presented spherical shape morphology with particle size of40?80 nm.The effect of Pae-GL SAMs on the intestinal absorption of Pae was studied and the results showed that the P_app of mixed solution of paeoniflorin and glycyrrhizic acid(MSPG)was increased to 1.86 times and 1.98 times in jejunum and ileum respectively as compared to paeoniflorin solution(PS),while the Pae-GL SAMs could further improve the P_app of paeoniflorin to 1.93 times and 2.61 times in the jejunum and ileum respectively,compared with MSPG.The pharmacokinetic results showed that compared with PS,C_max and AUC_0-t of the Pae-GL SAMs were increased to 2.17 times and 3.64 times,respectively,the MRT_0-t and t_1/2 were significantly longer and the CL was also significantly reduced.The MSPG could also improve the absorption of paeoniflorin,but there was no significant difference.The results indicated that the formation of GL SAMs significantly improved the oral absorption of paeoniflorin.2.Study on feasibility of applying ACQ Probe to identify the SGD-SAN in the decoctionACQ Probe was used to identify the SAN from SGD in vitro.The results showed that the Baishao single decoction(BSD),Gancao single decoction(GSD)and SGD all had fluorescence and their total fluorescence intensity(TRE)was in following order:GSD>SGD>BSD.The particle size of three kinds of decoctions after high-speed centrifugation were within 500 nm,with PDI of 0.4?0.6 and zeta potential within-10 m V.The results of TEM showed that BSD contained less SAN;GSD contained a lot of rod-shaped SAN,while SGD contained irregular spherical SAN.The SAN from three kinds of decoction all had strong water quenching sensitivity and could maintain good stability under gastrointestinal conditions that simulate the fasted state of rats.The results suggested that the fluorescence signal of ACQ probe could represent particle signal of SAN,indicating their structure integrity and identifying the SAN in vitro.3.Study on the correlation between the fluorescence and components of SGDThe TCM chromatographic fingerprints similarity evaluation software was used to analyze the similarity of HPLC fingerprints of BSD,GSD and SGD,and the results calculated 12,26 and 37 common peaks respectively.Meanwhile,the gallic acid,albiflorin,paeoniflorin,benzoylpaeoniflorin origined from Baishao and liquiritin apioside,liquiritin,isoliquiritin apioside,isoliquiritin,liquiritigenin,glycyrrhizic acid,isoliquiritigenin origined from Gancao were also identified.The three kinds of decoctions were divided into four parts by centrifugation at different speeds,and the Pearson correlation analysis was used to analyze the fluorescence intensity and the peak area of the HPLC spectrum.The results displayed that the peak areas of 26 common peaks of GSD were strongly positively correlated with the fluorescence intensity(R²>0.95,P<0.05),while there was weakly positive correlation between the peak areas of 12 common peaks of the BSD and fluorescence intensity(R²<0.9,P>0.05).The peak areas of 33 common peaks of the 37 common peaks of SGD were well strongly positive relevant with fluorescence intensity(R²>0.95,P<0.05).The results indicated that the main active components of Gancao such as liquiritin,glycyrrhizic acid,etc.participated in the formation of SGD-SAN,and the main active components of Baishao such as albiflorin,paeoniflorin,etc.could be encapsulated by SGD-SAN.4.Study on in vitro/in vivo absorption behavior of SGD-SAN in the decoctionThe ACQ probe was used to identify and monitor the behavior of SGD-SAN in the organism and tissue cells.The results showed that the GSD-SAN and SGD-SAN in the decoction could maintain in the rat abdomen for 8?12 h after oral administration with peak time at 6 h for GSD-SAN and 1 h for SGD-SAN,while the formation and retention of BSD-SAN in the decoction was too low to be detected.SAN in the decoction could be absorbed as intact particles and distributed mainly in the liver after oral administration of GSD and SGD with peak time at 8 h for GSD and 4 h for SGD.There was almost no intact particles absorption after oral administration of BSD.Although there was no significant difference,both of GSD and SGD had better absorption of SAN in the ileum than the jejunum,and SAN in the decoction could be absorbed by Caco-2 cells in the form of intact particles.The above results indicated that it was feasible to use the ACQ probe to identify and monitor the SGD-SAN in the organism and tissue cells.SAN in the decoction could be absorbed into the body in the form of intact particles and mainly distributed in the liver,and the absorption rate of SGD-SAN in the decoction was faster than GSD-SAN in the decoction.5.Effects of SGD-SAN on the encapsulation and in vitro release of main componentsThe ultrafiltration centrifugation method was used to study the distribution of the main components from SGD in the ultrafiltration tube.The results showed that the distribution of the Baishao components from BSD and MSSGD in the ultrafiltration tube was no difference.The concentration of the Baishao components from SGD in the lower layer of the ultrafiltration tube was significantly lower than that in the upper layer,indicating that the paeoniflorin and other three components did not participate in the formation of SAN,but could be encapsulated by SGD-SAN.Then the EE of Baishao components was calculated and the results showed the EE of gallic acid,albiflorin,paeoniflorin,benzoylpaeoniflorin were(17.73±0.26)%,(33.78±1.03)%,(33.61±0.90)%,(88.53±0.58)%,respectively.The concentrations of seven components of Gancao from GSD,MSSGD or SGD in the lower layer of the ultrafiltration tube were all significantly lower than that of the upper layer,indicating that Gancao components could be self-assembled to form SAN during the decocting process.The results of in vitro release study showed that the presence of SGD-SAN had a slow-release effect on the release of Baishao and Gancao components.The results further indicated that the active components of Gancao were involved in the formation of SAN,and the Baishao components such as paeoniflorin could be encapsulated by SGD-SAN,leading to a certain slow-release effect of paeoniflorin.6.Effect of SGD-SAN on the intestinal absorption and the anti-inflammatory effectSingle-pass intestinal perfusion was used to study the influence of the formation of SGD-SAN on the intestinal absorption of four components of Baishao and seven components of Gancao.In the Chapter 5,the drug concentration data before ultrafiltration centrifugation showed the contents of these 11 components were no significant difference between SGD and MSSGD,however,the intestinal absorption of these 11 components from SGD was significantly better than that of from the MSSGD,indicating that the formation of SGD-SAN was beneficial to the absorption of the components and the absorption-promoting ability of SAN in ileum segment was better than that in jejunum segment.The results of in vivo anti-inflammatory studies showed that SGD groups were more effective in inhibiting mouse ear swelling and improving subcutaneous tissue lesions,inhibiting rat cotton granuloma and transudate,and inhibiting the inflammatory factor(TNF-?,IL-1?,IL-6)secretion than those of MSSGD.The results indicated that the formation of SGD-SAN could promote the intestinal absorption of the active components,and further enhance its anti-inflammatory effect.7.Isolation,characterization and activity evaluation of SGD-SANThe SGD-SAN was isolated by high-speed centrifugation combined with suction filtration and then characterized.The results of particle size analysis showed that the average particle size of SGD-SAN were(246±8)nm with PDI of 0.36±0.08.The zeta potential was–(9.8±0.3)m V.The results of TEM and SEM showed that SGD-SAN was irregularly spherical.Elemental analysis showed that SGD-SAN contained C,N,O,Na,Mg,K.The polysaccharide and protein contents of SGD-SAN were(60.40±4.33)%and(30.96±1.62)%,respectively.The total contents of 11 components including paeoniflorin and glycyrrhizic acid were(11.00±1.15)%.LPS-induced RAW264.7 cell inflammation model was chosen to evaluate the activity of SGD-SAN.The results showed that SGD-SAN could significantly reduce the levels of NO,ROS,TNF-?,IL-6 and IL-1?in inflammatory cells.The cell uptake study was carried out by SGD-SAN incubating with RAW264.7 cells for2 h,and results showed that SGD-SAN could be taken up by the cells.These results indicated that SGD-SAN was mainly contained primary metabolites and active components,had the effect of inhibiting the release of inflammatory factors and could be taken up by macrophages in the form of intact particles.In summary,the formation of Pae-GL SAMs could effectively improve the oral absorption of paeoniflorin.The chemical components of SGD could self-assemble to form nanoparticles during the decocting process.The main active components of Gancao were involved in the formation of SAN and the main active components of Baishao could be encapsulated.SGD-SAN could be absorbed as intact particles and distributed mainly in the liver.The formation of SGD-SAN could delay the release of various components,promote the absorption of the components mainly in the ileum,and enhance the anti-inflammatory effect.SGD-SAN was mainly contained polysaccharides,proteins,paeoniflorin,glycyrrhizic acid and other active components.SGD-SAN had the effect of inhibiting the release of inflammatory factors.This study proved the feasibility of using ACQ probe to study the biological behavior of SAN from TCM decoction,provided concrete evidence for oral absorption of integral SAN.The intact particles absorption of SGD-SAN may be one of the mechanisms that the compatibility of SGD could promote the absorption of active component such as glycyrrhizic acid,paeoniflorin etc.This study enriched the scientific connotation of the compatibility theory of TCM compound,which was conducive to promoting the innovation and development of the research on TCM decoction.