High Hydrostatic Pressure Micellar Medium Extraction And Oral Drug Delivery System Of Artemisinin From Artemisia Annua L.

Artemisia annua L.is an annual herb of Artemisia in Asteraceae.The glandular hairs on the leaf surface contain antimalarial drug artemisinin(ART).ART and its derivative artesunate(AS),which were constructed by ultrasonic drug loading method to construct an oral pectincasein carrier loaded ART system(PC-ART)and pectin-casein carrier loaded AS system(PCAS),and unique micellar characteristics were found when the two were characterized.To evaluate the oral applicability of PC-ART and PC-AS,a series of in vitro and in vivo evaluations were carried out.At the same time,the toxicity of the two was tested.Finally,SLE mice were treated by oral administration for 8 weeks and the therapeutic effects of PC-ART and PC-AS on SLE were evaluated.(1)High hydrostatic pressure assisted APG1214(HHPE)was used to extract ART from the leaves of A.annua.The study used ART pre-column derivatization-HPLC method to quantitatively analyze the ART content in the leaf extract of A.annua.Choosed the green solvent APG1214,which had high saturation solubility for ART,high extraction yield,low price and eco-friendly.Using single-factor method and response surface method(RSM)analysis to optimize the extraction conditions,the optimal conditions for HHPE of ART were:the material particle size was 82 mesh,the material-to-liquid ratio was 1:27,the APG1214 concentration was 7.30%,and the pressure was 316 MPa.Under these conditions,the theoretical yield of ART was 6.96 mg/g,and the actual yield of ART was 6.78±0.13 mg/g verified by experiments.The two were basically the same,indicating the reliability of the model.Separated by ethyl acetate and recrystallized several times,ART crystals with a purity of 78.66% were finally obtained.In addition,the reusable characteristics of APG1214 proved the high efficiency and eco-friendliness of the HHPE.(2)To explore the mechanism of extracting ART from A annua leaves by HHPE.Among them,the extraction efficiency and energy consumption of HHPE,hydrothermal extraction,ultrasonic extraction and microwave extraction were compared.The results showed that ART was 89.18±1.65% obtained by high pressure-assisted APG1214 under the optimal conditions,which was higher than that of hydrothermal extraction(1.5 times),ultrasonic extraction(1.34times)and microwave extraction(1.27 times).However,the energy consumption of HHPE was only 1/86.7 of Soxhlet extraction,1/26 of hydrothermal extraction and 1/14.4 of ultrasonic and microwave extraction.Density functional theory(DFT)was used to find that APG1214 and ART were combined by hydrogen bonds to form supramolecular molecules.In addition,the extraction kinetics model revealed that the extraction process of ART by HHPE was mainly rapid washing,accompanied by slow diffusion,resulting in deformation and breakage of glandular trichomes(GT)stored in ART after extraction.Therefore,HHPE can become a green,sustainable and low-cost alternative for natural product extraction,providing a theoretical basis for industrial applications.(3)Oral pectin-casein carrier-loaded ART system(PC-ART)and pectin-casein carrier-loaded AS system(PC-AS)were constructed by ultrasonic drug loading method.Pectin-casein(PC)micelle carrier was synthesized by carbodiimide reaction and characterized.The results showed that the strength of amide bond vibration and the formation of critical micelle concentration(CMC)in infrared spectroscopy proved the success of the reaction between pectin and casein.PC-ART and PC-AS were established by ultrasonic drug loading method.The single-factor results showed that when the mass ratio of pectin to casein was 1:1,the percentage of ethanol was 60%,the ultrasonic loading time was 5 min,the power was 180 W,and the mass ratio of PC carrier to ART was 20:2,the drug loading and encapsulation efficiency of PC-ART reached the peak,the highest drug loading was 9.05%,and the encapsulation efficiency was 85.94%.For PC-AS,when the mass ratio of pectin to casein was 1:2,the ethanol percentage was 10%,the ultrasonic time was 20 min,the ultrasonic power was 300 W,the mass ratio of PC carrier to ART was 20:20,the drug loading and encapsulation efficiency of PC-AS reached the peak,the drug loading was 49.43 % and the encapsulation efficiency was 88.87 %.Finally,PC-ART and PC-AS were characterized.We found that the particle size of PC-ART(PC(1:1)=160 nm,PCART=100.2 nm)and PC-AS(PC(1:2)=202.4 nm,PC-AS=104.2 nm)decreased after drug loading,indicating that liposoluble drugs were encapsulated in the hydrophobic core by micelles,which changed the interaction force of micelles.In addition,the size of nanoparticles dispersed in distilled water(PC-ART=100.2 nm,PC-AS=104.2 nm)was larger than that of dried nanoparticles due to the presence of hydration layer(both are 60 nm).The infrared spectrum results showed that PC loaded ART and AS in the form of micelles at the center of micelles,so that the characteristic peaks of ART and AS were covered.(4)The oral applicability of PC-ART and PC-AS was analyzed by in vitro evaluation of saturated solubility,dissolution experiments and in vivo evaluation of bioavailability.The safety of PC-ART and PC-AS was evaluated using zebrafish and Caco-2 cells.The acute toxicity test of adult zebrafish,the long-term toxicity test of adult zebrafish and the cytotoxicity test of Caco-2 were carried out.In vitro evaluation results showed that PC-ART and PC-AS freeze-dried powder were well dispersed in distilled water,and the particle size and Zeta potential had no significant change within 10 days.The increase in water solubility of ART and AS loaded with pectin-casein makes the saturated solubility of PC-ART and PC-AS in deionized water,artificial gastric juice and artificial intestinal juice higher than that of the original drug.Moreover,PC-ART and PC-AS exist in artificial gastric juice and artificial intestinal juice in the form of micelles,which was manifested in the in vitro dissolution experiment that PC-ART and PC-AS dissolve less ART and AS in artificial gastric juice and artificial intestinal juice,while the dissolved water of the original drug is higher than that of PC-ART and PC-AS on average.The bioavailability test results showed that PC-ART and PCAS improved the bioavailability of the original drug,and the pectin-casein micelle drug delivery system promoted the absorption,distribution and elimination of the drug.The results showed that PC-ART and PC-AS did not cause significant damage to zebrafish in 96 h.In the28-day long-term toxicity test,the liver damage(ALT and AST)of PC-ART and PC-AS to zebrafish adult fish was less than that of the original drug.Moreover,PC-ART and PC-AS had less oxidative stress on zebrafish than ART and AS.In addition,Caco-2 cells treated with PCART and PC-AS still maintained high cell viability.The above results prove the safety of PCART and PC-AS.(5)MRL/lpr mice were orally administered with PC-ART and PC-AS for 8 consecutive weeks,and body weight,physiological status and urinary protein were monitored during the experiment.After the experiment,plasma was collected and 10% renal homogenate was prepared to detect SLE marker antibodies and inflammatory factors,and renal pathological sections were prepared to observe the degree of renal injury and immune complex deposition.The results showed that during administration,PC-ART and PC-AS controlled the weight gain of MRL/lpr mice caused by gland enlargement,and effectively reduced the urinary protein level.The decrease of serum creatinine(Cre)and blood urea nitrogen(BUN)indicates the recovery of renal function,SLE marker antibody(ANA),anti-ds DNA antibody(ds DNA-Ab)and inflammatory cytokines(IL-6).The decrease of IFN-γ level indicated the remission of SLE.In addition,PC-ART and PC-AS reduced renal injury and Ig G deposition of immune complex in MRL/lpr mice.In summary,PC-ART and PC-AS can effectively reduce the levels of SLE marker antibodies and inflammatory factors,reduce renal injury and immune complex deposition,restore renal function,reduce urinary protein levels,and have a positive effect on the treatment of SLE.In short,PC-ART and PC-AS with pectin-casein as the micelle shell improved the stability,water solubility,bioavailability and bioavailability of the drug,which was beneficial for oral administration to alleviate the symptoms of SLE and played a certain therapeutic effect.In summary,an efficient,eco-friendly and sustainable extraction method was used to obtain artemisinin from A.annua leaves,and artemisinin and its derivative artesunate were prepared into oral drugs with good water solubility and high bioavailability by using pectin-casein micelle carrier.These drugs were applied to the treatment of systemic lupus erythematosus,so that the active components of A.annua were processed and utilized with high value,which also provided certain data support for the extraction of effective components of plants and clinical medicine.