Preparation,Characterization and Function Evaluation of Porous Starch Microspheres Loaded with Artemisinin Extracted from Artemisia Annua

Author:Zhao Xue

Supervisor:yang feng jian


Degree Year:2019





Artemisinin is a sesquiterpene lactone compound containing a peroxy group and was first isolated from a medicinal plant Artemisia annua 1971.It is also the most effective therapeutic bioactive molecule against cerebral malaria and chloroquine-resistant malaria.Recent studies have indicated that artemisinin and its derivatives also have a variety of biological activities for other diseases,such as anti-tumor,anti-virus and immunosuppression.Especially,in the aspect of anti-tumor,artemisinin and its derivatives can target tumor cells that contain much higher iron ion content than normal cells,and generate free radicals through catalyzing the peroxidation bridge of artemisinin by iron ions,thereby inducing cancer cell death or inhibiting angiogenesis.Artemisinin and its derivatives have selective inhibitory effects on tumor cells,which can reduce toxic side effects on normal tissues,meanwhile reverse the multidrug resistance of tumors.There is no cross-resistance phenomenon between artemisinin and traditional chemotherapy drugs.Therefore,artemisinin has become a research hotspot in the field of anti-tumor bioactive molecules.However,artemisinin is difficult to be dissolved in water,because its water solubility is extremely poor;thus,it is incompatible with human blood and body fluid,leading to its low bioavailability.Uptaken artemisinin is quickly excreted through blood circulation,which seriously hinders its research and clinical application.Therefore,the main direction of developing artemisinin into a broad-spectrum anti-cancer bioactive molecule is to synthesize a series of its derivatives through modifying its functional group by glycosylation and salination.Its known derivatives include artemether,dihydroartemisinin,artesunate,etc.Although some of its derivatives have increased water solubility to a certain extent compared to artemisinin,its mechanisms of reproductive toxicity and genotoxicity in humans is still not clear enough,and safety concerns still exist.Therefore,the main content of this thesis is to develop porous starch microspheres loaded with artemisinin(ART-PS),through a porous starch bioactive molecular carrier with porous network structure,using the spatial confinement effect of carrier’s porous network structure,and the micro-crystalline state of the poorly soluble bioactive molecule highly dispered in the carrier system to improve the dissolution rate of artemisinin is the key to improve the bioavailability of artemisinin.Then,we carried out detailed research and comparison on the characterization,tissue distribution,in vitro safety,antimalarial activity and antitumor activity of our ART-PS,with original or regular artemisinin as a control.The research results we obtained are as follows:1.The experimental parameters affecting the loading amount and encapsulation efficiency were respectively determined by single factor experiments.We optimized the parameters to determine the optimal preparation conditions of ART-PS:the delivery of artemisinin powder concentration is 80 mg/mL;Maximum of 312.5 mg of artemisinin can be adsorbed in each 1 g of porous starch.The optimum adsorption time for adsorption of artemisinin by porous starch is 30 min.Under the optimal preparation conditions,the loading capacity and the encapsulation efficiency of ART-PS were 20.37%±0.61%and 81.86%±3.06%by HPLC.Therefore,it has the characteristics of high adsorption capacity and high drug loading capacity.2.The physicochemical properties of ART-PS were determined by scanning electron microscopy(SEM),Brunauer Emmett and Teller(BET),fourier-transform infrared spectroscopy(FTIR),X-ray diffraction(XRD),and differential scanning calorimetry(DSC)and thermo-gravimetry(TG).It was found that the porous starch had successfully loaded with artemisinin,and the artemisinin was adsorbed in the void of porous starch microspheres,which could be released after the porous starch was degraded by gastrointestinal enzymes in the body,thus exerting its efficacy and improving the water solubility of artemisinin.It was detected that porous starch was successfully loaded with artemisinin,its chemical structure had not changed,and its crystal structure was amorphous.3.The results showed that the saturated solubility of ART-PS was significantly higher than that of artemisinin active drugs.The saturated solubility of the porous starch microspheres-loading artemisinin in water,artificial gastric juice and artificial intestinal juice were 118.12μg/mL,64.58 μg/mL and 46.38μg/mL,respectively,while the saturated solubility of raw artemisinin in water,artificial gastric juice and artificial intestinal juice were 31.27 μg/mL,39.42 μg/mL and 26.94 μg/mL,respectively.The saturated solubility of the physical mixture in water,artificial gastric juice and artificial intestinal juice was 25.65 μg/mL,32.45 μg/mL and 18.90 μg/mL,and the saturation solubility of artemisinin piperaquine tablets in water,artificial gastric juice and artificial intestinal juice was 46.82 p.g/mL,59.71 jig/mL and 41.15μg/mL,respectively.Therefore,it has the characteristics of high saturated water solubility.4.The experimental results of ART-PS in vitro dissolution showed that the release effect of porous starch microspheres-loading artemisinin in three dissolution media was significantly improved compared with that of artemisinin active drugs and physical compounds,the dissolution of artemisinin and piperaquine tablets were similar.The final release cumulants of artemisinin active drug,porous starch microsphere loading artemisinin,physical mixture and artemisinin piperaquine tablets in water were 24.45%,98.67%,29.17%,and 85.38%respectively.The final release cumulants in the artificial gastric juice were 25.72%,92.21%,27.68%,and 49.46%respectively.The final release cumulants in the artificial intestinal fluid were 23.14%,88.41%,26.09%,and 84.74%respectively.Therefore,ART-PS has the better water solubility characteristic than the raw artemisinin drug.5.The bioavailability of ART-PS results show that the absorption rate of ART-PS in rats was significantly higher than that of raw artemisinin and artemisinin piperaquine tablets,in about 2 h after the treatment,blood drug concentration in rats reached the peak of 199.74μg/mL,and raw artemisinin and artemisinin piperaquine tablets in 4 h after the treatment,blood drug concentration reached the levels of highest 68.77 μg/mL and 72.63 μg/mL.Therefore,it has the characteristics of high bioavailability with high blood drug concentration.6.The drug content in heart,liver,spleen,lung,kidney and brain of 98 rats were measured.The results showed that the drug content of the porous starch microsphere-loading artemisinin was higher than that of the artemisinin active drug in all tissues,and the drug content maintained longer than that of the artemisinin active drug in the body.Therefore,ART-PS has better organization distribution characteristics.7.In the study on the safety of ART-PS in vitro,this paper compared the inhibitory effect of ART-PS with artemisinin active drugs on HL-7702 in human normal hepatocytes.The results showed that artemisinin does not have great killing effect on normal cells and has low cytotoxicity.Therefore,it has its own non-toxic safety characteristics in vitro.8.In the study of antimalarial activity of ART-PS,the SYBR Green I method was used respectively to investigate.The results show that the proliferation inhibition rate of artemisinin-based drugs for p.falciparum differed with solubility differences of these drugs.Among them,the IC50 values of ART-PS-c,ART-PS-h and APT-h were 9.23 nmol/L,13.99 nmol/L and 15.74 nmol/L.However,ART-h has very low solubility in water,and the statistical results show that it has no killing effect on malaria parasites.Therefore,it has higher antimalarial activity in term of IC50 than artemisinin piperaquine tablets and raw artemisinin.9.In the study of the anti-tumor activity of ART-PS,liver cancer cell line HepG2 was treated with two sets of schemes.MTT results indicated that,when combined with holo-transferrin,compared with raw artemisinin,ART-PS showed stronger inhibitory effects on tumor cells.Therefore,it has stronger anti-tumor activity than raw artemisinin when combined with holo-transferrin.