Preparation of Water-soluble Powders of Honokiol and Its Antitumor Activity in Vitro
Author:Wu Wei Wei
Supervisor:zhang xin xin zu yuan gang zhao xiu hua
With the development of cell biology and molecular biology,new approaches and targets for cancer treatment have been discovered,and new therapeutic drugs have been developed.The active ingredients of natural drugs have the advantages of natural,low toxicity,improving immunity and not easy to produce drug resistance,and play their unique advantages in the prevention and treatment of cancer.Honokiol is one of the main active ingredients of Magnolia officinalis,which is a unique Chinese medicine.It has many biological activities such as anti-inflammatory,anti-bacterial,anti-anxiety,anti-depression and anti-oxidation.In addition,in recent years,many researchers have found that the honokiol has good anti-cancer effects on many cancers,such as liver cancer,breast cancer,prostate cancer and glioma,indicating that it has certain application and development value in anti-cancer.However,honokiol,as a low solubility and high permeability drug,has a low solubility in water,which severely limits its absorption and utilization in the body and makes its pharmacodynamics not work properly.Therefore,improving the solubility of honokiol in water and its oral absorption is of great significance for its application and development in anticancer.Moreover,the different drug formulations may have different molecular mechanisms of anticancer.Therefore,in order to improve the solubility and bioavailability of honokiol,three water-soluble powders of honokiol and honokiol were prepared by three solubilization methods,and the best water-soluble powder was selected by comparing and analyzing the solubility and bioavailability of three water-soluble powders of honokiol and its inhibitory effect on tumor cells was investigated.The main contents and results are as follows:(1)In this paper,honokiol nanoparticles was successfully prepared by antisolvent precipitation method,and its physical and chemical properties,solubility and bioavailability in vivo were tested.The results showed that the chemical structure of honokiol nanoparticles was the same as that of free honokiol,and there was no change;the crystallinity of honokiol nanoparticles was lower than that of free honokiol,and it existed in amorphous form.In addition,the solubility of honokiol nanoparticles was significantly higher than that of free honokiol,which was about 46.52 mg/ml in artificial gastric juice(AGJ)(free honokiol was about 0.06 mg/ml)and 60.19 mg/ml in artificial intestinal juice(AIJ)(free honokiol was about 0.04 mg/ml).The in vitro release rate of honokiol nanopowder was 20.41 times and 26.2 times higher than that of free honokiol in AGJ and AIJ,respectively.In addition,the Cmax and AUC(O-T)values of honokiol nanoparticles in rats were significantly higher than those of the free honokiol,about 7.62 and 6.52 times of the free honokiol.(2)Honokiol solid dispersion was prepared by solvent evaporation method,which improved the solubility and oral bioavailability of honokiol.The best mass ratio of honokiol to PLX was 1:4.The results displayed that the morphology of solid dispersion was completely different from that of the free honokiol,and the intermolecular hydrogen bonds might be formed between honokiol and poloxamer 188 during the preparation process,and the honokiol existed in amorphous form in solid dispersion.Furthermore,the solubility of honokiol solid dispersion was significantly higher than that of free honokiol,was about 32.43 mg/ml in AGJ(free honokiol was about 0.06 mg/ml)and 34.41 mg/ml in AIJ(free honokiol was about 0.04 mg/ml).The in vitro release rate of honokiol solid dispersion was 28.01 times and 18.25 times higher than that of free honokiol in AGJ and AIJ,respectively.In addition,in vivo pharmacokinetic studies showed that the values of Cmax and AUC(O-T)of honokiol solid dispersion in rats were significantly higher than those of the free honokiol,which were about 6.98 and 4.41 times of the free honokiol.(3)Honokiol inclusion complex with high water solubility was successfully prepared by the combination method of grinding and co-precipitation with HP-β-CD as inclusion material.Phase equilibrium experiments showed that honokiol and HP-β-CD formed inclusion complexes with molar ratio of 1:1.The drug loading and encapsulation efficiency of the inclusion complexes were about 13.08±0.26%and 89.80±0.97%,respectively.Results of physicochemical characterization showed that the intermolecular hydrogen bonds might be formed between honokiol and HP-β-CD,and honokiol existed in an amorphous form in the inclusion complex.In addition,the solubility of inclusion complex was significantly higher than that of free honokiol,which was about 53.33 mg/mL in AGJ(free honokiol was about 0.06 mg/ml)and 62.22 mg/mL inAIJ(free honokiol was about 0.04 mg/ml).The in vitro release rate of inclusion complex was significantly higher than that of free honokiol,about 48 times and 38.76 times higher than that of free honokiol in AGJ and AIJ,respectively.Furthermore,the pharmacokinetic studies in vivo showed that the values of Cmax and AUC(O-T)of inclusion complex in rats were significantly higher than that of honokiol powder,about 8.12 times and 7.05 times higher than that of free honokiol.(4)Three water-soluble powders of honokiol were compared and analyzed in drug content,solubility and bioavailability.The results showed that the inclusion complex of honokiol had higher solubility and oral bioavailability than nanoparticles and solid dispersion,so it might have better anti-tumor effect.(5)In this paper,the antitumor effect of inclusion complex of honokiol in vitro was studied,and the possible mechanism of apoptotic effect of inclusion complex of honokiol on HepG2 cells in vitro was also discussed.The results showed that both free honokiol and inclusion complex of honokiol could inhibit growth and induce apoptosis of HepG2 cells.At the same concentration of honokiol and culture conditions,the inclusion complex of honokiol showed stronger cytotoxicity than free honokiol.CCK8 results showed that the inclusion complex of honokiol could strongly inhibit the growth of HepG2 cells,and the cell survival rate of inclusion complex group was significantly lower than that of free honokiol group with the same concentration.FCM results showed that the inclusion complex of honokiol could block the G0/G1 phase of HepG2 cells,and also induce apoptosis of HepG2 cells.The blocking and inducing effect of inclusion complex of honokiol was stronger than that of free honokiol.The Q-PCR and western blotting results showed that the inclusion complex of honokiol inhibited the expression of Bcl-2 and Bcl-xL genes in HepG2 cells,promoted the expression of p38-MAPK and caspase-3 genes in a dose-dependent manner.