Screening And Evaluation Of The Supramolecular Antitumor Complex Of Chinese Herbal Active Ingredients Curcumin

Turmeric,curcumae and rhizoma zedoariae are all belonging to curcuma plants.Traditional Chinese medicine holds that turmeric can promote Qi activity,dispel wind and promote blood circulation,dredge meridians to relieve pain.Curcumae can promote blood circulation and stop pain,promote Qi activity and resolve depression,clear away the heart-fire and cool blood,benefit bile and back the yellow function obviously.Rhizoma zedoariae has the effects of promoting Qi activity and relieving depression,breaking blood stasis and relieving pain.The pharmacological activities of the three Chinese medicines are mainly antitumor,antifungal,anti-inflammatory,hypolipidemic,gallbladder,liver protection and anti-thrombosis.Curcumin（CUR）is a kind of plant polypheno which is extracted from turmeric,curcumae and rhizoma zedoariae.Researches show that CUR possesses diverse pharmacological activities,suchasanticancer,hypoglycemiceffect,antiulcer,anti-inflammatory and antibacterial.It can be seen that CUR is the most important active pharmaceutical ingredients（API）in turmeric,curcumae and rhizoma zedoariae.The main advantage of CUR is being safe at high doses of 12 g/day in humans.However,the therapeutic effectiveness of curcumin is limited by very low solubility and poor bioavailability in the aqueous medium.Pharmaceutical co-crystals and co-amorphous are supramolecular complexes which are formed from API and the small molecule formers（co-amorphous former,CAF;co-crystal former,CCF）through non-covalent interactions involving hydrogen bonds and van der waals forces.Pharmaceutical co-crystals and co-amorphous keep the intact structure and pharmacological activities of API,while,create advantages on solubility,dissolution rates and even bioavailability.Supramolecular complexes are promising solid form and have attracted considerable attention for improving the physicochemical properties of APIs.In present dissertation,using CUR as model API,co-crystals and co-amorphous supramolecular complexes are screened based on the anticancer pharmacological activity.The objectives are to investigate the effect of binding between CUR and CCF on the properties of CUR,including solubility,dissolution rate,binding with BSA,pharmacokinetics and cell inhibiting activity.This research applies modern crystal engineering methods and theories to improve the physical and chemical properties of traditional Chinese medicine.The results offer the theorical support to develop CUR complex with definite anticancer mechanism.Part one:Screening and physiochemical property evaluation of the supramolecular complex based on curcuminObjective:Using CUR as model API,pharmaceutical co-crystals and co-amorphous complexes are screened to improve the solubility,dissolution rates of CUR.Methods:Using CUR as model API and the small molecule organic acids,amino acid,amide,piperazine and API（Ⅱ）with specific pharmac-ological activity as CCFs,co-crystals and co-amorphous supramolecular complexes are screened using solvent-assisted grinding method.The as-prepared CUR supramolecular complexes are characterized by powder x-ray diffraction（PXRD）,differential scanning calorimetry（DSC）,thermogra-vimetric analysis（TGA）,field emission scanning electron microcopy（FESEM）,infrared radiation（IR）,and solid-state ¹³C nuclear magnetic resona-nce（ssNMR）.Solubility and dissolution rate are measured using equilibrium solubility method and propeller method,respectively.Results:Based on PXRD and DSC analysis,one API-CAF co-amorphous（named CUR-piperazine co-amorphous）,two API-CCF co-crystals（named CUR-piperazine and CUR-isonicotine co-crystals）and one API（I）-API（Ⅱ）co-crystal（named CUR-gallic acid co-crystal）were determined.The results of IR and ssNMR indicated that the C=O???H-N hydrogen bonds in two API-CCF co-crystals were formed between the C=O group in CUR and the N-H groups in the piperazine or isonicotine,while,CUR-gallic acid co-crystal was sustained by C=O???H-O hydrogen bond between the phenol O-H group in CUR and the C=O group in gallic acid.The dissolution rates of CUR-based co-crystals were faster than that of CUR（P<0.05）.Conclusion:In vitro solubility and dissolution rate of CUR are improved by complexing with CCF.The screen of CUR-based co-crystal supramolecular complex has the potential to be a novel strategy to improve the in vitro physiochemical property.Part two:Study on interactions of CUR-based supramolecular complex with BSA using fluorescence spectroscopy and density functional theory simulation calculationObjective:The binding mechanisms of CUR-based co-crystal supramo-lecular complex with BSA are investigated at physiological pH condition.The variation and reason of the binding rule of CUR with BSA before and after formation of co-crystal complex are clarified at a molecular level.The results can provide academic reference to further investigation on storage,transpor-tation and action mechanism in vivo of CUR-base supramolecular complexes.Methods:The interactions of CUR and CUR-based supramolecular complexes with BSA at physiological pH are investigated by fluorescence spectroscopy,ultraviolet spectroscopy and density functional theory（DFT）simulation calculation.The fluorescence quenching data at 298,310 and 315K were analyzed and calculated using the Stern-Volme and modified Stern-Volme equations;the fluorescence quenching mechanism,binding constants K_b,binding sites and thermodynamic parameters of CUR-based supramo-lecular complexes with BSA are determined.Results:The binding constants K_bbetween CUR and BSA decreased with increasing temperature for CUR,CUR-piperazine and CUR-isonicotine co-crystals,indicating the involvement of static quenching mechanism due to CUR-BSA complex formation.However,the increase in temperature resulted in increase in K_b and decrease in quenching constant K_svv for CUR-gallic acid co-crystal,suggesting static quenching along with dynamic quenching mechanisms.The sign the thermodynamic parameters of binding reaction between CUR and BSA indicated that weak forces between CUR and BSA were both hydrogen bonds and/or van der Waals forces for CUR-piperazine and CUR-isonicotine co-crystals and hydrophobic interaction for CUR-gallic acid co-crystal.Comparing K_b of CUR-BSA and CUR-CCF-BSA,it showed that the introduction of CCF into CUR-CCF supramolecular complex changed the binding of CUR and BSA.The result of DFT calculation showed that hydrogen bonds in tryptophan-CUR-piperazine complex became weaker than that in tryptophan-CUR complex;the binding energy between tryptophan and CUR-piperazine complex(-17.9 k J·mol^-1)was also weaker than that between tryptophan and CUR(-26.1 kJ·mol^-1).The result of DFT simulation was consistent with experimental data from fluorescence quenching.Conclusion:The formation of API-CCF supramolecular complex may change the binding mechanism and binding forces of API with BSA,as a result,change in API transport in vivo.The binding mechanism and binding forces of API-CCF supramolecular complex with BSA vary with the kinds of CCF.In present study,in vivo API transport was simulated at a molecular level;the results were of significance for elucidating the action mechanism and pharmacokinetics of API-base co-crystal complexes and clarifying the nature of chemical interactions of biomacromolecule with small pharmaceutical molecules.Part three:Cytotoxicity assessment of CUR-based supramolecular complex and determination of pharmacokinetics parameters using UPLC-MS/MS methodObjective:The UPLC-MS/MS method used to determine the concentration of CUR in vivo is established.In vivo pharmacokinetics of CUR and CUR-based co-crystal complexes in experimental rats were evaluated.Selecting anticancer activity as investigating objective,the anticancer activities of CUR-based supramolecular complexes are evaluated and the corresponding anticancer model are established.Methods:In vivo pharmacokinetics of CUR and CUR-based co-crystal complexes in experimental rats were evaluated though CUR concentration determination using UPLC-MS/MS method.Cytotoxicity of CUR and CUR-based complexes to human colon cancer cell（HT-29）,lung cancer cell（A549）and hepatoma cell（Hepg）are evaluated by MTT assay.Results:Compared with intact CUR,the pharmacokinetics parameters C_max,T_max,AUC_0-t-t and AUC_0-∞of CUR-based co-crystals were improved（P<0.05）;C_maxax increased 30-fold,T_maxax shortened 6-fold,and bioavailability increased 6-fold.As for gallic acid,the C_max,T_max,AUC_0-t-t and AUC_0-∞of CUR-gallic acid co-crystal were lower than that of intact gallic acid,but the result was no statistical difference.The results of MTT assay showed that cytotoxicity of CUR-piperazine and CUR-isonicotine co-crystals to HT-29,A549 and Hepg cells were higher than these of intact CUR,although the results showed no statistical difference.Cytotoxicity of CUR-gallic acid co-crystal to A549 cells was higher than that of CUR at higher CUR concentration（no statistical difference）,while,co-crystal showed lower cytotoxicity to HT-29 and Hepg cells than CUR.Conclusion:The screen of CUR-based co-crystal supramolecular complex has the potential to be a novel strategy to improve the pharmac-okinetics parameters of C_max,T_maxax and bioavailability.Cell inhibitory rates of CUR-based supramolecular complex vary with the kinds of CCF and cell.Owing to the complexity of action mechanism and nature of multitarget,it is difficult to investigate different action mechanisms of Chinese herbal active ingredients in different cells.The results in present study offer the thought to this investigation and indicate the direction for further investigation of CUR-based supramolecular anticancer model,especially CUR-API（Ⅱ）antic-ancer model.