Synergistic Mechanism Of Vinegar-processed Frankincense Via Change Of Physical Properties And Regulation Of Transporter Expression

Traditional Chinese medicinal(TCM)processing,called Pao Zhi,is proven to increase medical potencies.The absorption of main active components of TCM is believed to be improved by Pao Zhi,yet the mechanism is still completely unclear.As an essential drug in TCM,frankincense is widely used for the treatment of inflammatory diseases such as rheumatism,rheumatoid arthritis,and osteoarthritis,and exhibiting notable anti-inflammatory properties.It is suggested that the effectivenes of frankincense was enhancenced after vinegar-processing.However,the main boswellic acids of frankincense showed low bioavailability,which has been a challenge in the successful formulation of therapeutic products.It is revealed that absorption of two kinds of boswellic acids have been improved after vinegar-processing.However,the mechanism of the absorption enhancement effect of processing is still completely unclear.The physical factors are key parameters in improving absorption of oral drugs.The absorption of drugs is influenced by transporters of liver and intestine.Several studies were carried out that MRP2(Multidrug resistance-associated protein 2)and OATP1B3(Organic anion transporting polypeptide 1B3)are responsible for improving absorption of boswellic acids.In addition,there are various nuclear receptors which control of transporters,such as CAR(Constitutive androstane receptor),FXR(Farnesoid X receptor)and PXR(Progesterone X receptor).The change of components of TCM has effects on the expression of transporters.Then they influence the absorption of components.In a conclusin,it is supposed that the change of physical properties and chemical components have effects on the expression of transporters after vinegar-proceesing of frankincense.They are closely related to the promotion of absorption of boswellic acids in vinegar-processed frankincense.The change of chemical composition of frankincense before and after vinegar processing was studied.It was found that the content of 9,11-dehydrogenation boswellic acid with π-π conjugate structure was greatly increased after vinegar processing,and it showed anti-inflammatory activity.The serum pharmacochemistry study also showed that the component was detected in the blood of rats with the intervention of processed frankincense.3-acetyl-9,11-dehydro-β-boswellic acid(ADHBA)is the most abundant ingredient of 9,11-dehydrogenated boswellic acid.Therefore,we compared the regulatory effects of ADHBA,frankincense and processed frankincense on drug transporters in the third and fourth chapters.The differences in the intestinal absorption characteristics and equilibrium solubilities between frankincense and processed frankincense were determined by the ultra-high-performance liquid chromatography coupled with a triple quadrupole electrospray tandem mass spectrometry(UHPLC-TQ-MS)method in this paper.And we investigated the physical properties of frankincense and processed frankincense,including parameters of micromeritics,rheology and biopharmaceutics.This paper also reviewed that frankincense,vinegar-processed frankincense and main differential components have effects on the expression of MRP2,OATP1B3,CAR,FXR and PXR which concern boswellic acids.It is revealed that there are differences on frankincense before and after vinegar-processing which control of nuclear receptors and thus modulating the activity of transporters.The main research contents and results are as follow:1.Study on the effects of vinegar processing on the intestinal absorption of main boswellic acids in frankincenseAccording to Pharmacopoeia of the People’s Republic of China(ChP)2015,the clinical application of processed frankincense is mainly used by its powder products.The everted rat gut sac was studied to determine the intestinal absorption rates of frankincense and processed frankincense both in powders and ethanol extracts.Main components of frankincense including 11-keto-β-boswellic acid(KBA),3-acetyl-11-keto-β-boswellic acid(AKBA),α-boswellic acid(α-BA),β-boswellic acid(β-BA),3-acetyl-α-boswellic acid(α-ABA)and 3-acetyl-β-boswellic acid(β-ABA)were simultaneously quantified using an UHPLC-TQ-MS method.The results suggested the proposed analysis method meet the requirements of the determination of the main boswellic acids.The absorption rates of KBA,AKBA,α-BA,β-BA in vinegar-processed frankincense are higher than in frankincense(P<0.01).In addition,the intestinal absorption rates of KBA,AKBA,α-BA,β-BA of frankincense and vinegar-processed frankincense in ethanol extracts are both higher than those in powders.However,by comparing the difference values,the values of KBA,AKBA,α-BA,β-BA in ethanol extracts are lower than those of powders(P<0.05).It is revealed low absorption of α-ABA and β-ABA.Thus,the absorption rates of α-ABA and β-ABA were not detected in frankincense and vinegar-processed frankincense.As a conclusion,vinegar-processing improved the absorption of main boswellic acids and the powders of frankincense produced much greater change after processing.2.Study on the effects of vinegar processing on the physical properties of drug concerns absorptionWe choose powders and ethanol extracts of frankincense and vinegar-processed frankincense as objects of study.To investigate the change of micromeritics,rheology and biopharmaceutics of frankincense before and after processing.Firstly,the surface morphology of frankincense and vinegar-processed frankincense was measured by scanning electron microscopy(SEM).The particle size,polymer dispersity index(PDI)and zeta potential of frankincense and vinegar-processed frankincense were determined by a laser particle size analyzer.In addition,the specific surface area,pore volume and porosity of frankincense and vinegar-processed frankincense were evaluated by a mercury intrusion porosimeter.Then,the viscosities of frankincense and vinegar-processed frankincense were measured by the viscosimeter in the simulate gastrointestinal tract.Finally,the established UHPLC-TQ-MS/MS method was used to determine the equilibrium solubilities of KBA,AKBA,α-BA,β-BA,α-ABA and β-ABA in frankincense and vinegar-processed frankincense at pH=2.0,7.0 and 7.8-8.0.The results showed that images of frankincense with a smooth surface,while the processed frankincense exhibited folds full of holes with a rough surface.Vinegar processing was resulting in the decrease of the particle size(P<0.05 or P<0.01),PDI.The zeta potential,specific surface area,pore volume and porosity of frankincense increased after vinegar processing(P<0.05 or P<0.01).In addition,the flow times of simulated gastric fluid and intestinal fluid of processed frankincense were lower than frankincense(P<0.05 or P<0.01).Thus,the viscosity of frankincense decreased after vinegar processing.Then the equilibrium solubilities of KBA,AKBA,α-BA,β-BA,α-ABA and β-ABA in vinegar-processed frankincense were higher than frankincense(P<0.05 or P<0.01)at pH=2.0,7.0 and 7.8-8.0.It is suggested that the vinegar-processing of powder which control of physical properties thus enhance the intestinal absorption of boswellic acids.3.Study on the effects of frankincense,vinegar-processed frankincense and major differential components on the transporters of drugTo investigate the differences between effects of frankincense and vinegar-processed frankincense on ulcerative colitis(UC)in rats,the disease activity index(DAI),colonic mucosal damage index(CMDI)scores,histological score(HS),pathological slide and inflammatory cytokines including tumor necrosis factor-α(TNF-α),interleukin-1β(IL-1β),interleukin-6(IL-6)were determined in the UC model.The method of reverse transcription-polymerase chain reaction(RT-PCR)and western blot(WB)were used to observe the expression of MRP2 in the liver of UC rats.We also determined the expression of MRP2 and OATP1B3 of frankincense,vinegar-processed frankincense and major differential components in L02 cells.The results showed that the UC model was established with 2,4,6-trinitrobenzenesulfonic acid solution(TNBS)/ethanol.Therapeutic effects of frankincense and vinegar-processed frankincense on UC rats were obvious.And therapeutic effects of vinegar-processed frankincense were better than frankincense.Expression of MRP2 in UC rats were lower than normal rats(P<0.05 or P<0.01),while expression increased in the frankincense and vinegar-processed frankincense(P<0.05 or P<0.01).And expression of vinegar-processed frankincense were higher than frankincense(P<0.05).In addition,compared to the blank group,the expression of MRP2 and OATP1B3 were increased in groups of frankincense,vinegar-processed frankincense,3-acetyl-9,11-dehydro-β-boswellic acid(ADHBA)and ADHBA+frankincense in L02 cells(P<0.05 or P<0.01).And groups of vinegar-processed frankincense and ADHBA+frankincense have the most significant effects(P<0.05 or P<0.01).It is revealed that frankincense which control of chemical components through the vinegar-processing and thus up-regulate the expression of MRP2 and OATP1B3.Then the intestinal absorption and liver recovery in the enterohepatic circulation were promoted.4.Study on the mechanism of the regulation of transporter expression via regulating nuclear receptors in frankincense,vinegar-processed frankincense and major differential componentsThe method of RT-PCR and WB were used to observe the expression of CAR,FXR and PXR in the liver of UC rats.We also determined the expression of CAR,FXR and PXR of frankincense,vinegar-processed frankincense and major differential components in L02 cells.In addition,receptor agonists including CITCO(C19H12N3OCl3S),GW4064(C28H22Cl3NO4)and dexamethasone(C22H29FO5)were used to investigate differences on expression of transporters in frankincense,vinegar-processed frankincense and major differential components.Expression of CAR,FXR and PXR in UC rats were lower than normal rats(P<0.05 or P<0.01),while expression increased in the frankincense and vinegar-processed frankincense(P<0.05 or P<0.01).And expression of vinegar-processed frankincense were higher than frankincense(P<0.05).In addition,compared to the blank group,the expression of CAR,FXR and PXR were increased in groups of frankincense,vinegar-processed frankincense,ADHBA and ADHBA+frankincense in L02 cells(P<0.05 or P<0.01).And groups of vinegar-processed frankincense and ADHBA+frankincense have the most significant effects(P<0.05 or P<0.01).In addition,the CAR,FXR and PXR were activated by agonists including CITCO,GW4064 and dexamethasone and thus up-regulate the expression of MRP2 and OATP1B3(P<0.05 or P<0.01)ConclusionThe absorption rates of main boswellic acids were increased after vinegar processing of frankincense.Therapeutic effects of vinegar-processed frankincense on UC rats were also enhanced.This paper was revealed that the physical properties—including the surface morphology,PDI,zeta potential,particle size,porosity,pore volume,specific surface area and viscosity—were changed.Then the equilibrium solubilities of KBA,AKBA,α-BA,β-BA,α-ABA and β-ABA were increased after vinegar processing of frankincense at pH=2.0,7.0 and 7.8-8.0,in which case the absorption of boswellic acids were improved.On the other hand,vinegar processing mainly contributes to the up-regulation of CAR,FXR and PXR,thereby resulting in an increase of the expression of MRP2 and OATP1B3.