| Aconite-liquorice is a common drug pair in Chinese medicine,and it is also a representative drug pair reflecting the compatibility of "mutual fear and mutual killing" in traditional Chinese medicine.The mechanism of detoxicification and efficacy enhancement is complicated,which is highly associated with multiple targets and time course.In the past,scientists mainly focusd on material basis of the detoxification mechanism of aconite-liquorice compatibility,for example,the chemical or physical reaction leads to the conversion of aconitine into other non-toxic or less toxic substances,resulting reduction of the toxicity of aconitine.In this study,we attepted to illustrate the impact of chemical ingredients on transporter’s function and activity,and activities of cytochrome,since transporter and drug metabolism enzymes play key roles in the process of drug in the body.Any changes,for instance inhibition or induction effects,can affect drug absorption,distribution,metabolism and therapeutic effects.Therefore,the effect of the main active ingredients of liquorice and its extract on the transporter,and drug metabolic enzymes,in combinations with changes of aconitine’s pharmacokinetic pattern and pharmacodynamics,were integrated to clarify the mechanisms of aconite-liquorice compatibility in efficacy enhancement and detoxification.Up-regulation of efflux transporters and drug metabolizing enzymes by major active components of liquoriceAs one of most widely used herbal medicine,liquorice exhibits diverse pharmacological activities,for instance,analgesic,antitussive,antiarrhythmic,anti-inflammatory,and immune regulation.Additionally,detoxification effects were observed in combination of liquorice with other herbal drugs.The mechanism of detoxification of liquorice has been extensively investigated through material basis and interference with CYPs though,investigations of its effect on transporters were very limited,according to the literature.The objective of this study was attempted to investigate the effect of active ingredients existing in liquorice on the efflux transporters as to clarify the potential mechanism of detoxification of liquorice.Multiple analytical approaches have been explored,including flow cytometry,fluorescent detection,RT-PCR,Western blot to measure the function,activity as well as mRNA/protein expression of efflux transporters on LS-180 cell model after treatments with active compounds of liquorice.Additionally,Caco-2 cell model was utilized to further investigate the potential impact of those ingredients on efflux transporter.Although up-regulation of mRNA expression of drug metabolism enzymes by active ingredients of liquorice is not less potent than expected,it has exhibited the potential in affecting the CYPs.The resulting data indicated that those active ingredients,including flavonoids(liquiritin,liquiritigenin,isoliquiritin,isoliquiritigenin and licochalcone A)and pentacyclic triterpene saponin(glycyrrhetinic acid)were able to upregulate the expression of efflux transporters,for example P-gp,BCRP and MRP2 to various levels.The gene expressions were approximately over 2.5 folds by comparison with that of control,and up to 13 folds and 16 folds for BCRP by isoliquiritin and isoliquiritigenin,and further confirmed by Western blot.The functional assay also supported up-regulation of efflux transporter by those ingredients.Flow cytometry study showed that the level of rhodamine123 as probe substrate in LS-180 cells decreased to approximately 50%after treatment with active ingredients of liquorice,compared with that of control.The fluorescent assay confirmed that change of rhodamine 123 was correlated with the concentrations of active ingredients given.The efflux transport of rhodamine 123 was enhanced in Caco-2 cell models as well.The study clarified potential detoxification mechanism of liquorice by up-regulating efflux transporter as to reduce absorption of xenobiotics across small intestinal membrane,which provided a new insight into pharmacological function of liquorice.The effects of the active components and extract of liquorice on transport of aconitum alkaloids and pharmacokinetic pattern in ratsAconite alkaloids are the main bioactive ingredients existing in aconitum,for instance aconitine,which exhibit potent analgesic,antirheumatic and other pharmacological effects.In this study,the effects of long-term treatment with liquorice on pharmacokinetics of aconitine in rats were investigated,meanwhile,an accurate,precise and sensitive LC-MS/MS analytical method which provided the technical support for detection of aconitum alkaloids was established.Pharmacokinetics of aconitine after oral administration of aconitine at 1.5 mg/kg either with pretreatment of liquorice water extract at 0.433 g/kg or 1.299 g/kg(crude drug)respectively for one week or not were studied.Additionally,LS-180 cells were utilized to explore the potential effects of bioactive ingredients of liquorice on transport of aconite alkaloids.The results revealed that exposure of aconitine after pretreatment with liquorice was altered remarkably.AUC decreased from 161±37.8 to 58.8±8.97 and 44.7±8.20ng/mL*h,respectively.Similarly,Cmax decreased from 26.2±5.19 to 11.8±1.15 and 6.86±0.600 ng/mL,respectively.Moreover,accumulation of aconitine and hypaconitine,not mesaconitine inside of LS-180 cells were reduced after pretreatment by comparison with control.In conclusion,the exposure of aconitine in vivo declined after pretreatment with liquorice extract,which may be highly associated with upregulated expression and/or function of P-gp.Identification of the uptake transporters of aconitum alkaloids and investigations of effects of liquorice on on tissue distribution of aconitine in liver and kidneyAconite as a commonly used herb has been extensively applied in treatment of rheumatoid arthritis,pain relief,as well as cardiotonic actions.Aconitum alkaloids have been demonstrated to be the most potent ingredients in aconite,in terms of efficacy against diseases,but are also highly toxic.Apart from the neurological and cardiovascular toxicity exposed,the damage of aconitum alkaloids on hepatocytes and nephrocyte in long-term use should also be carefully considered.This study attempted to investigate the role of uptake transporters mediating the transport process of aconitum alkaloids into liver and kidney.The resulting data revealed that hOATP1B1,1B3,hOCT1 and hOAT3 were mainly involved in uptake of aconitum alkaloids.Additionally,inhibitory effect of bioactive ingredients of liquorcie on uptake transporters were screened and further confirmed by IC50 values determined.The in vitro study suggested that liquorice might lower toxicity of aconite by reducing its exposure in liver and/or kidney through inhibition of uptake transporters.Eventually,the in vivo study was indicative of detoxification of liquorice by decreasing the exposure of aconitine as representative compound in liver after co-administration,even though the exposure in kidney altered was less significant.In summary,hOATPIB1,1B3,hOCT1 and hOCT3 were determined as the key uptake transporters mediating the transport process of aconitum alkaloids into liver and/or kidney,and liquorice may alleviate the toxicity caused by reduction of exposure through inhibition of those key uptake transporters.Study on synergistic mechanism of aconite and liquoriceAconite(Wutou in Chinese)is a commonly used medicinal plant which is derived from the processed parent roots of Aconitum carmichaeli Debx.which exhibits many pharmacological actions.Wutou-Gancao herb-pair is extensively used to attenuate the toxicity and enhance the efficacy of aconite.In this study,potential synergic mechanism of the herb pair was investigated by utilizing multiple approaches.In silico and Caco-2 cell model were applied to study the potential binding mode of bioactive ingredients existing in liquorice with P-gp,as well as the inhibition effects on P-gp by using rhodamine 123 as probe substrate.Additionally,anti-inflammatory activity of aconitine combined with active ingredients of liquorice were evaluated in rats through adjuvant induced model.Pharmacokinetic patterns of aconitine after co-administration with active ingredients of liquorice were also investigated.The in silico docking study revealed the potential binding mode with outward facing conformation of P-gp.The resulting data obtained from transport of rhodamine 123 across Caco-2 cell monolayer further indicated that function of P-gp was inhibited by chemicals in liquorice.The synergic effect was therefore proposed to be attributed to inhibition of P-gp by liquorice since aconitine has been demonstrated to be the substrate of P-gp.Anti-inflammatory effect of aconitine(1 mg/kg)in rats was enhanced in combination with bioactive ingredients of liquorice(10 mg/kg).In the meanwhile,the exposure of aconitine in vivo were altered,in terms of Cmax and AUC.For instance,the Cma and AUC were increased to 1.9 and 1.3 folds,respectively,when used in combination with liquiritigenin.The resulting findings revealed that potential synergic mechanism of Wutou-Gancao herb-pair by inhibiting function of key efflux transporter P-gp to enhance the exposure of aconitine in systematic circulation,and further the anti-inflammatory effect,which help clarifying the compatibility rationale of these two herbs.The therapeutic effect of aconite-liquorice on adjuvant induced arthritis(AIA)Aconite has the effect of anti-rheumatoid arthritis,but due to its potential toxicity,the aconite is always used with liquorice to reduce its toxicity.In this chapter,the adjuvant induced arthritis model was established successfully by intracutaneous administration of Freund’s complete adjuvant to evaluate the effect of aconite-liquorice drug pair on rheumatoid arthritis.During continuous lavage for 30 days,changes of volume of rat hind paw,arthritis’ index,body weight after dosing were recorded.Blood samples were also collected for evaluation of IL-1β,IL-6 and TNF-α,as well as pathological examination.The results showed that the rats’ hind paw volume of the aconite-liquorice group were significantly lower than that of the model group,and the rats’ weight were also increased,which was better than the aconite single decoction.The levels of cytokines determined by Elisa further confirmed the effect of aconite-liquorice.In addition,the degree of joint lesion was also significantly alleviated in the aconite-liquorice group compared with the model group via pathological examination.Therefore,the comprehensive analysis showed that aconite-liquorice drug pair exhibited therapeutic effect against rat rheumatoid arthritis and presented advantages in treatment by comparison with aconite single decoction. |
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