Inhibitory effect of selected herbal supplements on CYP450-mediated metabolism : an in vitro approach

White, Charlize (2016-03)

Thesis (MSc)--Stellenbosch University, 2016.

Thesis

ENGLISH ABSTRACT: INTRODUCTION Herbal products are popularly used as complementary and alternative medicines to treat a variety of conditions. Often patients use them in conjunction with conventional medicines. Herbal products contain many pharmacologically active phytochemicals that may interfere with the absorption, distribution, metabolism, and elimination of medicines. This interaction can lead to an increase of the plasma concentrations of other medicines to toxic levels, or to their decrease below therapeutic levels, resulting in lack of efficacy. The liver cytochrome P450 (CYP) enzymes are responsible for the metabolism of a large majority of medicines. In order to provide more information on the potential interaction between African herbal medicines and conventional medicines, the present study has investigated the inhibition of selected CYP enzymes by three popular South African medicinal plants, Buchu, African ginger, and Warburgia salutaris. METHODS Buchu capsules, African ginger, and Warburgia salutaris tablets were obtained in a local pharmacy. 60% methanol/water extracts were prepared and analysed by GC-MS to reveal the composition of the volatile components of each product. Fluorogenic inhibition assays were conducted using Vivid® recombinant CYP screening kits according to the manufacturer’s protocol. This protocol included the pre-incubation of herbal extracts, recombinant CYP isoform and cofactor solution. The metabolic reaction was initiated by the addition of CYP-specific substrate and NADP+; the solution was incubated for 30 minutes at 37°C, after which fluorescence was measured using a microplate reader. The percentage remaining activity was calculated and used to determine the IC50 values of each herbal product. Time - dependent inhibition (TDI) was evaluated using the normalized ratio, NADP+-, concentration -, and time - dependent approaches. RESULTS The GC-MS analysis revealed monoterpenes, sesquiterpenes, and alkane hydrocarbons in the volatile component. Warburgia salutaris, African ginger, and Buchu inhibited CYP2C19 with IC50 values of 5.88 μg/ml, 32.38 μg/ml, and 53.52 μg/ml, respectively. Likewise, the IC50 values of 5.64 μg/ml, 1.09 μg/ml, and > 100 μg/ml were obtained for inhibition of CYP3A4 by Warburgia salutaris, African ginger, and Buchu, respectively. Using the normalized ratio, Warburgia salutaris and African ginger showed time- and concentration - dependent inhibition of CYP1A2, and Buchu showed intermediate TDI effects that were not concentration dependent. All three extracts showed TDI of CYP3A4; the inhibition displayed by Buchu and Warburgia salutaris was NADP+ dependent. African ginger was the only extract to show NADP+ dependent inhibition of CYP1A2. A kinetic TDI assay Stellenbosch University https://scholar.sun.ac.za iii showed that the IC50 value of African ginger decreased over time, indicating TDI. Warburgia salutaris was not a time-dependent inhibitor of CYP3A4, and Buchu may have a limited time-dependent inhibitory effect. CONCLUSION Warburgia salutaris, African ginger, and Buchu have the potential to cause clinically relevant herb-drug interaction, if sufficient concentrations are achieved in vivo. Further studies are needed to confirm this finding.

AFRIKAANSE OPSOMMING: INLEIDING Kruie produkte word gebruik as komplementêre en alternatiewe medisynes om ‘n verskeidenheid van gesondheidstoestande te behandel. Pasiënte gebruik dit dikwels tesame met konvensionele medisynes. Kruie produkte bevat verskeie farmakologiese aktiewe plant chemikalieë wat met die absorpsie, distribusie, metabolisme en eliminasie van medisyne inmeng. Hierdie interaksie kan aanleiding gee tot ‘n toename in plasma konsentrasies van die ander medisynes tot toksiese vlakke of tot hul afname na onder terapeutiese vlakke wat dan aanleiding gee tot ‘n gebrek aan doeltreffendheid. Die lewer sitochroom P450 (CYP) ensiemes is verantwoordelik vir die metabolisme van die meeste medisynes. Die huidige studie is onderneem in ‘n poging om meer inligting aangaande die potensiële interaksies tussen Afrika kruie medisynes en konvensionele medisynes te bepaal. Die inhibisie van geselekteerde CYP ensiemes deur drie gewilde Suid Afrikaanse medisinale plante, Buchu, Afrika gemmer en Warburgia salutaris is ondersoek. METODES Buchu kapsules, Afrika gemmer, en Warburgia salutaris tablette is by ‘n plaaslike apteek bekom. 60% methanol/water ekstrakte is voorberei en die samestelling van die vlugtige komponete van elke produk is deur die analises met GC-MS bepaal. Fluoroserende inhibisie bepalings is uitgevoer deur gebruik te maak van Vivid® rekombinante CPY siftings toetse. Hierdie protokol sluit in die pre-inkubasie van die kruie ekstrakte, rekombinate CYP isoform en ko-faktor oplossing. Die metaboliese reaksie word geaktiveer deur die byvoeging van CYP-spesifieke substrate en NADP+; die oplossing is vir 30 minute by 37oC geinkubeer, waarna fluorosensie deur middel van ‘n mikroplaatleser gemeet is. Die persentasie oorblywende aktiwiteit is bereken en daarna gebruik om die IC50 waardes van elke kruie produk te bepaal. Die tydafhanklike inhiberende uitwerking (TAI) is bereken deur gebruik te maak van die genormaliseerde verhouding, NADP+-, konsentrasie-, en tyd afhanklike benaderings. RESULTATE Die GC-MS analises het monoterpiene, sekwiterpiene, en alkaan koolwaterstowwe aangetoon. Warbugia salutaris, Afrika gemmer, en Buchu het CYP2C19 geinhibeer met IC50 waardes van 5.88 ug/ml, 32.38 ug/ml, en 53.52 ug/ml onderskeidelik. Eweneens is IC50 waardes van 5.64 ug/ml, 1.09 ug/ml en > 100 ug/ml onderskeidelik verkry met inhibisie van CYP3A4 deur Warburgia salutaris, Afrika gemmer en Buchu. Deur gebruik te maak van die genormaliseerde verhouding wys Warburgia salutaris en Afrika gemmer tyd en konsentrasie-afhanklike inhibisie van CYP1A2. Buchu wys intermediêre TDI effekte wat nie konsentrasie afhanklik is nie. Al drie ekstrakte het ‘n TDI van Stellenbosch University https://scholar.sun.ac.za v CYP3A4 aangedui; die inhibisie aangetoon deur Buchu en Warburgia salutaris was NADP+ afhanklik. Afrika gemmer was die enigste ekstrak wat NADP+ afhanklike inhibisie van CYP1A2 aangetoon het. ‘n Kinetiese TDI toets het gewys dat die IC50 waarde van Afrika gemmer oor tyd afneem wat TDI aandui. Warburgia salutaris is nie ‘n tyd-afhanklike inhibitor van CYP3A4 nie en Buchu kan dalk ‘n beperkte tyd-afhanklike inhibitoriese effek hê. GEVOLGTREKKING Warburgia salutaris, Afrika gemmer en Buchu het die potensiaal om klinies relevante kruie-medisyne interaksies te veroorsaak indien genoegsame konsentrasies in vivo bereik word. Verdere studies is nodig om hierdie bevindinge te bevestig.

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