Thesis (PhD)--Stellenbosch University, 2013. / ENGLISH ABSTRACT: Introduction
Earlier studies have shown the popularity of herbal products among people as traditional,
complementary or alternative medication. One of the major clinical risks in the concomitant
administration of herbal products and prescription medicine is pharmacokinetic herb-drug interaction
(HDI). This is brought about by the ability of phytochemicals to inhibit or induce the activity of
metabolic enzymes and transport proteins. The aim of this study was to investigate the potential of the
crude extracts of popular medicinal herbs used in South Africa to inhibit major cytochrome P450
(CYP) enzymes and transport proteins through in vitro assessment.
Methods
Medicinal herbs were obtained from traditional medical practitioners and 15 were selected for this
study. The selected herbal products were extracted and incubated with human liver microsomes to
monitor the following reactions as markers for the metabolic activities of the respective CYP:
phenacetin O-deethylation (CYP1A2), diclofenac 4‟-hydroxylation (CYP2C9), S-mephenytoin 4‟-
hydroxylation (CYP2C19) and testosterone 6β-hydroxylation (CYP3A4). In addition, the influence of
Lessertia frutescens (formerly Sutherlandia frutescens) and Hypoxis hemerocallidea was investigated
on more isozymes: coumarin 7-hydroxylation (CYP2A6), bupropion hydroxylation (CYP2B6),
paclitaxel 6α-hydroxylation (CYP2C8), bufuralol 1‟-hydroxylation (CYP2D6), chlorzoxazone 6-
hydroxylation (CYP2E1) and midazolam 1‟-hydroxylation (CYP3A4/5). The generation of the CYPspecific
substrates/metabolites were monitored and quantified with the aid of LC-MS/MS. The
metabolic clearance of midazolam using cryopreserved hepatocytes was monitored in the presence of
Lessertia frutescens and Hypoxis hemerocallidea. The potential of both to inhibit human ATP-binding
cassette (ABC) transporter activity was assessed using recombinant MDCKII and LLC-PK1 cells overexpressing
human breast cancer resistant protein (BCRP) and human P-glycoprotein (P-gp),
respectively. Similarly, the potential for interactions with human organic anion transporting polypeptide
(OATP1B1 and OATP1B3) was assessed using recombinant HEK293 cells over-expressing
OATP1B1 and OATP1B3, respectively. Results
Bowiea volubilis, Kedrostis Africana, Chenopodium album, Lessertia frutescens (methanolic extract),
Hypoxis hemerocallidea, Spirostachys africana and Lessertia frutescens (aqueous extract), in
ascending order of potency demonstrated strong inhibition of CYP1A2 activity (IC50 = 1-100 g/mL).
Similarly, Emex australis, Alepidea amatymbica, Pachycarpus concolor, Lessertia frutescens,
Capparis sepiaria, Kedrostis africana and Pentanisia prunelloides inhibited CYP2C9 with IC50 less
than 100 g/mL. The following demonstrated strong inhibition of CYP2C19 with IC50 values less than
100 g/mL: Acacia karroo, Capparis sepiaria, Chenopodium album, Pachycarpus concolor,
Ranunculus multifidus, Lessertia frutescens and Zantedeschia aethiopica. CYP3A4 was inhibited by
Lessertia frutescens, Hypoxis hemerocallidea, Spirostachys Africana, Bowiea volubilis, Zantedeschia
aethiopica, Chenopodium album, Kedrostis Africana, Acacia karroo, Emex australis, Pachycarpus
concolor, Ranunculus multifidus, Capparis sepiaria and Pentanisia prunelloides. Time-dependent
(irreversible) inhibition of CYP3A4/5 (KI = 296 μg/mL, kinact = 0.063 min-1) and delay in the
production of midazolam metabolites in the human hepatocytes, leading to a 40% decreased
midazolam upscaled in vivo clearance, was observed with Lessertia frutescens. Further, Lessertia
frutescence inhibited the activity of P-gp (IC50 = 324.8 μg/mL), OATP1B1 (IC50 = 10.4 μg/mL) and
OATP1B3 (IC50 = 6.6 μg/mL). Hypoxis hemerocallidea inhibited the activity of OATP1B1 (IC50 = 118.7
μg/mL) and OATP1B3 (IC50 = 290.1 μg/mL) with no potent inhibitory effects on P-gp. None of the two
inhibited the activity of BCRP within the tested concentrations.
Conclusion
The result indicates the potential for HDI between the selected medicinal herbs and the substrates of
the enzymes investigated in this study, if sufficient in vivo concentrations are achieved. / AFRIKAANSE OPSOMMING: Inleiding
Vroeëre studies het aangedui dat die gebruik van plantaardige produkte as tradisionele, aanvullende
en alternatiewe medikasie baie gewild is. Een van die grootste kliniese risiko‟s geassosieer met die
gelyktydige gebruik van plantaardige produkte met voorskrifmedikasie is farmakokinetiese kruiegeneesmiddel
interaksies (HDI). Hierdie interaksies word veroorsaak deur die vermoë van
plantchemikalieë om die aktiwiteit van metaboliese ensieme en transportproteïene te inhibeer of te
induseer. Die doel van hierdie studie is om ondersoek in te stel na die moontlikheid van onsuiwer
ekstrakte van gewilde Suid-Afrikaanse medisinale kruie om die belangrikste sitochroom P450 (CYP)-
ensieme en transportproteïene te inhibeer. Hierdie ondersoek sal plaasvind deur middel van in vitrostudies.
Metodes
Medisinale kruie is verkry vanaf tradisionele genesers, waaruit ʼn totaal van 15 kruie geselekteer is vir
gebruik tydens hierdie studie. Die geselekteerde kruie is geëkstraheer en met menslike
lewermikrosome geïnkubeer om die volgende reaksies as merkers vir die metaboliese aktiwiteit van
die onderskeie CYP-ensieme te moniteer: fenasetien-O-deëtilasie (CYP1A2), diklofenak-4‟-
hidroksilasie (CYP2C9), S-mefenitoïen-4‟-hidroksilasie (CYP2C19) en testosteroon-6β-hidroksilasie
(CYP3A4). Afgesien van die voorafgaande, is ook die invloed van Lessertia frutescens en Hypoxis
hemerocallidea op verskeie ander iso-ensieme ondersoek. Hierdie iso-ensieme is soos volg:
koumarien-7-hidroksilasie (CYP2A6), bupropioonhidroksilasie (CYP2B6), paklitaksiel-6α-hidroksilasie
(CYP2C8), bufuralol-1‟-hidroksilasie (CYP2D6), chloorsoksasoon-6-hidroksilasie (CYP2E1) en
midasolaam-1‟- hidroksilasie (CYP3A4/5). Die produksie van CYP-spesifieke substrate/metaboliete is
gemoniteer en deur middel van LC-MS/MS-analises gekwantifiseer. Die metaboliese opruiming van
midasolaam deur middel van krio-gepreserveerde hepatosiete is gemoniteer in die teenwoordigheid
van Lessertia frutescens en Hypoxis hemerocallidea. Die moontlikheid van beide om menslike ATPbindingskasset
(ABC)-transporteerderaktiwiteit te inhibeer is bepaal deur die gebruik van
rekombinante MDCKII- en LLC-PK1-selle wat onderskeidelik menslike borskanker-weerstandige
proteïen (BCRP) en menslike P-glikoproteïen (P-gp) potensieel. Op ʼn soortgelyke wyse is die
moontlikheid vir interaksies met menslike organiese anion-transportpolipeptiede (OATP1B1 en
OATP1B3) bepaal deur rekombinante HEK293-selle te gebruik wat onderskeidelik OATP1B1 en
OATP1B3 potensieel. Resultate
Bowiea volubilis, Kedrostis Africana, Chenopodium album, Lessertia frutescens (metanol-ekstrak),
Hypoxis hemerocallidea, Spirostachys africana en Lessertia frutescens (water-ekstrak), in
toenemende potensie, het sterk inhibisie van CYP1A2-aktiwiteit (IC50 = 1-100 g/mL) getoon. In
ooreenstemming met die voorafgaande resultate het Emex australis, Alepidea amatymbica,
Pachycarpus concolor, Lessertia frutescens, Capparis sepiaria, Kedrostis africana en Pentanisia
prunelloides CYP2C9 met IC50–waardes van minder as 100 g/mL geïnhibeer. Die volgende het
sterk inhibisie van CYP2C19 met IC50-waardes van minder as 100 g/mL getoon: Acacia karroo,
Capparis sepiaria, Chenopodium album, Pachycarpus concolor, Ranunculus multifidus, Lessertia
frutescens en Zantedeschia aethiopica. CYP3A4 is deur Lessertia frutescens, Hypoxis
hemerocallidea, Spirostachys Africana, Bowiea volubilis, Zantedeschia aethiopica, Chenopodium
album, Kedrostis Africana, Acacia karroo, Emex australis, Pachycarpus concolor, Ranunculus
multifidus, Capparis sepiaria en Pentanisia prunelloides geïnhibeer. Tydafhanklike (onomkeerbare)
inhibisie van CYP3A4/5 (KI = 296 μg/mL, kinact = 0.063 min-1) en vertraging in die produksie van
midasolaammetaboliete in menslike hepatosiete wat aanleiding gee tot ʼn 40% afname in midasolaam
bepaal in vivo opruiming, is waargeneem met Lessertia frutescens. Lessertia frutescens het ook die
aktiwiteit van P-gp (IC50 = 324.8 μg/mL), OATP1B1 (IC50 = 10.4 μg/mL) en OATP1B3 (IC50 = 6.6
μg/mL) geïnhibeer. Hypoxis hemerocallidea het die aktiwiteit van OATP1B1 (IC50 = 118.7 μg/mL) en
OATP1B3 (IC50 = 290.1 μg/mL) geïnhibeer met geen betekenisvolle effekte op P-gp nie. Geen een
van die twee het die aktiwiteit van BCRP geïnhibeer binne die konsentrasies waarin getoets is nie.
Gevolgtrekking
Die resultate van hierdie studie dui aan dat wanneer voldoende in vivo-konsentrasies bereik word, die
moontlikheid vir kruie-geneesmiddel interaksies tussen die geselekteerde medisinale kruie en
ensiemsubstrate ʼn werklikheid word.
| Identifer | oai:union.ndltd.org:netd.ac.za/oai:union.ndltd.org:sun/oai:scholar.sun.ac.za:10019.1/85850 |
| Date | 12 1900 |
| Creators | Fasinu, Pius Sedowhe |
| Contributors | Rosenkranz, Bernd, Bouic, Patrick J., Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Medicine. Division of Clinical Pharmacology. |
| Publisher | Stellenbosch : Stellenbosch University |
| Source Sets | South African National ETD Portal |
| Language | en_ZA |
| Detected Language | English |
| Type | Thesis |
| Format | 249 p. : ill. |
| Rights | Stellenbosch University |
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