In vitro hepatotoxicity and herb-drug interactions of selected African plant extracts

Abstract

Herbal remedies are an important and often-used commodity in developing countries, such as those in Africa. There is a long-standing belief that these medicinal preparations are more effective and safe than allopathic medications due to their natural origins. However, very little information is available to describe the toxicological nature of African herbal remedies, especially with regards to their hepatotoxic effects, or ability to alter the pharmacokinetic profiles of other compunds. The aim of this in vitro study was to assess the hepatotoxic potential of a panel of selected African herbal remedies, as well as their potential to induce drug-herb interactions. Crude hot water and methanol extracts were prepared from seventeen African plants using brewing and ultrasonic maceration techniques, respectively. Phytochemical screening was done to determine the broad constituency of the extracts using thin layer chromatography, biochemical reactions and free radical scavenging. Cytotoxicity against the HepG2 hepatocarcinoma and Caco-2 colon carcinoma cell lines were determined using the sulforhodamine B staining assay. Acokanthera oppositifolia, Boophane disticha, Moringa oleifera, Solanum aculeastrum, Tabernaemontana elegans, Terminalia sericea and Ziziphus mucronata were selected for further hepatotoxic assessment using a mixture of spectrophotometric, fluorometric, chemiluminescent and flow cytometric assays. Oxidative stress (reactive oxygen species, glutathione and lipid peroxidation levels), mitochondrial membrane potential, fatty acid accumulation and caspase-3/7 activation was assessed using fluorometric assays, while adenosine triphosphate levels were assessed using a chemiluminescent assay. The effect of the extracts on cellular kinetics and mode of cell death was determined using flow cytometric techniques. Drug-herb interactions of the crude extracts were assessed by measuring their effect on P-glycoprotein activity, nevirapine permeability and cytochrome P450 (CYP2B6, CYP2D6 and CYP3A4) enzyme activity. Phytochemical analyses successfully identified the broad constituency and antioxidant profiles of the crude extracts, which correlated well with that already described in literature. Phytochemical classes that were most prominent in the majority of extracts were the alkaloids, flavonoids, glycosides, phenolic acids and saponins. These results were used to determine potential contributing factors during the hepatotoxicity and drug-herb interactions assays. Thirteen extracts displayed activity against the HepG2 cell line, while twelve were active against the Caco-2 cell line. Cytotoxicity was in generally more potent against the Caco-2 cell line, indicating a potential susceptibility of the intestinal tract towards these herbal remedies. A high risk of cytotoxicity was identified for extracts such as A. oppositifolia, S. aculeastrum and T. elegans. Further hepatotoxic assessment indicated that the majority of extracts depolarised the mitochondrial membrane, however oxidative stress was rarely induced. Steatotic changes were evident as shown by the increased retention of fatty acids. Cytotoxicity was mostly a mixture of antiproliferative effects, and worryingly, the induction of necrotic cell death. Although the methanol extracts tended to be more potent than the hot water extracts, the latter induced detrimental effects as well, albeit at a higher relative concentration. The most prominent hepatotoxic effects were observed after exposure to T. elegans, with a halfmaximal inhibitory concentration of 3.07 ?g/mL. Only the methanol extract of S. aculeastrum displayed prominent P-glycoprotein inhibitory activity (HepG2 = 2.92-fold, Caco-2 = 1.29-fold). Five extracts (hot water extracts of Burkea africana and Senecio latifolius, and the methanol extracts of Mundulea sericea, Rauvolfia caffra and Solanum aculeastrum) were selected for assessment of their ability to modulate nevirapine transport across the Caco-2 cell line. All five extracts decreased nevirapine efflux, indicating a propensity for increasing its bioavailability. Due to the lack of P-glycoprotein inhibitory activity from the majority, it appears that this reduced efflux is not necessarily P-glycoprotein-dependent. Altered membrane fluidics and inhibition of other membrane transporters are suggested as potential contributing factors. The majority of extracts displayed prominent CYP450 inhibitory activity (CYP3A4>CYP2B6>CYP2D6). Most extracts displayed a higher selectively towards CYP3A4, which highlights the caution required. More than 50% of drugs currently used on the market are metabolised by the CYP3A4 isoform, and thus the risk is high when comparing it to the low concentrations required to elicit an effect. Both extracts of B. africana, the hot water extract of T. sericea and the methanol extract of Z. mucronata displayed non-selective inhibition across all three isoforms, indicating a possible affinity for a structurally-conserved site across CYP450 enzymes. Due to the genetic makeup of the African populace, which has a significant proportion of CYP450 alleles displaying reduced or inactived enzyme activity, risk is thus high for attenuating their metabolic functions. The methanol extract displayed the highest inhibitory activity against CYP3A4, with a half-maximal inhibitory concentration <1.2 ?g/mL. It is evident throughout the study that there is a high risk of hepatotoxicity or drug-herb interactions. This is specifically observed when taking into account the low amount of extract required to reach the inhibitory concentrations determined in vitro. Inhibition of intestinal P-glycoprotein transporters and CYP450 enzymes appear to be more at risk than that of the liver. This is one of the few studies delving into the toxicological natures of African herbal remedies, specifically in light of hepatotoxicity and herb-drug interactions, and thus serves as a foundation for further assessment.