Suparmi

In Indonesia, the market demand for herbal products keeps growing, and as a result, herbal products increasingly provide economic and perceived clinical benefits. A risk and benefit assessment are crucial to be performed to support the safe use of herbal products although the consumers perceive herbal product as “safe” and “natural” and thus “healthy” (Chapter 1). The aim of the present thesis was to perform an assessment of potential risks and some benefits of herbal products available in the Indonesian market. The model compounds chosen included especially naturally occurring genotoxic and carcinogenic botanical constituents including alkenylbenzenes (ABs) and pyrrolizidine alkaloids (PAs). Beneficial effects focussed on potential PPARγ activation by the carotenoids bixin and crocetin. Existing but also novel testing strategies were used to evaluate the relevance of effects at estimated human intake levels.

The consumer risks of jamu, Indonesian traditional herbal medicines, was assessed focussing on the presence of AB-containing botanical ingredients (Chapter 2). Methyleugenol, appeared to be a major AB present, being detected in 91.3% of the collected jamu samples. Quantification of AB levels and exposure resulting from use of the respective jamu products resulted in Margin of Exposure (MOE) values generally <10,000, indicating a priority for risk management when assuming daily consumption during a lifetime. Using Haber’s rule, it was estimated that two weeks consumption of these jamu only once would not raise a concern (MOE >10,000). However, when considering use for two weeks every year during a lifetime, 5 samples still raised a concern. It is concluded that the consumption of AB-containing jamu can be of concern especially when consumed daily for longer periods of time.

Based on these results it was anticipated that methyleugenol may also be present and pose a risk in Indonesian instant herbal beverages. Chapter 3 analysed methyleugenol in many instant herbal beverages containing various mixed herbs collected on the Indonesian market by a targeted sampling strategy. Interestingly, eugenol was detected in a few samples at a level that resulted in an estimated daily intake (EDI) lower than the acceptable daily intake (ADI) of 2.5 mg/kg bw thus not raising a concern for human health. The MOE for methyleugenol intake by targeted consumers, including both adults and children, revealed that several of the herbal products targeted at adults would require a risk management action, while only a few samples targeted at children pointed at a priority for risk management, when the respective herbal beverages would be used every day during a lifetime. However, when assuming the consumption for 2 weeks, every year during a lifetime and using Haber’s rule then all MOE values were estimated to be > 10,000 indicating no priority for risk management. An overview of the current product registration type of these samples indicated that herbal beverages registered as household food (labelled as ‘Depkes RI P-IRT’), would raise a concern when people would consume them every day during a lifetime. The study provided data that can support establishment of a maximum permitted level (MPL) for methyleugenol in herbal beverages in Indonesia.

Another group of genotoxic compounds potentially present in botanicals and botanical preparations and raising a health concern are PAs. In Chapter 4, the occurrence and accompanying risks of PAs in Indonesian jamu were evaluated. PAs were detected in 97.1% of the jamu containing PA-producing botanicals and in 74% of the jamu samples that had no PA-producing botanicals listed on their label. This latter point shows contamination with PA-producing plants due to co-harvesting of PA-containing weeds during cultivation or harvesting of the materials. Short-time, 4 days up to 2 weeks, consumption of jamu, is unlikely to result in acute toxic effects, although one sample would exceed an intake of 10 µg PAs/kg bw/day which may cause hepatic veno-occlusive disease (HVOD) and PA-induced liver injury (PA-ILI) in humans. When evaluating the potential risk for genotoxicity and carcinogenicity via the MOE approach, MOE values below 10,000 were obtained for 46.6% of the samples, indicating a priority for risk management when assuming daily lifelong consumption. Assuming consumption for two weeks every year during a lifetime, and using Haber's rule, 37% of the jamu samples containing PA-producing botanicals still raised a concern, while the jamu consisting of non-PA-producing botanicals would be of low concern. This study provided data that can support risk management actions in Indonesia to minimize the potential health risk for jamu consumers due to the occurrence of toxic PAs in these products.

Exposure to these PAs, including monocrotaline via herbal product consumption is of concern because of their hepatotoxicity and the fact that they are genotoxic carcinogens. Considering that only for a limited number of 1,2-unsaturated PAs in vivo toxicity data are available, hampering risk assessment where differences in relative potency between different PAs are considered, alternative testing strategies including read-across and quantitative in vitro to in vivo extrapolation (QIVIVE) become important for risk analysis. In Chapter 5, a combination of in vitro-physiologically based kinetic (PBK) modeling-facilitated reverse dosimetry was used to predict the in vivo acute liver toxicity of the PA monocrotaline and to characterize the influence of its metabolism on its relative toxic potency compared to lasiocarpine and riddelliine. In the absence of data on acute liver toxicity of monocrotaline upon oral exposure, the predicted dose-response curve for acute liver toxicity in rats and the resulting benchmark dose lower and upper confidence limits for 10% effect (BMDL10 and BMDU10) were compared to data obtained in studies with intraperitoneal or subcutaneous dosing regimens. This indicated the predicted BMDL10 value to be in line with no-observed-adverse-effect levels (NOAELs) derived from available in vivo studies. The predicted BMDL10-BMDU10 of 1.7-6.3 mg/kg bw/day also matched the oral dose range of 1-3 mg PA/kg bw/day at which adverse effects in human are reported. A comparison to the oral toxicity of the related pyrrolizidine alkaloids (PAs) lasiocarpine and riddelline revealed that, although in the rat liver hepatocytes study monocrotaline was less toxic than lasocarpine and riddelliine, due to its relatively inefficient clearance the in vivo acute liver toxicity was predicted to be comparable. It was concluded that the combined in vitro-PBK modeling approach can provide insight in monocrotaline-induced acute liver toxicity in rats thereby filling existing gaps in the database on PA toxicity. Furthermore, the results reveal that the kinetic and metabolic properties of PAs can vary substantially and should be considered when considering differences in relative potency between different PAs.

The combined in vitro-PBK modeling approach was also applied for benefit analysis of the therapeutic effect of bixin and crocetin in type 2 diabetes mellitus suggested to occur via PPARγ activation. Chapter 6 investigated whether at realistic dietary intake from botanical preparations bixin and crocetin could induce PPARγ-mediated gene expression in humans. Concentration-response curves obtained from in vitro PPARγ-reporter gene assays were converted to in vivo dose-response curves using PBK modeling-facilitated reverse dosimetry, from which the benchmark dose resulting in a 50% effect above background level (BMD50) values were predicted and subsequently compared to dietary exposure levels. In the PPARγ reporter gene assay bixin and crocetin activated PPARγ-mediated gene transcription in a concentration-dependent manner with similar potencies. Due to differences in kinetics, the predicted BMD50 values for in vivo PPARγ activation was about 30-fold different, amounting to 115 and 3,505 mg/kg bw for crocetin and bixin, respectively. Human dietary and/or supplemental estimated daily intakes may reach these BMD50 values for crocetin but not for bixin, pointing at better possibilities for in vivo PPARγ activation by crocetin. The results presented further show that based on a combined in vitro-in silico approach, it could be estimated whether at realistic dietary intakes plasma concentrations of bixin and crocetin are likely to reach concentrations that activate PPARγ-mediated gene expression, without the need for a human intervention study.

The results obtained in the present thesis support the conclusion that there is a need for risk management formulating regulatory actions to minimize the potential health risks for consumers in Indonesia due to the occurrence of toxic ABs, PAs and AAs in herbal products (Chapter 7). It is important to note that this conclusion holds for herbal products collected by targeted sampling, and not for all herbal products on the Indonesian market. Several methodological considerations are formulated that need to be considered when performing the risk and benefit analysis to avoid over-/under-estimation of risks or benefits. Furthermore, the relevance of risk assessment to support risk management action is highlighted, and considerations regarding the potential for the application of PBK model based QIVIVE for predicting beneficial as well as adverse effects, without the need for animal experiments and/or human intervention studies are formulated.

Altogether, it can be concluded that the risk assessment using the MOE approach combined with Haber’s rule can be used to prioritize risk management actions to prevent the adverse health effects of consuming Indonesian herbal products containing genotoxic carcinogens. In addition, a novel testing strategy, combining in vitro and PBK modeling-facilitated reverse dosimetry was found to facilitate risk and benefit assessment of botanical compounds without the need for animal experiments and/or human intervention studies.