In vitro bioassays to evaluate beneficial and adverse health effects of botanicals: promises and pitfalls

Drug Discovery Today
Volume 22, Number 8
August 2017

REVIEWS

Reviews
KEYNOTE REVIEW

Teaser In vitro assays are widely and effectively used to test the efficacy and adverse effects of botanicals and botanical preparations. The pitfalls of these assays are however often incorrectly and inaccurately taken into account hampering adequate extrapolation to in vivo situations often resulting in false interpretations. These pitfalls as well as strategies to overcome them are discussed.

In vitro bioassays to evaluate beneficial and adverse health effects of botanicals: promises and pitfalls Gerhard Prinsloo1,2, Georgia Papadi1,5, Mebrahtom G. Hiben1,3, Laura de Haan1, Jochem Louisse1, Karsten Beekmann1, Jacques Vervoort2,4 and Ivonne M.C.M. Rietjens1 1

Division of Toxicology, Wageningen University, Stippeneng 4, 6708 WE Wageningen, The Netherlands Department of Agriculture and Animal Health, University of South Africa, Private bag x 6, Florida, South Africa 3 Department of Pharmacognosy, School of Pharmacy, College of Health Sciences, Mekelle University, Mekelle, Ethiopia 4 Laboratory of Biochemistry, Wageningen University, Stippeneng 4, 6708 WE Wageningen, The Netherlands 5 Department of Biological Applications & Technology, University of Ioannina, Greece 2

This review provides an update on the promises and pitfalls when using in vitro bioassays to evaluate beneficial and adverse health effects of botanicals and botanical preparations. Important issues addressed in the paper are: (i) the type of assays and biological effects available; (ii) falsepositives, false-negatives and confounding factors; (iii) matrix and combination effects; (iv) extrapolation of in vitro data to the in vivo situation; (v) when (not) to use bioassays; and (vi) identification of active constituents. It is concluded that in vitro bioassays provide models to detect beneficial as well as adverse activities, but that linking these observations to individual ingredients and extrapolations to the in vivo situation is more complicated than generally anticipated. Introduction The use of botanicals and botanical preparations to pursue supposed beneficial health effects was of importance historically, just as it is now. In addition, a number of plant-derived food items form an integral part of regular human diets or can be developed as so-called novel foods. However, there is also increasing awareness among safety experts and regulators of risks that are associated with the use of botanicals and botanical ingredients in food [1,2]. It is clear that ‘natural’ does not equate to ‘safe’ and that, in modern society, adverse health effects can occur as a result of (mis)use of botanicals and botanical ingredients. With the growing awareness of these issues, efforts to ensure safety of botanicals and botanical ingredients increase as well. Several guidance documents on safety assessment of botanicals and botanical preparations to be used as ingredients in food and food supplements have been published [1]; although, at present, relevant legislative frameworks for risk assessment are not available yet. It is well recognised that botanical Corresponding author: Prinsloo, G. ([email protected]) 1359-6446/ã 2017 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.drudis.2017.05.002

Gerhard Prinsloo is a senior lecturer in the Department of Agriculture and Animal Health at the University of South Africa. His research focuses on cultivation of indigenous plants used as food, medicine and cosmeceuticals, and the associated effects of the environment on the chemical profile. Metabolomic analysis is applied to identify the changes in the metabolome as a result of external factors and the effects on the beneficial properties of the plants. The ultimate aim is to develop guidelines for commercial production without compromising the safety and efficacy of the cultivated material. Georgia Papadi received her BSc and MSc degrees from the Department of Biochemistry and Biotechnology, University of Thessaly, Greece, focusing on molecular diagnostics. Currently, she is a PhD candidate at the Division of Toxicology of Wageningen University, The Netherlands, and the Department of Biological Applications & Technology, University of Ioannina, Greece. Her research interests involve the potential health benefits and risks of botanicals and botanical preparations. Mebrahtom G. Hiben is assistant professor of pharmacognosy at the Department of Pharmacognosy, School of Pharmacy, College of Health Sciences, Mekelle University, Mekelle, Ethiopia. Currently, he is a PhD student at the Division of Toxicology, Wageningen University, Wageningen, The Netherlands. His research interest focuses on the potential benefits and safety aspects of natural products, mainly of plant origin. Laura de Haan is a technician at the division Toxicology at the University Wageningen and Research. Her work focuses on the use of cell culture and molecular biology techniques in the elucidation of mechanisms

www.drugdiscoverytoday.com

1187

Drug Discovery Today
Volume 22, Number 8
August 2017

REVIEWS

Reviews
KEYNOTE REVIEW

important in the toxic effects of various compounds found in the human diet. She contributes to the research of many PhD students and researchers and is co-author on many publications.

GLOSSARY

Jochem Louisse is assistant professor at the division of Toxicology of Wageningen University, The Netherlands. He holds a PhD from Wageningen University and has worked as a postdoctoral researcher at the European Centre for the Validation of Alternative Methods (EURL ECVAM) of the Joint Research Centre of the European Commission. His research focuses on the development of non-animal-based testing methods that can be applied in toxicological risk assessment, including the development of human stem-cell-based tissue models for toxicity testing and the application of PBK-modelling-based reverse dosimetry to translate in vitro concentration-response data to predicted in vivo dose-response data.

ADME Absorption, distribution, metabolism and excretion EFSA European Food Safety Authority EpRE Electrophile-responsive element IC50 The concentration of an inhibitor that reduces the activity by half MIC Minimum inhibitory concentration PBK Physiologically based kinetic PPARa Peroxisome-proliferator-activated receptor a PPARg Peroxisome-proliferator-activated receptor g PCA Principle component analysis

Karsten Beekmann is assistant professor at the Division of Toxicology at Wageningen University, The Netherlands. His research focuses on the metabolism of foodborne xenobiotics. The emphasis of his current work is on the role of the gut microbiota in toxicology, studying the gut microbial metabolism of foodborne xenobiotics and the consequences of the metabolism for their biological activities. Dr Beekmann also works on in vitro models that can be used to generate data to describe gut microbial metabolism in PBK models. Jacques Vervoort has a PhD in analytical biochemistry on protein˘vitamin-B2 interactions. He studies protein structure and function, protein˘ligand interactions and metabolism of exogenous and endogenous molecules. He is an author of over 300 scientific publications and has a h-factor (Web of Science) of 46. Major topics in his research are proteomics and metabolomics as well as bioinformatics tools to speed up identification of unknowns. Ivonne M.C.M. Rietjens is head of the division of Toxicology at Wageningen University (WU), The Netherlands. She is a member of the Royal Netherlands Academy of Arts and Sciences (KNAW) and of many national and international committees. She has been promotor of 92 PhD students (26 ongoing). She is author of over 400 scientific publications and has a h-factor (WoS) of 46. Major topics of research focus on risk assessment of natural toxins, physiologically based kinetic (PBK) models for low-dose and in vitro to in vivo extrapolation, alternative methods for animal testing and development of mode-of-action-based bioassays.

or botanical extracts before testing in vivo. Bioassays detecting beneficial as well as adverse health effects can be of use. Whereas in the following sections bioassays for beneficial and adverse effects are presented, it is important to realise that this division might not always be as obvious as presented. Depending on the conditions, a biological effect that is considered adverse can become beneficial and vice versa. An example is the activation of electrophile-responsive element (EpRE)-mediated gene expression, reflecting induction of detoxifying cancer-protective enzymes but at the same time reflecting exposure to a possible toxic electrophile. Other examples are bioassays for detection of estrogenicity reflecting endocrine activity that could be considered adverse but can be beneficial in, for example, post-menopausal women. This should be kept in mind for the overviews presented in the following sections.

In vitro bioassays to detect beneficial effects

and botanical extracts can have a history of use that might support their safety but also that, for botanicals that do not have such a history of use or for preparations for which intended uses will substantially increase historical intake levels, additional data to support their safety are required [1]. Often isolation and identification of active ingredients can lead to drug development; however, the use of botanicals, botanical preparations and botanical ingredients, and effective ways to better judge possible beneficial health effects and related health claims, would facilitate the risk:benefit assessment of their use, and this is discussed in this review. It is obvious, especially given the ethical and financial limitations of in vivo studies with experimental animals and humans, that the use of in vitro bioassays in studies to evaluate beneficial and adverse health effects of botanicals and botanical preparations provides a solution to identify potential chemicals of interest, although they obviously will present pitfalls at the same time. The aim of the present review is to present an updated overview and give examples of the promises and pitfalls related to the use of in vitro bioassays to evaluate potential beneficial as well as adverse health effects of botanicals, botanical preparations and their active ingredients.

The type of assays and parameters available: relationship with biological health effects Bioassays can be used to detect possible biological health effects using easy and cost-effective methods, which is an important advantage when screening and evaluating botanical preparations 1188

www.drugdiscoverytoday.com

Table 1 provides an overview of some in vitro bioassays that detect possible beneficial health effects. Examples of bioassays frequently used to detect beneficial effects of botanicals and botanical ingredients include assays for antioxidant activity [3] and assays for antimicrobial, antidiabetes, anti-inflammation, anticholinesterase and antiglycation activity [4–10]. Also, bioassays for morededicated parameters can be of use including for example reporter gene assays for activation of EpRE-mediated gene expression, reflecting induction of detoxifying cancer-protective enzymes [11–15], activation of peroxisome-proliferator-activated receptor g(PPARg) which plays a crucial part in adipogenesis related to improved insulin sensitivity in patients with type 2 diabetes mellitus [16–19] and activation of PPARa, which is an important factor in lipid metabolism and has been related to improved lipid plasma profiles with increased high-density lipoprotein (HDL) cholesterol and decreased levels of low-density lipoprotein (LDL) cholesterol and triglycerides [20–24]. The existence of bioassays that can monitor the activation of these specific cellular signalling pathways enables the screening of a high number of samples in a high-throughput manner to determine potential beneficial effects of the botanical extracts. Fig. 1 shows, as an example, the induction of EpRE-mediated gene expression as detected in a bioassay with reporter gene EpRE-LUX cells exposed to methanolic extracts of onion (dotted bar), Braeburn apple (vertically striped bar), broccoli (horizontally striped bar) and Brussels sprouts (diagonally striped bar) [25]. The assay enables testing at different concentrations of various extracts to characterise the relative effectivity of the botanical extract.

Drug Discovery Today
Volume 22, Number 8
August 2017

REVIEWS

TABLE 1

Examples of bioassays to detect possible beneficial effects of botanical extracts. Endpoint detected

Refs

DPPH radical scavenging

a

Antioxidant activity

[3,122]

Hydrogen peroxide scavenging (H2O2) assay

Antioxidant activitya

[3,123]

Nitric oxide scavenging activity

a

Antioxidant activity

[3,124]

Trolox equivalent antioxidant capacity (TEAC) method/ABTS radical cation decolorisation assay

Antioxidant activitya

[3,125]

Total radical-trapping antioxidant parameter (TRAP) method

Antioxidant activitya

[3,126]

Ferric reducing-antioxidant power (FRAP) assay

Antioxidant activitya

[3,127]

a

[3,128]

Oxygen radical absorbance capacity (ORAC) method

Antioxidant activity

Advanced glycation end products (AGEs) inhibition assay

Antiglycation activity

[8–10]

PPARg CALUX

assay

Peroxisome proliferator-activated receptor g mediated effects

[19]

PPARa CALUX1 assay

Peroxisome proliferator-activated receptor a mediated effects

[25]

EpRE LUX/Nrf2 CALUX1 assay

Activation of the Nrf2 oxidative response pathway

[13,14]

In vitro glucose uptake in C2C12 myocytes and 3T3-L1 adipocytes

Antidiabetes

[6]

Enzyme assays (some examples)

Antiviral activity using reverse transcriptase (RT), integrase (IN) and protease (PR), anticholinesterase activity and tyrosinase assay

[129–135]

1

Disc diffusion or microtitre assays

Antibacterial and antifungal activity using various test organisms

[4]

Antiplasmodial

Antimalarial activity

[136]

Cox1 and Cox 2

Inflammation

[5,137]

Antimycobacterial

AntiTB activity

[138]

a

The overview presents only a selection of the many antioxidant assays available. An extended overview can be found in the review presented by Alam et al. [3].

In vitro bioassays to detect adverse effects Table 2 presents an overview of in vitro bioassays that can be used to detect possible adverse health effects. The presence of possible genotoxic ingredients in botanicals and botanical extracts is an issue of particular concern, given the general absence of regulatory

RLU exposed/RLU solvent control

20 18 16 14 12 10 8 6 4 2 0 0 5 30 45

0 5 30 45

0 5 10 15 30

0 5 10 15 30

Concentration (g FW/l) Drug Discovery Today

FIGURE 1

EpRE-mediated gene expression upon exposure to different concentrations of methanolic extracts of onion (dotted bars), Braeburn apple (vertically striped bars), broccoli (horizontally striped bars) and Brussels sprouts (diagonally striped bars). Concentrations of the extracts are expressed as gram fresh weight per litre (gFW/l). Adapted, with permission, from [25].

requirements for putting botanicals and botanical preparations on the market and the fact that several botanicals are known to contain natural ingredients that are genotoxic and/or carcinogenic. A compendium of compounds with possible toxic properties has been published by the European Food Safety Authority (EFSA) compiled from information from various countries and European and international organisations [26]. An inventory of botanical ingredients that are of possible concern for human health because of their genotoxic and/or carcinogenic properties that can be present in botanicals and in the modern food chain revealed several compounds of concern [27,28]. Use of bioassays to detect these potential adverse health effects in the field of botanicals and botanical preparations is possible but complicated. The EFSA published a guidance on the safety assessment of botanicals and botanical preparations also commenting on the issue of genotoxicity [1]. For genotoxicity testing of botanicals and botanical preparations, in vitro tests at the gene and chromosome levels are required. Specific tests include the Ames test, a bacterial reverse mutation assay [29], in vitro tests in mammalian cells for the detection of chromosomal aberrations [30], an in vitro micronucleus assay in mammalian cells [31] and the mouse lymphoma tk assay in mammalian cells [32]. Additional tests to consider to assess possible adverse effects are reporter gene assays, such as the p53 pathway (p53 CALUX1) assay for genotoxicity [14], or assays that detect for example the endocrine activity of a sample. The latter can be detected in assays like the reporter gene assays for estrogen or androgen activity, such as the so-called ER- and AR-CALUX1 assays detecting the potential for estrogen receptor or androgen receptor (ant)agonism [33–35] and the TR-CALUX1 assay to detect thyroid hormone receptor www.drugdiscoverytoday.com

1189

Reviews
KEYNOTE REVIEW

Bioassay

Drug Discovery Today
Volume 22, Number 8
August 2017

REVIEWS

TABLE 2

Examples of bioassays to detect possible adverse effects of botanical extracts.

Reviews
KEYNOTE REVIEW

Bioassay

Endpoint detected

References

Bacterial reverse mutation test (Ames test) (OECD guideline 471) Mammalian chromosome aberration test (OECD guideline 473) Mammalian cell micronucleus assay (OECD guideline 487) Mammalian cell gene mutation test (HPRT test) (OECD 476) Mammalian cell gene mutation assay suing the thymidine kinase gene (mouse lymphoma tk assay)(OECD 490) p53 CALUX1 DR CALUX1 ER CALUX1 AR CALUX1 assay PR CALUX1 assay GR CALUX1 assay TR CALUX1 assay RAR CALUX1 assay TCF CALUX1 AP1 CALUX1 Hif1a CALUX1 ER stress CALUX1 kB CALUX1 P21 CALUX1

Genotoxicity Genotoxicity Genotoxicity Genotoxicity Genotoxicity

[29] [30] [31] [139] [32]

Genotoxicity Dioxin- receptor activation Estrogen-receptor-mediated effects Androgen-receptor-mediated effects Progesterone-receptor-mediated effects Glucocorticoid-receptor-mediated effects Thyroid hormone-receptor-mediated effects Retinoid acid-receptor-mediated effects wnt/TCF pathway activation AP1 pathway activation / cell cycle control Chemical hypoxia response Endoplasmic reticulum stress response NF-kB-mediated signalling Transcription of p21 inhibitor of cell cycle progression

[14] [140] [33] [34] [34] [34] [36,37] [37,141] [37,141] [37,141] [37,141] [37,141] [37,141] [37,141]

interaction [36,37]. Although responses as measured in reporter gene assays presented in Table 2 are not always directly related to an adverse cellular response, they indicate the presence of chemicals that can lead to adverse effects, because they relate to the transcriptional regulation in response to a toxicological insult [38] and are therefore particularly suitable for screening of botanicals and botanical preparations [39].

better prediction of a complex biological response than single assays. An example can be found in a study where chemicals were tested in a battery of assays, including 24 CALUX1 transcriptional activation assays, and several other bioassays to detect reproductive toxicants [37].

Omics technologies

Table 3 provides an overview of the pitfalls and possible solutions when using bioassays, and the possible confounding factors that can cause false-positive or false-negative outcomes. An important consideration when testing botanical samples is whether the compound of concern will actually be present in the botanical sample at a concentration that will allow its detection in a bioassay. Given the fact that the OECD guidelines for the bacterial reverse mutation assay [29] indicate that: ‘‘the recommended maximum test concentration for soluble non-cytotoxic substances is 5 mg/plate or 5 ml/plate”, the actual ingredient of genotoxic concern should be relatively potent or present at relatively high levels in the extract to actually induce a positive response in this test. Especially for compounds of concern it remains an important challenge to establish whether their concentration in a complex mixture is high enough to detect their biological effect. It makes regulatory bodies require extensive chemical characterisation of a botanical mixture under consideration down to a level where the amount of unknowns would be low enough to not raise any concern, for example because exposure to these unknowns would remain below the so-called threshold of toxicological concern (TTC) [48]. Also, the presence of other compounds in the sample that induce cytotoxicity in the test system can limit the concentrations of active compounds in question that can be tested, or requires additional sample clean-up. Another factor to consider when one wants to eliminate or reduce the occurrence of false-negatives or false-positives is to ensure the inclusion of biotransformation enzymes required to convert the botanical ingredients to the

More recently, omics-based studies have emerged for testing the potential biological activity of botanicals and botanical preparations [40,41]. These technologies can predict the beneficial as well as the adverse profiles of the test substances in a more untargeted holistic way. It is likely that these omics technologies will accelerate and facilitate the rate of discovery and analysis of the biological effects of complex botanical mixtures in the near future [41,42]. They might also be of use to overcome another pitfall in the field related to the fact that it must be assured that the scope of possible health effects analysed by the selected bioassays must be wide enough to include diverse and even unknown modes-of-action. Whether to choose an in vitro bioassay for a dedicated selected possible health effect or rather apply a more holistic omics-based approach will depend on whether a random screening or a knowledge-based targeted screening is required. Both approaches can be effective, where initial screening of samples of unknown biological effect might benefit from use of omics-based technologies; specific questions on safety aspects or identification of ingredients with a specific beneficial effect could benefit from dedicated assays [43– 47]. In the field of drug discovery where it is recognised that drugs including botanicals and their extracts can exert their effect through multiple target interactions, the focus changes from single to multi-target testing [42,47]. Multi-target testing can include omics-based approaches but also the combination of a large panel of bioassays, especially when the mode-of-action, beneficial or adverse, of the botanical under investigation is not known [39]. A battery of complementary test systems can provide a 1190

www.drugdiscoverytoday.com

False-positives and false-negatives: confounding factors

Drug Discovery Today
Volume 22, Number 8
August 2017

REVIEWS

TABLE 3

Bioassays

Potential pitfall

Solution

Refs

Bacterial Reverse Mutation (AMES) Assay

Concentration of compound of concern in botanical preparation too low

Chemical characterization of botanical preparation to a level where the unknowns will be below the TTC

[29]

Bacterial Reverse Mutation (AMES) Assay

Extracts with high levels of histidine give false positives

Consideration of this effect especially with herbal preparations that might contain high histidine levels

[39]

Bacterial Reverse Mutation (AMES) Assay

Interference of (iso)flavonoids, chlorophyll, fatty acids and tannins

Removal of (iso0flavonoids), chlorophyll, fatty acids and tannins before testing

[39]

Cellular in vitro models

Inactive form of ingredient in samples resulting in false negatives

Addition of biotransformation enzymes

[55]

Luciferase reporter gene assays

Compounds stabilizing the firefly luciferase reporter protein give false positives

Use of the cytotox CALUX1 cell line which has invariant expression of the luciferase reporter gene to investigate and exclude this effect

[14,57,58]

In vitro reverse transcriptase, DNA polymerase and topoisomerase I and II assays

Presence of magnesium or manganese divalent ions used as co-factor and providing false positives

Consideration of these effects especially with herbal preparations that might contain metal ions

[60,64–66]

In vitro reverse transcriptase, DNA polymerase and topoisomerase I and II assays

Palladium and iron inhibitreverse transcriptase. Tannins in botanical preparations bind to the proteins

Consideration of these effects especially with herbal preparations that might contain metal ions. Removal of tannins before testing.

[60,64–66]

Noncellular microsomal radiometric aromatase assay

Interference of fatty acids

Removal of fatty acids before testing

[61]

Microtitre antimicrobial assays

Photosynthetic pigments

Removal of pigments and use of suitable colour reagent

[4,67]

Microtitre antimicrobial assays

Inoculum size and concentration of calcium and magnesium in the growth medium affect certain organisms

Consideration of the effect of metal ions in the herbal preparation and use of colony forming units (CFU) and McFarland standards

[4,67]

MTT or the resazurin assays

Constituents with antioxidant activity result in too high MTT activity

Use another assay for cell viability

[69]

actual metabolite that raises the genotoxic and carcinogenic concern or to inactivate an ingredient of concern. This can be illustrated by the group of alkenylbenzenes, including compounds like estragole, methyleugenol, safrole and others that are known to cause hepatocarcinogenicity by a genotoxic mode-of-action [49–51]. High levels of these compounds can be found in several herbs and spices such as nutmeg (Myristica fragrans), star anise (Illicium verum), cinnamon (Cinnamomum), tarragon (Artemisia dracunculus), sweet basil (Ocimum basilicum) and fennel (Foeniculum vulgare), and they are present in their essential oils as well as in food products derived from these botanicals such as basil-based pesto sauce, cola flavoured beverages and herbal food supplements [49,50,52–54]. Because bioactivation of these alkenylbenzenes to a

OCH3

DNA-reactive 10 -sulfooxymetabolite requires the activity of cytochrome P450s and sulfotransferases (Fig. 2), enzymes that might be absent in various cellular in vitro models for genotoxicity testing like the Ames test, the compounds test negative for genotoxicity. This holds even when adding S9 for metabolic activation because S9 does not contain the sulfotransferase enzymes and cofactor for sulfation. However, when the Salmonella typhimurium TA100 strain used in the Ames test (in the presence of S9) was genetically modified to express sulfotransferase enzymes, methyleugenol tested positive for genotoxicity and was shown to result in DNA adduct formation [55]. False-positives can also occur as a result of inaccurate reflection of ADME (see Glossary for common terms used in this review)

OCH3

P450s OH

Estragole

1ʹ-Hydroxy-estragole

OCH3

SULTs OSO3-

1ʹ-Sulfooxy-estragole Drug Discovery Today

FIGURE 2

Bioactivation of alkenylbenzenes (taking estragole as the model compound) by cytochrome P450 (CYP) and sulfotransferase (SULT) enzymes to the ultimate carcinogenic 10 -sulfooxyestragole. www.drugdiscoverytoday.com

1191

Reviews
KEYNOTE REVIEW

Summary of some important potential pitfalls for commonly used bioassays and possible solutions.

REVIEWS

Reviews
KEYNOTE REVIEW

characteristics that would result in inactivation of active ingredients in the in vivo situation. An example of this can be found when testing soy isoflavones for estrogenicity in a bioassay. The parent aglycone will test positive but this might not result in directly relevant information for the in vivo situation given that conjugation by, for example, glucuronosyltransferases will produce a conjugate with reduced activity [56]. Another important reason for possible false-positives relates to artefacts described for the bioassays using luciferase as the reporter gene [57]. Compounds present in botanical preparations and extracts, including for example several (iso)flavonoids, have been observed to lead to stabilisation of the firefly luciferase reporter enzyme increasing the bioluminescent signal during the cell-based assay [57]. This artefact is probably the result of the direct interaction of the compound with the firefly luciferase reporter enzyme thereby increasing its half-life and stabilising the enzyme activity [57,58]. To overcome this problem, the effect of the test compound or botanical extract can be assessed in the cytotox CALUX1 cell line, which has invariant expression of the luciferase reporter gene protein [14]. When using a luciferase reporter gene assay to detect possible biological activities of botanical samples it is essential to test the same samples in this cytotox CALUX1 assay as well. A decrease in luciferase activity in this bioassay indicates a cytotoxic effect, whereas an increase in luciferase activity in the cytotox CALUX1 cells could indicate stabilisation of the luciferase enzyme and possible false-positives for reporter gene expression in the CALUX1 assay of interest. An artefact causing false-positives in the Ames test for genotoxicity can arise for extracts that contain high levels of histidine. Because the test is based on detecting mutated revertants that can grow and form colonies in medium lacking histidine, adding a botanical test sample with histidine will cause the bacteria to form colonies without a mutation, causing false-positives. The presence of flavonoids, such as quercetin, in plant extracts can lead to positive results in the Ames test [59] and these are considered false-positive because the compounds are generally not genotoxic in mammalian cells and in vivo. Botanical extracts can also contain other constituents causing confounding of the bioassay results. Examples are the presence of fluorescent constituents like (iso)flavones and chlorophyll which will hamper assays based on fluorescence detection, as well as the interference of fatty acids and tannins [39]. Tannins with their high affinity for proteins have been shown to inhibit reverse transcriptase, DNA polymerase and the enzymes topoisomerase I and II [60]. Fatty acids can for instance interfere with the noncellular microsomal radiometric aromatase assay which determines in vitro and in vivo aromatase enzyme activity, the rate limiting step in estrogen biosynthesis. Fatty acids have been investigated for use as nutritional supplements and, although they can be biologically relevant, their almost ubiquitous occurrence in plant extracts and frequent positive response in noncellular assays necessitates the removal of fatty acids during screening and large-scale bioassay-guided fractionation [61]. Photosynthetic pigments often create challenges in determining the minimum inhibitory concentration (MIC) values in microtitre assays and are known to create false-positives in, for example, the cyclooxygenase (Cox)-1 anti-inflammatory bioassay [4,62,63]. 1192

www.drugdiscoverytoday.com

Drug Discovery Today
Volume 22, Number 8
August 2017

Using assays testing biological activity based on enzyme activity detection methods should also be approached with careful consideration to prevent false-positives or negatives. The reverse transcriptase enzyme(RT) is often used in determining antiviral activity. It has been found that HIV-1 RT uses magnesium (Mg) or manganese (Mn) divalent ions as a co-factor and activity is therefore optimal with Mg ions present, but a marginal increase in these ions might influence the enzyme activity resulting in false-positives [64]. By contrast, the presence of palladium (Pd) and iron (Fe) is known to induce irreversible inhibition of RT resulting in inaccurate data [65,66]. It has also been reported that the inoculum size in antimicrobial assays has a large effect on MIC values and some organisms are also affected by the calcium (Ca) and Mg concentrations in the growth medium in the widely used microplate method [4,67]. Another example is the presence of constituents with antioxidant activity which can cause artefacts in assays using redox-sensitive readouts including assays for cytotoxicity like the MTT or the resazurin assays [68], or bioassays based on peroxidase-catalysed quantitative conversion of coloured products that can be measured by absorbance or fluorescence including for example commercially available kits for detection of free fatty acids or triglycerides [69]. Antioxidants such as flavonoids inhibit peroxidase activity thereby creating false bioassay results [69].

Matrix and combination effects In addition to the matrix-derived constituents in extracts that can result in false-positives or -negatives, as presented in the previous section, other matrix-derived effects can also occur. These include effects on bioaccessibility, combination effects that influence ADME characteristics and thereby modify the biological effects of constituents in the in vivo situation or combination effects that result in additive, synergistic or antagonistic activities on the biological effect studied. Matrix-derived effects on bioaccessibility of compounds and/or from interactions with processes underlying the actual transport across the cellular membrane can influence the outcome of a bioassay when testing a complex extract. An example of such a matrix interaction can be found in the interaction of chlorophyllin with aflatoxin B1, resulting in lower bioavailability of aflatoxin when in a matrix of green leafy vegetables because of formation of a strong noncovalent complex [70–72]. Chlorophyllin was also reported to bind to planar aromatic carcinogens such as benzo(a) pyrene (B[a]P) thereby significantly reducing B[a]P-DNA adduct formation in normal human mammary epithelial cells [73]. Other examples can be found in matrix effects on the bioavailability of ferulic acid, b-carotene and other carotenoids, isoflavones, polycyclic aromatic hydrocarbons (PAHs) including B[a]P, dioxins and dibenzofurans, polyphenols including green tea catechins, and coumarins [74]. Also, effects of flavonoids on the activity of membrane-bound active ATP-binding cassette (ABC) transporter proteins could influence the cellular uptake and bioavailability of other constituents present in a botanical extract thereby influencing the biological effect detected, because intracellular concentrations can be increased or decreased depending on whether the ABC transporter is involved in cellular uptake or efflux of the active ingredients [75]. Matrix-derived combination effects can also result from interactions at the level of ADME characteristics. For example, the

Drug Discovery Today
Volume 22, Number 8
August 2017

REVIEWS

Extrapolation of in vitro data to the in vivo situation

The effect of hydrolysis

(a) 30

(b) 30

RLU exposed/RLU solvent control

Hydrolysis of botanical extracts can result in hydrolysis of glycosidic bonds of phenolic compounds present, resulting in formation of nonconjugated analogues able to induce a specific biological response to an even larger extent than the nonhydrolysed extract. This effect is illustrated by the example where botanical extracts were tested in a bioassay for PPARg-mediated gene expression using hydrolysed and nonhydrolysed extracts (Fig. 3). This reflects at the same time a possible pitfall for use of bioassays to detect biological activities of botanicals and their extracts. Although hydrolysis of the samples tested can affect the biological effect studied, it is essential to ascertain that the method of hydrolysis is biologically compatible so that hydrolysis would be expected to occur in vivo under real-life conditions to a similar extent. Sambucus nigra secondary metabolites such as quercetin-3-O-rutinoside, quercetin-3-O-glucoside, kaempferol-3O-rutinoside, isorhamnetin-3-O-rutinoside, isorhamnetin-3-Oglucoside and 5-O-caffeoylquinic acid do not activate PPARg whereas some of their aglycones are potent agonists of PPARg, which highlights the role of transformation in the gut after absorption [81]. Hydrolysis of the major soy isoflavones, genistin and daidzin, is known to be essential to liberate their estrogenic potential, but does not occur under conditions encountered in the gastrointestinal tract without the activity of specialised enzymes and especially of the gut microbiota. In particular, the role of the

25

20

15

10

5

25

20

15

10

5

0

0 1 5 10 15 20

1 5 10 15 20

1 5 10 15 20

Concentration (g FW/l)

1 5 10 15 20

1 5 10 15 20

1 5 10 15 20

1 5 10 15 20

1 5 10 15 20

Concentration (g FW/l) Drug Discovery Today

FIGURE 3

PPARg-mediated gene expression induced upon exposure to nonhydrolysed (a) and hydrolysed (b) methanolic extracts of onion (dotted bars), Braeburn apple (vertically striped bars), broccoli (horizontally striped bars) and Brussels sprouts (diagonally striped bars). Adapted, with permission, from [25]. www.drugdiscoverytoday.com

1193

Reviews
KEYNOTE REVIEW

When using the results obtained in an in vitro bioassay to draw conclusions for the in vivo situation it is of the utmost importance to consider the type of extract tested, its pre-treatment and the relevance of this pre-treatment and the extract as tested for the in vivo situation.

RLU exposed/RLU solvent control

flavonoid nevadensin present in basil was shown to reduce the DNA adduct formation by the genotoxic carcinogens methyleugenol and estragole also present in basil [76–78], whereas a mace extract containing malabaricone C appeared to have a similar effect on DNA adduct formation of safrole, also present in mace [79]. Further studies showed that these effects were caused by inhibition of the sulfotransferases required for bioactivation of methyleugenol, estragole and safrole to their ultimate carcinogenic 10 -sulfooxymetabolites (Fig. 2) [76–79]. Proof of the importance of combination effects for the ultimate outcome of exposure to a botanical extract comes from studies reporting the loss of biological activity upon sample fractionation indicating that the overall activity appears to depend on the complex interplay between more than one ingredient in the botanical preparation. Bioassay-guided fractionation of Malva parviflora constituents responsible for its Cox-1 anti-inflammatory activity resulted in loss of activity upon fractionation, but the activity was regained again after combining different fractions [63]. Similar observations and loss of activity was reported upon fractionation of an active Rosa canina extract [80]. Bioassay-guided fractionation of a Sambucus nigra (elderflower) extract that activated PPARg in a bioassay resulted in the identification of two wellknown PPARg agonists: a-linolenic acid and linoleic acid, as well as the flavanone naringenin, but these chemicals alone did not account for all the PPARg activation and the activity might be ascribed to the effects of other compounds present in the plant and/or to combination effects that are lost when testing individual constituents [81]. Thus, a desired biological response could be due to not one but to a mixture of active botanical ingredients and their interplay [82]. This can also explain that, although the relative proportion of individual ingredients can vary from batch to batch, the bioactivity of the mixture as a whole can remain similar [39].

Drug Discovery Today
Volume 22, Number 8
August 2017

REVIEWS

Another pitfall could be that upon consumption the deconjugated active ingredients can be metabolised upon and during uptake in the gastrointestinal tract and in the liver before entering the systemic circulation. This could result in metabolites with modified biological activity. Without taking into account these potential consequences of metabolism for the biological activity of botanical ingredients, it is difficult to extrapolate results obtained in in vitro bioassays to the in vivo situation. For example, compounds such as kaempferol, apigenin and galangin are only present in low concentrations in plasma because they are present nearly exclusively as conjugated glucuronides in the systemic circulation [87–89]. This was further demonstrated with kaempferol and quercetin in providing anxiolytic activity only upon oral intake. These flavonoids are therefore considered to act as prodrugs that are transformed into their active hydroxyphenylacetic acid metabolites [90]. To include metabolic activity in in vitro bioassays, incubation of the preparations tested with S9-liver homogenates, liver microsomes, cryopreserved hepatocytes or liver slices, and/or fresh tissue

samples including the relevant cofactors, have to be combined with the bioassays testing the primary constituents but also their potential metabolites. However, even under these conditions in vitro bioassays provide in vitro concentration response data whereas in vivo judgement of beneficial effects and safety requires in vivo dose–response curves.

Physiologically based kinetic modelling A current way forward solving this issue is the use of so-called physiologically based kinetic (PBK) modelling which allows prediction of the consequences of ADME of active ingredients for the ultimate plasma and tissue levels of a compound of interest. A PBK model is a set of mathematical equations that together describe the ADME characteristics of a compound within an organism [91]. PBK models can predict the actual concentrations of the relevant metabolite in plasma or any tissue of interest, based on the dose level of an ingredient as it occurs in a botanical or botanical preparation. PBK models can, for example, predict the level of the biologically active free aglycone of (iso)flavonoids upon exposure to a conjugated form occurring in a botanical preparation [92]. By using the PBK models in the reverse order, in vitro effective concentrations detected in bioassays can be converted to effective in vivo dose levels. Fig. 4 presents an example predicting whether intake of a botanical supplement containing the soy isoflavone genistein is expected to result in an estrogenic response in vivo. When human PBK models are used for such extrapolations the resulting dose–response curves even relate to the human population [93]. Only when being able to combine the data from an in vitro bioassay on a botanical ingredient with measured or predicted levels of the relevant biological metabolite in the in vivo situation can the possible health effects of a botanical be evaluated. Such an approach will also overcome the disadvantage that testing compounds at high concentrations in in vitro bioassays could represent unrealistic physiological conditions [46]. Papers on botanicals and their ingredients often claim interesting effects without considering whether the concentrations actually needed to induce the effect make any sense compared with what can be expected in the

200

PBK-modelling-based 150

Reverse dosimetry

100 50 0 0.001

0.1 1 0.01 Concentration genistein (µM)

10

Response (% of max estradiol)

Metabolism of compounds

Response (% of max estradiol)

Reviews
KEYNOTE REVIEW

gut microbiota in the conversion of active ingredients in the gastrointestinal tract can be significant, and metabolites formed by the gut microbiota can perform an essential role in the ultimate biological effect of a botanical ingredient. An example is the formation of (S)-equol as a metabolite of the soy isoflavone, daidzein, by the gut microbiota which has been reported to be more active as an estrogen-active compound than daidzein itself [83]. Another example is the formation of enterodiol and enterolactone from ingested lignans through the gut microbiota [84,85]; increased plasma concentrations of enterodiol and enterolactone are associated with lower prevalence of cardiovascular disease and certain cancers. The purgative effect of sennosides on the intestinal flora could be influenced by the different bacteria possessing various glucosidase activities. Bacteria possessing ß-glucosidase activity produce rheinanthrone which is oxidised to sennidin, which is most probably responsible for the laxative properties [86].

200 150 100 50 0 0.1

1 10 Oral dose genistein (mg/kg bw)

100

Drug Discovery Today

FIGURE 4

PBK-modelling-based reverse dosimetry converting in vitro bioassay data for activity of genistein in an ER-CALUX1 assay [56] into a predicted in vivo dose– response curve, which reveals that intake of food supplements containing genistein (intake levels estimated to range from 0.43–13 mg/kg bw) would probably result in estrogenic effects in relevant target organs [142]. 1194

www.drugdiscoverytoday.com

Drug Discovery Today
Volume 22, Number 8
August 2017

When (not) to use bioassays It is important to know when to use and when not to use bioassays. Situations where not to use a bioassay can occur for example when detecting constituents in botanicals and botanical extracts for which far-more-efficient chemical analyses might be available. This is true for example when testing for the presence of heavy metals or pesticides. For pesticides efficient and high-throughput

multiresidue methods exist, based on LC- or GC-MS methods [105–108] that are far more efficient than reported bioassays [109–111]. A major advantage of the use of bioassays to analyse botanicals and botanical preparations is that, in contrast to chemical analyses, bioassays provide a unique platform to detect unknown congeners with a relevant biological activity. In this way, novel bioactive ingredients, or novel compounds of concern, can be identified. For example, the discovery of the vinca alkaloids from Vinca rosea, also known as Catharanthus roseus, resulted from the observation that the extract of the plant produced prolongation of life in mice with P-1534 leukaemia and fractionation of the plant was undertaken. Several alkaloids were identified by bioassay-guided fractionation and yielded the compounds vincristine and vinblastine [112]. Vinblastine is a constituent of several chemotherapy regimens and vincristine is marketed under the brand name Oncovin1 and used in the treatment of various cancers. Another reason for using bioassays can be when a large number of samples should be analysed but the chemical analytical method for the respective analysis would be expensive and time consuming. Because in vitro bioassays are suitable for high-throughput applications, bioassays are currently applied in various regulatory protocols for safety testing. The use of DR-CALUX1 to detect dioxin-like toxicity is a good example [113]. The respective bioassay can be applied for first-tier screening after which suspected samples could be tested further by analytical methods to identify and quantify the constituents in the extract responsible for the observed biological activity. In this safety testing framework, it is also an advantage that bioassays will detect unknown congeners with a biological activity of interest, and are able to quantify the biological potential of complex mixtures as a whole. Current stateof-the-art bioassays have various advantages over analytical chemical techniques that favour their use, although they can often not elucidate the exact quantities and individual congeners present in an extract. To this end, bioassays need to be followed up by analytical methods, including bioassay-guided fractionation, to eventually identify the active constituents and their levels present. It is also important to distinguish between bioguided fractionation of an active extract to discover a lead molecule and the quantification of a well-known active principle in a botanical extract; the latter mostly being used in quality control in safety consideration in contrast to the identification of new constituents as discussed in the next section.

Identification of active constituents For botanicals and botanical extracts the active constituents might be unknown even when the extract induces a response in a bioassay of interest. Active ingredient(s) of interest can be isolated through bioassay-guided fractionation [39]; although several drawbacks hampering this approach have been identified including for example loss of activity upon separation, instability of the isolated compounds or poor solubility of the separated constituents in the assay medium [39,114]. Another issue encountered when identifying and subsequently quantifying compounds of interest relates to the fact that especially botanicals and botanical preparation can show wide variability in the levels of the active ingredients. The presence of active ingredients can vary with soil and climate conditions, geographical origin, time of harvest and harvesting and processing practices; and a botanical extract might www.drugdiscoverytoday.com

1195

Reviews
KEYNOTE REVIEW

systemic circulation as well as in tissues [94]. It has been suggested to define clinically relevant concentrations at IC50 of <50 or <100 mg/ml for extracts and at <5 or <25 mM for individual compounds [39,46,94,95]. Use of PBK models will provide detailed information on the levels that are systemically available upon exposure to specific dose levels and could provide insight in these aspects as illustrated by the example presented in Fig. 4. Obviously, this requirement will not easily be fulfilled for complex mixtures such as botanicals and botanical preparations containing large numbers of active ingredients for which PBK models or in vivo plasma levels will be largely unavailable. However, taking the importance of ADME characteristics in the human body into account, is important before drawing any conclusion on the relevance of a biological effect for human health. This is an issue often ignored in current research on biological activity of botanicals and botanical ingredients. An example of this can be found in the many claims related to the antioxidant effects of botanicals. Testing botanical extracts in bioassays for antioxidant activity will generally result in positive outcomes. However, that does not necessarily infer that botanicals will exert such health effects in vivo, mainly because the bioavailability of the active ingredients in vivo will be limited owing to limited uptake and/or modification of the active congener to a metabolite without this activity [96,97]. Using quercetin as an example, although some conjugated metabolites can retain up to 50% of the antioxidant activity of the aglycone [98,99], the concentrations of quercetin and its conjugates that can be found in the systemic circulation are generally considered negligible when compared with the antioxidant activity of the well-known cellular antioxidants like vitamin C, vitamin E and glutathione (GSH) present at much higher concentrations [100,101]. In some cases, however, the activity measured in an in vitro bioassay correlates with the in vivo effect, such as the correlations between the relative potency of a series of estrogen agonists in sensitive bioassays like the ER-CALUX1 and the in vivo potency observed in the uterotrophic bioassay for estrogenicity in rodents: a short-term screening assay for estrogenic properties [102], indicating the relevance of this in vitro bioassay to predict the in vivo effects [103]. It is clear that, to apply this PBK modelling, the chemical identity of the compound of interest needs to be identified. This is not generally the case and especially difficult for botanicals with only limited chemical identification and botanicals containing unknown active ingredients. Nevertheless, current developments in analytical chemistry and also in the field of PBK modelling, where more generic models are being developed [104], illustrate the future promises of the approach. It can be concluded that bioassays provide models to detect activity of biological extracts and their ingredients but that relating these observations to the in vivo situation is more complicated than generally acknowledged.

REVIEWS

REVIEWS

Reviews
KEYNOTE REVIEW

have to be quantified and characterised chemically and biologically to ensure a consistent level of ingredients and the biological activity of interest. Identification of specific molecules in natural products is challenging because of the complexity of the matrix. The number of secondary metabolites is not known but a conservative estimate is that more than one-billion different molecules can be present in nature [115]. Hitherto, 120 million organic and inorganic molecules are present in the CAS database [116]. Identification of specific molecules in complex biological samples can only be achieved either through isolation and/or identification using MS and NMR methods [117] (untargeted approach) or by using reference molecules that in GC-MS- or LC-MS-based methods not only show the exact retention times as the molecule of interest but also have the identical fragmentation patterns [117]. In recent years dereplication in product discovery has become important. In these studies, the quick identification of known compounds can help to focus on identification of new unknown molecules in complex mixtures. Identification of the new unknown molecules can only be achieved either through isolation or LC-MS-based identification with reference molecules. Fragmentation is preferably achieved using an orbitrap FT-MS system with iontrap fragmentation resulting in fragmentation trees [118,119]. The latter method using reference molecules is considered to be a targeted approach. It must be mentioned that many articles that claim MS-based ‘identification’ of characteristic compounds of interest should not be classified as such, because the molecules of interest have been annotated or have a tentative identification at best. Without NMR data for almost all secondary metabolites, identification based on LC-MS data is impossible [117]. Compounds of interest can be selected based on chemometric methods in which raw data from measurements, especially from LC-MS or NMR origin, are aligned, normalised and analysed for variables that are discriminating between the NMR or LC-MS measurements. In most cases principle component analysis (PCA) is used to select for the discriminating peaks or resonances of the LC-MS or NMR datasets [120]. These discriminating peaks reflecting components or metabolites present in the sample can be annotated and subsequently identified after MS- or NMR-guided purification, followed by identification using MS and NMR methods [117]. When obtaining NMR and LC-MS data for the same samples, chemometric combination of the data-matrices is possible, improving the identification of individual components without painstaking purification [121]. However, this procedure is hampered by the differences in sensitivity between NMR-based and LC-MS-based detection methods.

Concluding remarks The aim of the present review is to provide an update on the promises and pitfalls when using in vitro bioassays to evaluate beneficial and adverse health effects of botanicals. Use of in vitro assays is needed given the ethical and financial constraints of using in vivo studies for large numbers of preparations. Based on the overview and the promises and pitfalls presented, it is concluded that bioassays provide models to detect beneficial as well as adverse activities of complex botanical extracts and preparations, but that relating these observations to individual ingredients and to the in vivo situation is more complicated than generally acknowledged. 1196

www.drugdiscoverytoday.com

Drug Discovery Today
Volume 22, Number 8
August 2017

The rapid increase in global consumption of botanicals and botanical ingredients, and the fact that botanicals and botanical preparations are widely marketed often without adequate safety or efficacy testing [2], indicate that there is an urgent need for efficient methods to elucidate their possible health effects, including beneficial and adverse effects. Given the high number of preparations and their complex nature and variability in quality and content, effective, cheap and high-throughput methods that can detect overall bioactivity of complex mixtures including unknown active principles are required. Bioassays can provide such tools, whether used for a dedicated selected possible health effect or when applied in a more holistic omics-based approach or in a multi-assay test battery. It is important, however, to be aware of the potential pitfalls of bioassay-based studies on botanicals and their extracts including the action of confounding factors, use of physiologically unrealistic concentrations and the absence of adequate ADME characteristics including the absence of the role of the gut microbiota, all able to generate false-negative or falsepositive results. It should be realised that methods able to convert data from in vitro bioassays to the in vivo situation will be essential to take full advantage of the bioassay-based concentration response data generated. PBK-modelling-based reverse dosimetry could provide such an approach although its application to complex mixtures might provide a real challenge. It is also of interest to note that some of the pitfalls encountered for testing effects of botanicals in in vitro bioassays equally apply to in vivo bioassays such as rodent assays, and studies using human volunteers, and this also holds for some of the solutions. Thus, interspecies and inter-individual ADME differences, including differences in gut microbiota and biotransformation enzymes, complicate extrapolation in both cases almost equally. Also, the fact that botanicals are complex mixtures of often highly metabolised compounds complicates in vitro as well as in vivo studies and requires consideration of matrix effects, interactions, identification of active ingredients and consideration of actual levels of ingredients of interest. The fact that in vitro assays allow highthroughput efficient screening can actually be of help to elucidate and solve some of these issues. Fig. 5 further illustrates this use of bioassays in a screening strategy to detect and identify active ingredients of interest. In addition, in vitro bioassays can help to overcome the pitfall of in vivo rodent bioassays where rodents might not be adequate models for humans. In fact, data from in vitro bioassays can be converted to human in vivo dose–response curves when using human PBK models for the reverse dosimetry, thus providing a means to actually predict whether effects observed in vitro can be expected in vivo in the human population. An example of such an approach can be found in a recent study describing PBK modelling of hesperidin metabolism and its use to predict in vivo effective doses in humans [93]. Finally, given that beneficial as well as adverse effects can be detected, a potential risk:benefit evaluation could be of interest. It could be of importance to consider that when botanical extracts or their active ingredients prove to be genotoxic this disadvantage will generally outweigh the benefits of the botanical, whereas drug-related applications can accept a certain risk to generate the benefit. Altogether, it can be concluded that in vitro bioassays can provide unique possibilities to evaluate beneficial and adverse

Drug Discovery Today
Volume 22, Number 8
August 2017

REVIEWS

- Selection of botanical or herb

Identify constituents - Analytical techniques (LC-MS, GC-MS, NMR, etc.) - Compound databases Massbank, MAGMA, HMDB etc.

Bioactivity of identified compounds

Reviews
KEYNOTE REVIEW

- Preparation method

Bioassays [pe: Ames, MTT, microtitre, RT, luciferase reporter gene assays (EpRE, ER, AR), antioxidant, Cox assays, etc.]

Bioassay guided fractionation

Consideration of pitfalls and confounding factors resulting in false-positives and-negatives

Identification of bioactive ingredients Drug Discovery Today

FIGURE 5

Proposed use of bioassays in a screening strategy.

health effects of botanicals and that awareness of their pitfalls will contribute to their future adequate application.

Acknowledgements Georgia Papadi, Jacques Vervoort and Ivonne M.C.M. Rietjens acknowledge financial support from the SOIT foundation (the

Foundation for Stimulation of Innovation in Toxicology). The University of South Africa is thanked for funding through the Vision Keepers Programme. No funders were responsible for the study design, data collection, analysis or interpretation of data, writing of the report or the decision to submit the paper for publication.

References 1 EFSA (2009) Guidance on safety assessment of botanicals and botanical preparations intended for use as ingredients in food supplements. EFSA J. 7, 1–19 2 Speijers, G. et al. (2010) Safety assessment of botanicals and botanical preparations used as ingredients in food supplements: testing an European Food Safety Authority-tiered approach. Mol. Nutr. Food Res. 54, 175–185 3 Alam, M.N. et al. (2010) Review on in vivo and in vitro methods evaluation of antioxidant activity. Saudi Pharm. J. 21, 143–152 4 Eloff, J.N. (1998) A sensitive and quick microplate method to determine the minimal inhibitory concentration of plant extracts for bacteria. Planta Med. 64, 711–713 5 Ja¨ger, A.K. et al. (1996) Screening of Zulu medicinal plants for prostaglandinsynthesis inhibitors. J. Ethnopharmacol. 52, 95–100 6 van de Venter, M. et al. (2008) Antidiabetic screening and scoring of 11 plants traditionally used in South Africa. J. Ethnopharmacol. 119, 81–86 7 Zschocke, S. and van Staden, J. (2000) Cryptocarya species – substitute plants for Ocotea bullata? A pharmacological investigation in terms of cyclooxygenase-1 and -2 inhibition. J. Ethnopharmacol. 71, 473–478 8 Matsuura, N. et al. (2002) Screening system for the Maillard reaction inhibitor from natural product extracts. J. Health Sci. 48, 520–526

9 Ratnasooriya, W.D. et al. (2014) In vitro antiglycation and cross-link breaking activities of Sri Lankan low-grown orthodox orange pekoe grade black tea (Camellia sinensis L.). Trop. J. Pharm. Res. 13, 567–571 10 Se´ro, L. et al. (2013) Tuning a 96-well microtiter plate fluorescence-based assay to identify AGE inhibitors in crude plant extracts. Molecules 18, 14320– 14339 11 Talalay, P. et al. (2003) Importance of phase 2 gene regulation in protection against electrophile and reactive oxygen toxicity and carcinogenesis. Adv. Enzyme Regul. 43, 121–134 12 Lee, J.-S. and Surh, Y.-J. (2005) Nrf2 as a novel molecular target for chemoprevention. Cancer Lett. 224, 171–184 13 Boerboom, A. et al. (2006) Newly constructed stable reporter cell lines for mechanistic studies on electrophile-responsive element-mediated gene expression reveal a role for flavonoid planarity. Biochem. Pharmacol. 72, 217–226 14 van der Linden, S.C. et al. (2014) Development of a panel of high-throughput reporter-gene assays to detect genotoxicity and oxidative stress. Mutat. Res. Genet. Toxicol. Environ. Mutagen. 760, 23–32 15 Giudice, A. and Montella, M. (2006) Activation of the Nrf2-ARE signaling pathway: a promising strategy in cancer prevention. Bioessays 28, 169–181

www.drugdiscoverytoday.com

1197

REVIEWS

Reviews
KEYNOTE REVIEW

16 Tontonoz, P. et al. (1994) Adipocyte-specific transcription factor ARF6 is a heterodimeric complex of two nuclear hormone receptors, PPAR gamma and RXR alpha. Nucleic Acids Res. 22, 5628–5634 17 Barak, Y. et al. (1999) PPARg is required for placental, cardiac, and adipose tissue development. Mol. Cell. 4, 585–595 18 Tontonoz, P. and Spiegelman, B.M. (2008) Fat and beyond: the diverse biology of PPARgamma. Annu. Rev. Biochem. 77, 289–312 19 Gijsbers, L. et al. (2011) Stable reporter cell lines for peroxisome proliferatoractivated receptor g (PPARg)-mediated modulation of gene expression. Anal. Biochem. 414, 77–83 20 Kersten, S. et al. (1999) Peroxisome proliferator-activated receptor alpha mediates the adaptive response to fasting. J. Clin. Invest. 103, 1489–1498 21 Idzior-Walus, B. et al. (2000) Effects of comicronised fenofibrate on lipid and insulin sensitivity in patients with polymetabolic syndrome X. Eur. J. Clin. Invest. 30, 871–878 22 Reddy, J.K. and Hashimoto, T. (2001) Peroxisomal beta-oxidation and peroxisome proliferator-activated receptor alpha: an adaptive metabolic system. Annu. Rev. Nutr. 21, 193–230 23 Koh, K.K. et al. (2005) Beneficial effects of fenofibrate to improve endothelial dysfunction and raise adiponectin levels in patients with primary hypertriglyceridemia. Diabetes Care 28, 1419–1424 24 Remick, J. et al. (2010) Fibrate therapy: an update. Cardiol Rev. 16, 129–141 25 Gijsbers, L. (2013) In vitro reporter gene assays for assessment of PPAR- and Nrf2mediated health effects of tomato and its bioactive constituents. In Dissertation. Wageningen University 159 p, ISBN 978-94-6173-509-6 http://library.wur.nl/ WebQuery/edepot/248963 26 EFSA (2012) Compendium of botanicals reported to contain naturally occuring substances of possible concern for human health when used in food and food supplements. EFSA J. 10, 2663 27 Rietjens, I.M.C.M. et al. (2015) Matrix-derived combination effects influencing absorption, distribution, metabolism and excretion (ADME) of food-borne toxic compounds: implications for risk assessment. Toxicol. Res. 4, 23–35 28 Van den Berg, S.J.P.L. et al. (2011) Levels of genotoxic and carcinogenic ingredients in plant food supplements and associated risk assessment. Food Nutr. Sci. 2, 989– 1010 29 OECD (1997) OECD test no. 471: bacterial reverse mutation test. Organ. Econ. Cooperation Dev. Publ. http://www.oecd-ilibrary.org/environment/ test-no-471-bacterial-reverse-mutation-test_9789264071247-en 30 OECD (2014) OECD test no. 473: in vitro mammalian chromosomal aberration test. Organ. Econ. Co-operation Dev. Publ. 473, 1–20. http://www.oecd.org/ chemicalsafety/risk-assessment/1948434.pdf 31 OECD (2014) OECD TG487: in-vitro mammalian cell micronucleus test. Organ. Econ. Co-operation Dev. Publ. https://ntp.niehs.nih.gov/iccvam/suppdocs/feddocs/ oecd/oecd-tg487-2014-508.pdf 32 OECD (2015) OECD test no. 490: in vitro mammalian cell gene mutation tests using the thymidine kinase gene. Organ. Econ. Co-operation Dev. Publ. https://ntp. niehs.nih.gov/iccvam/suppdocs/feddocs/oecd/oecd-tg487-2014-508.pdf 33 Legler, J. et al. (1999) Development of a stably transfected estrogen receptormediated luciferase reporter gene assay in the human T47D breast cancer cell line. Toxicol. Sci. 48, 55–66 34 Sonneveld, E. et al. (2005) Development of androgen- and estrogen-responsive bioassays, members of a panel of human cell line-based highly selective steroidresponsive bioassays. Toxicol. Sci. 83, 136–148 35 van der Burg, B. et al. (2010) Optimization and prevalidation of the in vitro ERalpha CALUX method to test estrogenic and antiestrogenic activity of compounds. Reprod. Toxicol. 30, 73–80 36 Freitas, J. et al. (2011) Detection of thyroid hormone receptor disruptors by a novel stable in vitro reporter gene assay. Toxicol. In Vitro 25, 257–266 37 Piersma, A.H. et al. (2013) Evaluation of an alternative in vitro test battery for detecting reproductive toxicants. Reprod. Toxicol. 38, 53–64 38 Jennings, P. et al. (2013) An overview of transcriptional regulation in response to toxicological insult. Arch. Toxicol. 87, 49–72 39 Agarwal, A. et al. (2014) Use of in vitro bioassays for assessing botanicals. Curr. Opin. Biotechnol. 25, 39–44 40 Ngo, L.T. et al. (2013) 21st century natural product research and drug development and traditional medicines. Nat. Prod. Rep. 30, 584–592 41 Buriani, A. et al. (2012) Omic techniques in systems biology approaches to traditional Chinese medicine research: present and future. J. Ethnopharmacol. 140, 535–544 42 Ulrich-Merzenich, G. et al. (2007) Measurement of synergistic effects of a phytopharmaceutical by microarray-analysis. Planta Med. 73 SL_026 43 Slish, D.F. et al. (1999) Ethnobotany in the search for vasoactive herbal medicines. J. Ethnopharmacol. 66, 159–165

1198

www.drugdiscoverytoday.com

Drug Discovery Today
Volume 22, Number 8
August 2017

44 Piersen, C.E. et al. (2004) Chemical and biological characterization and clinical evaluation of botanical dietary supplements: a phase I red clover extract as a model. Curr. Med. Chem. 11, 1361–1374 45 Patwardhan, B. and Mashelkar, R.A. (2009) Traditional medicine-inspired approaches to drug discovery: can Ayurveda show the way forward? Drug Discov. Today 14, 804–811 46 Butterweck, V. and Nahrstedt, A. (2012) What is the best strategy for preclinical testing of botanicals? A critical perspective. Planta Med. 78, 747–754 47 Medina-Franco, J.L. et al. (2013) Shifting from the single to the multitarget paradigm in drug discovery. Drug Discov. Today 18, 495–501 48 Smith, R.L. et al. (2005) A procedure for the safety evaluation of natural flavor complexes used as ingredients in food: essential oils. Food Chem. Toxicol. 43, 345– 363 49 SCF (2001) Opinion of the Scientific Committee on Food on Estragole (1-allyl-4methoxybenzene). Eur. Comm. Heal. Consum. Prot. Dir. https://ec.europa.eu/food/ sites/food/files/safety/docs/sci-com_scf_out104_en.pdf 50 SCF (2001) Opinion of the Scientific Committee on Food on Methyleugenol (4allyl-1,2-dimethoxybenzene). Eur. Comm. Heal. Consum. Prot. Dir. https://ec. europa.eu/food/sites/food/files/safety/docs/sci-com_scf_out102_en.pdf 51 SCF (2002) Opinion of the Scientific Committee on Food on the safety of the presence of safrole (1-allyl-3,4- methylene dioxy benzene) in flavourings and other food ingredients with flavouring properties. Eur. Comm. Heal. Consum. Prot. Dir. https://ec.europa.eu/food/sites/food/files/safety/docs/sci-com_scf_out116_en.pdf 52 Choong, Y.-M. and Lin, H.-J. (2001) A rapid and simple gas chromatographic method for direct determination of safrole in soft drinks. J. Food Drug Anal. 9, 27– 32 53 Siano, F. (2003) Determination of estragole, safrole and eugenol methyl ether in food products. Food Chem. 81, 469–475 54 Raffo, A. et al. (2013) Quantitation of tr-cinnamaldehyde, safrole and myristicin in cola-flavoured soft drinks to improve the assessment of their dietary exposure. Food Chem. Toxicol. 59, 626–635 55 Herrmann, K. et al. (2012) Identification of human and murine sulfotransferases able to activate hydroxylated metabolites of methyleugenol to mutagens in Salmonella typhimurium and detection of associated DNA adducts using UPLC-MS/ MS methods. Mutagenesis 27, 453–462 56 Islam, M.A. et al. (2015) Deconjugation of soy isoflavone glucuronides needed for estrogenic activity. Toxicol. In Vitro 29, 706–715 57 Sotoca, A.M. et al. (2010) Superinduction of estrogen receptor mediated gene expression in luciferase based reporter gene assays is mediated by a posttranscriptional mechanism. J. Steroid Biochem. Mol. Biol. 122, 204–211 58 Auld, D.S. et al. (2008) A specific mechanism for nonspecific activation in reportergene assays. ACS Chem. Biol. 3, 463–470 59 Resende, F.A. et al. (2012) Mutagenicity of flavonoids assayed by bacterial reverse mutation (Ames) test. Molecules 17, 5255–5268 60 Collins, R.A. et al. (1998) Removal of polyphenolic compounds from aqueous plant extracts using polyamide minicolumns. IUBMB Life 45, 791–796 61 Balunas, M.J. et al. (2006) Interference by naturally occurring fatty acids in a noncellular enzyme-based aromatase bioassay. J. Nat. Prod. 69, 700–703 62 Murillo-Amador, B. et al. (2013) Physiological, morphometric characteristics and yield of Origanum vulgare L. and Thymus vulgaris L. exposed to open-field and shade-enclosure. Ind. Crops Prod. 49, 659–667 63 Shale, T.L. et al. (2005) Variation in antibacterial and anti-inflammatory activity of different growth forms of Malva parviflora and evidence for synergism of the antiinflammatory compounds. J. Ethnopharmacol. 96, 325–330 64 Bolton, E.C. et al. (2002) Inhibition of reverse transcription in vivo by elevated manganese ion concentration. Mol. Cell. 9, 879–889 65 Filler, A. and Lever, A.M. (1997) Effects of cation substitutions on reverse transcriptase and on human immunodeficiency virus production. AIDS Res. Hum. Retroviruses 13, 291–299 66 Bessong, P. and Obi, C.L. (2006) Review: ethno-pharmacology of human immundeficiency virus in South Africa. African J. Biotechnol. 5, 1693–1699 67 Cos, P. et al. (2006) Anti-infective potential of natural products: how to develop a stronger in vitro ‘‘proof-of-concept”. J. Ethnopharmacol. 106, 290–302 68 Shoemaker, M. et al. (2004) Reduction of MTT by aqueous herbal extracts in the absence of cells. J. Ethnopharmacol. 93, 381–384 69 Hoek-van den Hil, E.F. et al. (2012) Interference of flavonoids with enzymatic assays for the determination of free fatty acid and triglyceride levels. Anal. Bioanal. Chem. 402, 1389–1392 70 Breinholt, V. et al. (1995) Mechanisms of chlorophyllin anticarcinogenesis against aflatoxin B1: complex formation with the carcinogen. Chem. Res. Toxicol. 8, 506– 514 71 Breinholt, V. et al. (1995) Dietary chlorophyllin is a potent inhibitor of aflatoxin B1 hepatocarcinogenesis in rainbow trout. Cancer Res. 55, 57–62

72 Breinholt, V. et al. (1999) Chlorophyllin chemoprevention in trout initiated by aflatoxin B(1) bath treatment: an evaluation of reduced bioavailability vs. target organ protective mechanisms. Toxicol. Appl. Pharmacol. 158, 141–151 73 Keshava, C. et al. (2009) Chlorophyllin significantly reduces benzo[a]pyrene [BP]DNA adduct formation and alters Cytochrome P450 1A1 and 1B1 expression and EROD activity in normal human mammary epithelial cells (NHMECs). Environ. Mol. Mutagen. 50, 134–144 74 Rietjens, I.M.C.M. et al. (2002) The pro-oxidant chemistry of the natural antioxidants vitamin C, vitamin E, carotenoids and flavonoids. Environ. Toxicol. Pharmacol. 11, 321–333 75 Brand, W. et al. (2006) Flavonoid-mediated inhibition of intestinal ABC transporters may affect the oral bioavailability of drugs, food-borne toxic compounds and bioactive ingredients. Biomed. Pharmacother. 60, 508–519 76 Al-Subeihi, A.A.A. et al. (2013) Inhibition of methyleugenol bioactivation by the herb-based constituent nevadensin and prediction of possible in vivo consequences using physiologically based kinetic modeling. Food Chem. Toxicol. 59, 564–571 77 Alhusainy, W. et al. (2010) Identification of nevadensin as an important herbbased constituent inhibiting estragole bioactivation and physiology-based biokinetic modeling of its possible in vivo effect. Toxicol. Appl. Pharmacol. 245, 179– 190 78 Alhusainy, W. et al. (2013) In vivo validation and physiologically based biokinetic modeling of the inhibition of SULT-mediated estragole DNA adduct formation in the liver of male Sprague–Dawley rats by the basil flavonoid nevadensin. Mol. Nutr. Food Res. 57, 1969–1978 79 Martati, E. et al. (2014) Malabaricone C-containing mace extract inhibits safrole bioactivation and DNA adduct formation both in vitro and in vivo. Food Chem. Toxicol. 66, 373–384 80 Deliorman Orhan, D. et al. (2007) In vivo anti-inflammatory and antinociceptive activity of the crude extract and fractions from Rosa canina L. fruits. J. Ethnopharmacol. 112, 394–400 81 Christensen, K.B. et al. (2010) Identification of bioactive compounds from flowers of black elder (Sambucus nigra L.) that activate the human peroxisome proliferatoractivated receptor (PPAR)g. Phyther. Res. 24, S129–S132 82 Bovee, T.F.H. et al. (2015) Are effects of common ragwort in the Ames test caused by pyrrolizidine alkaloids? Mutat. Res. 778, 1–10 83 Magee, P.J. (2010) Is equol production beneficial to health? Proc. Nutr. Soc. 70, 10– 18 84 Kuijsten, A. et al. (2005) Pharmacokinetics of enterolignans in healthy men and women consuming a single dose of secoisolariciresinol diglucoside. J. Nutr. 135, 795–801 85 Hullar, M.A.J. et al. (2015) Enterolignan-producing phenotypes are associated with increased gut microbial diversity and altered composition in premenopausal women in the United States. Cancer Epidemiol. Biomarkers Prev. 24, 546–554 86 Kobashi, K. et al. (1980) Metabolism of sennosides by human intestinal bacteria. Planta Med. 40, 225–236 87 Barrington, R. et al. (2009) Absorption, conjugation and efflux of the flavonoids, kaempferol and galangin, using the intestinal CaCo-2/TC7 cell model. J. Funct. Foods 1, 74–87 88 Hollman, P. (2004) Absorption, bioavailability, and metabolism of flavonoids. Pharm. Biol. 42, 74–83 89 Chen, J. et al. (2003) Metabolism of flavonoids via enteric recycling: role of intestinal disposition. J. Pharmacol. Exp. Ther. 304, 1228–1235 90 Vissiennon, C. et al. (2012) Route of administration determines the anxiolytic activity of the flavonols kaempferol, quercetin and myricetin – are they prodrugs? J. Nutr. Biochem. 23, 733–740 91 Rietjens, I.M.C.M. et al. (2011) Tutorial on physiologically based kinetic modeling in molecular nutrition and food research. Mol. Nutr. Food Res. 55, 941–956 92 Boonpawa, R. et al. (2014) A physiologically based kinetic (PBK) model describing plasma concentrations of quercetin and its metabolites in rats. Biochem. Pharmacol. 89, 287–299 93 Boonpawa, R. et al. (2017) Physiologically based kinetic modeling of hesperidin metabolism and its use to predict in vivo effective doses in humans. Mol. Nutr. Food Res. http://dx.doi.org/10.1002/mnfr.201600894 94 Cos, P. et al. (2006) Anti-infective potential of natural products: how to develop a stronger in vitro ‘‘proof-of-concept”. J. Ethnopharmacol. 106, 290–302 95 Gertsch, J. (2009) How scientific is the science in ethnopharmacology? Historical perspectives and epistemological problems. J. Ethnopharmacol. 122, 177–183 96 Beekmann, K. et al. (2012) A state-of-the-art overview of the effect of metabolic conjugation on the biological activity of flavonoids. Food Funct. 3, 1008–1018 97 Beekmann, K. et al. (2015) The effect of glucuronidation on isoflavone induced estrogen receptor (ER)a and ERb mediated coregulator interactions. J. Steroid Biochem. Mol. Biol. 154, 245–253

REVIEWS

98 Manach, C. et al. (2004) Polyphenols: food sources and bioavailability. Am. J. Clin. Nutr. 79, 727–747 99 Manach, C. et al. (1998) Quercetin is recovered in human plasma as conjugated derivatives which retain antioxidant properties. FEBS Lett. 426, 331–336 100 Fraga, C.G. et al. (2014) In vitro measurements and interpretation of total antioxidant capacity. Biochim. Biophys. Acta 1840, 931–934 101 Hollman, P.C.H. et al. (2011) The biological relevance of direct antioxidant effects of polyphenols for cardiovascular health in humans is not established. J. Nutr. 141, 989–1009S 102 OECD (2007) OECD test no. 440: uterotrophic bioassay in rodents. Organ. Econ. Cooperation Dev. Publ. https://ntp.niehs.nih.gov/iccvam/suppdocs/feddocs/oecd/ oecdtg440.pdf 103 Wang, S. et al. (2014) Towards an integrated in vitro strategy for estrogenicity testing. J. Appl. Toxicol. 34, 1031–1040 104 Wetmore, B.A. et al. (2012) Integration of dosimetry, exposure, and high-throughput screening data in chemical toxicity assessment. Toxicol. Sci. 125, 157–174 105 Fernandez Moreno, J.L. et al. (2008) Multiresidue method for the analysis of more than 140 pesticide residues in fruits and vegetables by gas chromatography coupled to triple quadrupole mass spectrometry. J. Mass Spectrom. 43, 1235–1254 106 Tuan, S.-J. et al. (2009) Multiresidue analysis of 176 pesticides and metabolites in pre-harvested fruits and vegetables for ensuring food safety by gas chromatography and high performance liquid chromatography. J. Food Drug Anal. 17, 163–177 107 Wong, J.W. et al. (2011) Multiresidue pesticide analysis by capillary gas chromatography-mass spectrometry. Methods Mol. Biol. 747, 131–172 108 Munoz, E. et al. (2012) Multiresidue method for pesticide residue analysis in food of animal and plant origin based on GC or LC and MS or MS/MS. J. AOAC Int. 95, 1777–1796 109 Dewey, J.E. (1958) Pesticide residues, utility of bioassay in the determination of pesticide residues. J. Agric. Food. Chem. 6, 274–281 110 Needham, P.H. (1960) An investigation into the use of bioassay for pesticide residues in foodstuffs. Analyst 85, 792–809 111 Bowman, M.C. et al. (1982) Stressed bioassay systems for rapid screening of pesticide residues. Part II: determination of foliar residues for safe reentry of agricultural workers into the field. Arch. Environ. Contam. Toxicol. 11, 447–455 112 Johnson, I.S. et al. (1963) The Vinca alkaloids: a new class of oncolytic agents. Cancer Res. 23, 1390–1427 113 Behnisch, P.A. et al. (2002) Screening of dioxin-like toxicity equivalents for various matrices with wildtype and recombinant rat hepatoma H4IIE cells. Toxicol. Sci. 69, 125–130 114 Weller, M.G. (2012) A unifying review of bioassay-guided fractionation, effectdirected analysis and related techniques. Sensors (Basel) 12, 9181–9209 115 Kind, T. and Fiehn, O. (2007) Seven golden rules for heuristic filtering of molecular formulas obtained by accurate mass spectrometry. BMC Bioinformatics 8, 105 116 Chemical abstract service. Available at: https://www.cas.org/content/at-a-glance 117 Moco, S. et al. (2007) Metabolomics technologies and metabolite identification. Trends Anal. Chem. 26, 855–866 118 van der Hooft, J.J.J. et al. (2012) Spectral trees as a robust annotation tool in LC-MS based metabolomics. Metabolomics 8, 691–703 119 Rojas-cherto, M. et al. (2012) Metabolite identification using automated comparison of high-resolution multistage mass spectral trees. Anal. Chem. 84, 5524–5534 120 Yi, L. et al. (2016) Chemometric methods in data processing of mass spectrometrybased metabolomics: a review. Anal. Chim. Acta 914, 17–34 121 Moco, S. et al. (2008) Intra- and inter-metabolite correlation spectroscopy of tomato metabolomics data obtained by liquid chromatography-mass spectrometry and nuclear magnetic resonance. Metabolomics 4, 202–215 122 Manzocco, L. et al. (1998) Antioxidant properties of tea extracts as affected by processing. LWT Food Sci. Technol. 31, 694–698 123 Ruch, R.J. et al. (1989) Prevention of cytotoxicity and inhibition of intercellular communication by antioxidant catechins isolated from Chinese green tea. Carcinogenesis 10, 1003–1008 124 Marcocci, L. et al. (1994) The nitric oxide-scavenging properties of Ginkgo Biloba extract EGb 761. Biochem. Biophys. Res. Commun. 201, 748–755 125 Seeram, N.P. et al. (2006) Catechin and caffeine content of green tea dietary supplements and correlation with antioxidant capacity. J. Agric. Food. Chem. 54, 1599–1603 126 Ghiselli, A. et al. (1995) A fluorescence-based method for measuring total plasma antioxidant capability. Free Radic. Biol Med. 18, 29–36 127 Oxidants and Antioxidants Part A. Benzie, I.F.F. and Strain, J.J., eds), 1999.Elsevier 128 Prior, R.L. et al. (2003) Assays for hydrophilic and lipophilic antioxidant capacity (oxygen radical absorbance capacity (ORAC(FL))) of plasma and other biological and food samples. J. Agric. Food. Chem. 51, 3273–3279

www.drugdiscoverytoday.com

1199

Reviews
KEYNOTE REVIEW

Drug Discovery Today
Volume 22, Number 8
August 2017

REVIEWS

Reviews
KEYNOTE REVIEW

129 Eldeen, I.M.S. et al. (2011) In vitro antibacterial, antioxidant, total phenolic contents and anti-HIV-1 reverse transcriptase activities of extracts of seven Phyllanthus sp. South African J. Bot. 77, 75–79 130 Kapewangolo, P. et al. (2013) Inhibition of HIV-1 enzymes, antioxidant and antiinflammatory activities of Plectranthus barbatus. J. Ethnopharmacol. 149, 184–190 131 Klos, M. et al. (2009) In vitro anti-HIV activity of five selected South African medicinal plant extracts. J. Ethnopharmacol. 124, 182–188 132 Tshikalange, T.E. et al. (2008) In vitro anti-HIV-1 properties of ethnobotanically selected South African plants used in the treatment of sexually transmitted diseases. J. Ethnopharmacol. 119, 478–481 133 Curto, E.V. et al. (1999) Inhibitors of mammalian melanocyte tyrosinase: in vitro comparisons of alkyl esters of gentisic acid with other putative inhibitors. Biochem. Pharmacol. 57, 663–672 134 Ellman, G.L. et al. (1961) A new and rapid colorimetric determination of acetylcholinesterase activity. Biochem. Pharmacol. 7, 88–95 135 Momtaz, S. et al. (2008) Inhibitory activities of mushroom tyrosine and DOPA oxidation by plant extracts. South African J. Bot. 74, 577–582 136 Chukwujekwu, J.C. et al. (2014) Antiplasmodial, HIV-1 reverse transcriptase inhibitory and cytotoxicity properties of Centratherum punctatum Cass. and its fractions. South African J. Bot. 90, 17–19

1200

www.drugdiscoverytoday.com

Drug Discovery Today
Volume 22, Number 8
August 2017

137 Aremu, A.O. et al. (2010) In vitro antimicrobial, anthelmintic and cyclooxygenaseinhibitory activities and phytochemical analysis of Leucosidea sericea. J. Ethnopharmacol. 131, 22–27 138 Newton, S.M. et al. (2002) The evaluation of forty-three plant species for in vitro antimycobacterial activities; isolation of active constituents from Psoralea corylifolia and Sanguinaria canadensis. J. Ethnopharmacol. 79, 57–67 139 OECD (1997) In vitro mammalian cell gene mutation test. Organ. Econ. Co-operation Dev. Publ. http://dx.doi.org/10.1787/9789264071322-en 140] Aarts, J.M.M.J.G. et al. (1995) Species-specific antagonism of Ah receptor action by 2,20 ,5,50 -tetrachloro- and 2,20 ,3,30 ,4,40 -hexachlorobiphenyl. Eur. J. Pharmacol. Environ. Toxicol. Pharmacol. 293, 463–474 141 van der Burg, B. et al. (2013) A panel of quantitative Calux1 reporter gene assays for reliable high-throughput toxicity screening of chemicals and complex mixtures. In High-Throughput Screening Methods in Toxicity Testing (Steinberg, P.O., ed.), pp. 519–532, John Wiley & Sons 142 Boonpawa, R. et al. (2017) In vitro–in silico based analysis of the dose-dependent in vivo estrogenicity of the soy phytoestrogen genistein in humans. Br. J. Pharmacol. (accepted)