Induction of peroxisome proliferator activated receptor γ (PPARγ) mediated gene expression and inhibition of induced nitric oxide production by Maerua subcordata (Gilg) DeWolf.

BACKGROUND: The health benefits of botanicals is linked to their phytochemicals that often exert pleiotropic effects via targeting multiple molecular signaling pathways such as the peroxisome proliferator-activated receptors (PPARs) and the nuclear factor kappaB (NFκB). The PPARs are transcription factors that control metabolic homeostasis and inflammation while the NF-κB is a master regulator of inflammatory genes such as the inducible nitric-oxide synthase that result in nitric oxide (NO) overproduction. METHODS: Extracts of Maerua subcordata (MS) and selected candidate constituents thereof, identified by liquid chromatography coupled to mass spectroscopy, were tested for their ability to induce PPARγ mediated gene expression in U2OS-PPARγ cells using luciferase reporter gene assay and also for their ability to inhibit lipopolysaccharide (LPS) induced NO production in RAW264.7 macrophages. While measuring the effect of test samples on PPARγ mediated gene expression, a counter assay that used U2OS-Cytotox cells was performed to monitor cytotoxicity or any non-specific changes in luciferase activity. RESULTS: The results revealed that the fruit, root, and seed extracts were non-cytotoxic up to a concentration of 30 g dry weight per litre (gDW/L) and induced PPARγ mediated gene expression but the leaf extract showed some cytotoxicity and exhibited minimal induction. Instead, all extracts showed concentration (1-15 gDW/L) dependent inhibition of LPS induced NO production. The root extract showed weaker inhibition. Among the candidate constituents, agmatine, stachydrine, trigonelline, indole-3-carboxyaldehyde, plus ethyl-, isobutyl-, isopropyl, and methyl-isothiocyanates showed similar inhibition, and most showed increased inhibition with increasing concentration (1-100 µM) although to a lesser potency than the positive control, aminoguanidine. CONCLUSION: The present study demonstrated for the first time the induction of PPARγ mediated gene expression by MS fruit, root, and seed extracts and the inhibition of LPS induced NO production by MS fruit, leaf, root, and seed extracts and some candidate constituents thereof.

The role of metabolism in the developmental toxicity of polycyclic aromatic hydrocarbon-containing extracts of petroleum substances.

In vitro assays presently used for prenatal developmental toxicity (PDT) testing only assess the embryotoxic potential of parent substances and not that of potentially embryotoxic metabolites. Here we combined a biotransformation system, using hamster liver microsomes, with the ES-D3 cell differentiation assay of the embryonic stem cell test (EST) to compare the in vitro PDT potency of two 5-ring polycyclic aromatic hydrocarbons (PAHs), benzo[a]pyrene (BaP) and dibenz[a,h]anthracene (DBA), and dimethyl sulfoxide extracts from five PAH-containing petroleum substances (PS) and a gas-to-liquid base oil (GTLb), with and without bioactivation. In the absence of bioactivation, DBA, but not BaP, inhibited the differentiation of ES-D3 cells into beating cardiomyocytes in a concentration-dependent manner. Upon bioactivation, BaP induced in vitro PDT, while its major metabolite 3-hydroxybenzo[a]pyrene was shown to be active in the EST as well. This means BaP needs biotransformation to exert its embryotoxic effects. GTLb extracts tested negative in the EST, with and without bioactivation. The PS-induced PDT in the EST was not substantially changed following bioactivation, implying that metabolism may not play a crucial role for the PS extracts under study to exert the in vitro PDT effects. Altogether, these results indicate that although some PAH require bioactivation to induce PDT, some do not and this latter appears to hold for the (majority of) the PS constituents responsible for the in vitro PDT of these complex substances.

Hazard assessment of Maerua subcordata (Gilg) DeWolf. for selected endpoints using a battery of in vitro tests.

ETHNOPHARMACOLOGICAL RELEVANCE: Maerua subcordata (Gilg) DeWolf is a medicinal and wild food plant growing mainly in east Africa. Especially its root tuber is widely used in traditional medicine to treat several infectious and chronic diseases but also in some toxicity implications like use as abortifacient. AIM OF THE STUDY: the present study applied in silico and in vitro tests to identify possible hazards of M. subcordata (fruit, leaf, root, seed) methanol extracts focussing on developmental toxicity. MATERIALS AND METHODS: Ames test, estrogen receptor alpha (ERα) assay, aryl hydrocarbon receptor (AhR) assay, embryonic stem cell test (EST), and zebrafish embryotoxicity test (ZET) were employed. Besides, a Derek Nexus toxicity prediction was performed on candidate structures obtained from metabolomics profiling of the extracts using liquid chromatography coupled to multistage mass spectroscopy (LC/MSn) and a MAGMa software based structural annotation. RESULTS: Glucosinolates, which degrade to isothiocyanates, and biogenic amines were among the candidate molecules identified in the extracts by LC/MSn - MAGMa software structural annotation. Isothiocyanates and some other candidate molecules suggested a positive mutagenicity alert in Derek toxicity predictions. All the extracts showed negative mutagenicity in the Ames test. However, the Derek predictions also identified endocrine and developmental toxicity as possible endpoints of concern. This was further assessed using in vitro tests. Results obtained reveal that leaf extract shows AhR and ERα agonist activities, inhibited differentiation of ES-D3 stem cells into contracting cardiomyocytes in the EST (p < 0.001) as well as inhibited hatching (p < 0.01) and showed acute toxicity (p < 0.01) in the ZET. Also, the fruit extract showed toxicity (p < 0.05) towards zebrafish embryos and both fruit and seed extracts showed AhR agonist activities while root extract was devoid of activity in all in vitro assays. CONCLUSION: The leaf extract tests positive in in vitro tests that may point towards a developmental toxicity hazard. The current evaluations did not raise concerns of genotoxicity or developmental toxicity for the fruit, seed and root extracts. This is important given the use of especially these parts of M. subcordata, in traditional medicine and/or as (famine) food.

Effects of Maerua subcordata (Gilg) DeWolf on electrophile-responsive element (EpRE)-mediated gene expression in vitro.

Plant extracts and phytochemicals may prevent chronic diseases via activation of adaptive cellular stress response pathways including induction of antioxidant and phase II detoxifying enzymes. The regulatory regions of these inducible genes encode the electrophile-response element (EpRE). This study tested the EpRE induction ability of Maerua subcordata (fruit, leaf, root, seed) methanol extracts and selected candidate constituents thereof, identified by liquid chromatography coupled with multistage mass spectroscopy, employing an EpRE luciferase reporter gene assay using hepa-1c1c7 mouse hepatoma cells. A parallel Cytotox CALUX assay using human osteosarcoma U2OS cells was used to monitor any non-specific changes in luciferase activity or cytotoxicity. Results showed that fruit, root, and seed extracts were non-cytotoxic up to a concentration of 30 gram dry weight per litre but the leaf extract exhibited some cytotoxicity and that the leaf (despite some cytotoxicity), fruit, and seed extracts showed strong induction of EpRE mediated gene expression while induction by the root extract was minimal. Selected candidates included glucosinolates, isothiocyanates, and some biogenic amines. Subsequent studies showed that methyl-, ethyl-, isopropyl-, isobutyl- isothiocyanates, and sec-butyl thiocyanate as well as glucobrassicin induced concentration (1-100 µM) dependent EpRE-mediated gene expression while the biogenic amines stachydrine and trigonelline acted as inhibitors of EpRE-mediated gene expression at 100 µM. The identification of glucolepidiin, glucobrassicin, glucocapparin, stachydrine, and trigonelline in all extracts was confirmed using standards and based on multiple reaction monitoring; yet, glucobrassicin level in the root extract was negligible. In conclusion, this study provided a first report on EpRE mediated gene expression effects of M. subcordata; and despite detection of different glucosinolates in all extracts, those containing glucobrassicin particularly displayed high EpRE induction. Because EpRE inducers are cytoprotective and potential chemopreventive agents while inhibitors are suggested adjuvants of chemotherapy, results of this study imply that process manipulation of this plant may result in herbal preparations that may be used as chemopreventive agents or adjuvants of chemotherapies.

Conversion of major soy isoflavone glucosides and aglycones in in vitro intestinal models.

SCOPE: This study compares conversion of three major soy isoflavone glucosides and their aglycones in a series of in vitro intestinal models. METHODS AND RESULTS: In an in vitro human digestion model isoflavone glucosides were not deconjugated, whereas studies in a Caco-2 transwell model confirmed that deconjugation is essential to facilitate transport across the intestinal barrier. Deconjugation was shown upon incubation of the isoflavone glucosides with rat as well as human intestinal S9. In incubations with rat intestinal S9 lactase phlorizin hydrolase, glucocerebrosidase, and cytosolic broad-specific ß-glucosidase all contribute significantly to deconjugation, whereas in incubations with human intestinal S9 deconjugation appeared to occur mainly through the activity of broad-specific ß-glucosidase. Species differences in glucuronidation and sulfation were limited and generally within an order of magnitude with 7-O-glucuronides being the major metabolites for all three isoflavone aglycones and the glucuronidation during first pass metabolism being more efficient in rats than in humans. Comparison of the catalytic efficiencies reveals that deconjugation is less efficient than conjugation confirming that aglycones are unlikely to enter the systemic circulation. CONCLUSION: Altogether, the data point at possible differences in the characteristics for intestinal conversion of the major soy isoflavones between rat and human, especially with respect to their deconjugation.

An n-3 PUFA-rich microalgal oil diet protects to a similar extent as a fish oil-rich diet against AOM-induced colonic aberrant crypt foci in F344 rats.

The chemopreventive effects of high fat microalgal oil diet on azoxymethane (AOM)-induced colonic aberrant crypt foci (ACF) were studied in male Fischer 344 rats following 8 weeks of dietary treatment. These effects were compared to the effects of high fat fish oil and high fat corn oil diets to determine whether microalgal oil is a good alternative for fish oil regarding protection against colorectal cancer. Despite the difference in fatty acid composition and total amount of n-3 polyunsaturated fatty acids (PUFAs) between microalgal oil and fish oil, both these oils gave the same 50% reduction of AOM-induced ACF when compared to corn oil. To determine whether oxidative stress could play a role in the chemoprevention of colorectal cancer by n-3 PUFAs, feces and caecal content were examined using the TBA assay. The results showed that lipid peroxidation does occur in the gastrointestinal tract. As several lipid peroxidation products of n-3 PUFAs can induce phase II detoxifying enzymes by an EpRE-mediated pathway, the in vivo results suggest that this route may contribute to n-3 PUFA-mediated chemoprevention. All in all, n-3 PUFA-rich oil from microalgae is as good as fish oil regarding chemoprevention in the colon of the rat.

The pro-oxidant chemistry of the natural antioxidants vitamin C, vitamin E, carotenoids and flavonoids.

Natural antioxidants like vitamin C, vitamin E, carotenoids, and polyphenols like flavonoids, are at present generally considered to be beneficial components from fruit and vegetables. The anti-oxidative properties of these compounds are often claimed to be responsible for various beneficial health effects of these food ingredients. Together these studies provide the basis for the present rapidly increasing interest for the use of natural antioxidants as functional food ingredients and/or as food supplements. However, at higher doses or under certain conditions antioxidant-type functional food ingredients may exert toxic pro-oxidant activities. The present manuscript gives an overview of especially this pro-oxidative chemistry and toxicity of well-known natural antioxidants including vitamin C, vitamin E, carotenoids and flavonoids.