Phytochemical characterization and bioactivity toward breast cancer cells of unhydrolyzed and acid-hydrolyzed extracts of Fagonia indica.

Phytochemicals from the genus, Fagonia, have been attracting increasing attention due to their potential beneficial effects on human health. Fagonia species contain various types of phytochemicals such as flavonoids, alkaloids, saponins, terpenoids, coumarins and tannins. In this study, we investigated the phytochemical composition of unhydrolyzed and acid-hydrolyzed extracts of Fagonia indica and their bioactivity toward breast cancer MCF-7 cells in vitro. The results revealed that F. indica contains phytochemicals consistent with the reported phytochemical composition of this Fagonia species, with greater amounts of aglycones detected in the hydrolyzed extract. The crude extract of F. indica without acid hydrolysis was found to be ineffective in inhibiting the growth of MCF-7 cells at doses below 1000 µg/mL. However, after acid hydrolysis (to mimic gastro-intestinal hydrolysis), the F. indica extract became growth-inhibitory to MCF-7 cells as low as 10 µg/mL and the cytotoxicity increased with increasing dose and time of treatment. The results suggest that F. indica extracts contain phytochemicals in glycosidic forms whose aglycones are active as anti-proliferative agents toward breast cancer cells in vitro.

Computation-Assisted Identification of Bioactive Compounds in Botanical Extracts: A Case Study of Anti-Inflammatory Natural Products from Hops.

The slow pace of discovery of bioactive natural products can be attributed to the difficulty in rapidly identifying them in complex mixtures such as plant extracts. To overcome these hurdles, we explored the utility of two machine learning techniques, i.e., Elastic Net and Random Forests, for identifying the individual anti-inflammatory principle(s) of an extract of the inflorescences of the hops (Humulus lupulus) containing hundreds of natural products. We fractionated a hop extract by column chromatography to obtain 40 impure fractions, determined their anti-inflammatory activity using a macrophage-based bioassay that measures inhibition of iNOS-mediated formation of nitric oxide, and characterized the chemical composition of the fractions by flow-injection HRAM mass spectrometry and LC-MS/MS. Among the top 10 predictors of bioactivity were prenylated flavonoids and humulones. The top Random Forests predictor of bioactivity, xanthohumol, was tested in pure form in the same bioassay to validate the predicted result (IC50 7 µM). Other predictors of bioactivity were identified by spectral similarity with known hop natural products using the Global Natural Products Social Networking (GNPS) algorithm. Our machine learning approach demonstrated that individual bioactive natural products can be identified without the need for extensive and repetitive bioassay-guided fractionation of a plant extract.

Vitamin C Activates the Folate-Mediated One-Carbon Cycle in C2C12 Myoblasts.

Vitamin C (L-ascorbic acid, AA) is an essential cellular antioxidant and cofactor for several α-ketoglutarate-dependent dioxygenases. As an antioxidant, AA interacts with vitamin E to control oxidative stress. While several reports suggest an interaction of AA with folate (vitamin B9) in animals and humans, little is known about the nature of the interaction and the underlying molecular mechanisms at the cellular level. We used an untargeted metabolomics approach to study the impact of AA on the metabolome of C2C12 myoblast cells. Compared to untreated cells, treatment of C2C12 cells with AA at 100 µM resulted in enhanced concentrations of folic acid (2.5-fold) and 5-methyl-tetrahydrofolate (5-methyl-THF, 10-fold increase) whereas the relative concentrations of 10-formyl-tetrahydrofolate decreased by >90% upon AA pretreatment, indicative of increased utilization for the biosynthesis of active THF metabolites. The impact of AA on the folate-mediated one-carbon cycle further manifested itself as an increase in the levels of methionine, whose formation from homocysteine is 5-methyl-THF dependent, and an increase in thymidine, whose formation from deoxyuridine monophosphate (dUMP) is dependent on 5,10-methylene-THF. These findings shed new light on the interaction of AA with the folate-mediated one-carbon cycle and partially explain clinical findings that AA supplementation enhances erythrocyte folate status and that it may decrease serum levels of homocysteine, which is considered as a biomarker of cardiovascular disease risk.

Phytochemical characterization of Tabernanthe iboga root bark and its effects on dysfunctional metabolism and cognitive performance in high-fat-fed C57BL/6J mice.

Preparations of the root bark of Tabernanthe iboga have long been used in Central and West African traditional medicine to combat fatigue, as a neuro-stimulant in rituals, and for treatment of diabetes. The principal alkaloid of T. iboga, ibogaine, has attracted attention in many countries around the world for providing relief for opioid craving in drug addicts. Using a plant metabolomics approach, we detected five phenolic compounds, including 3-O-caffeoylquinic acid, and 30 alkaloids, seven of which were previously reported from T. iboga root bark. Following a report that iboga extracts contain insulinotropic agents, we aimed to determine the potential alleviating effects of the water extract of iboga root bark on high-fat diet (HFD)-induced hyperglycemia as well as its effects on cognitive function in male C57BL/6J mice. Feeding a HFD to mice for 10 weeks produced manifestations of metabolic syndrome such as increased body weight and increased plasma levels of glucose, triacylglycerols, total cholesterol, LDL-cholesterol, insulin, leptin, and pro-inflammatory mediators (IL-6, MCP-1, ICAM-1), as compared to mice fed a low-fat diet (LFD). Supplementation of HFD with iboga extract at ibogaine doses of 0.83 (low) and 2.07 (high) mg/kg/day did not improve these HFD-induced metabolic effects except for a reduction of plasma MCP-1 in the low dose group, indicative of an anti-inflammatory effect. When the HFD mice were tested in the water maze, the high-dose iboga extract caused hippocampus-dependent impairments in spatial learning and memory, as compared to mice receiving only a HFD.

Vitamin C deficiency activates the purine nucleotide cycle in zebrafish.

Vitamin C (ascorbic acid, AA) is a cofactor for many important enzymatic reactions and a powerful antioxidant. AA provides protection against oxidative stress by acting as a scavenger of reactive oxygen species, either directly or indirectly by recycling of the lipid-soluble antioxidant, α-tocopherol (vitamin E). Only a few species, including humans, guinea pigs, and zebrafish, cannot synthesize AA. Using an untargeted metabolomics approach, we examined the effects of α-tocopherol and AA deficiency on the metabolic profiles of adult zebrafish. We found that AA deficiency, compared with subsequent AA repletion, led to oxidative stress (using malondialdehyde production as an index) and to major increases in the metabolites of the purine nucleotide cycle (PNC): IMP, adenylosuccinate, and AMP. The PNC acts as a temporary purine nucleotide reservoir to keep AMP levels low during times of high ATP utilization or impaired oxidative phosphorylation. The PNC promotes ATP regeneration by converting excess AMP into IMP, thereby driving forward the myokinase reaction (2ADP → AMP + ATP). On the basis of this finding, we investigated the activity of AMP deaminase, the enzyme that irreversibly deaminates AMP to form IMP. We found a 47% increase in AMP deaminase activity in the AA-deficient zebrafish, complementary to the 44-fold increase in IMP concentration. These results suggest that vitamin C is crucial for the maintenance of cellular energy metabolism.

Xanthohumol and related prenylated flavonoids inhibit inflammatory cytokine production in LPS-activated THP-1 monocytes: structure-activity relationships and in silico binding to myeloid differentiation protein-2 (MD-2).

Xanthohumol (XN) is a prenylated chalcone-type flavonoid found in hops and beer. Our objective of this study was to determine the anti-inflammatory activities of XN, isoxanthohumol (IX), and 15 related prenylated chalcones and flavanones, as well as their structure-activity relationships. The anti-inflammatory activities of the flavonoids were measured by their ability to inhibit lipopolysaccharide (LPS)-induced cytokine production in human monocytic THP-1 cells. The position, number, and length of the prenyl groups had a marked influence on the inhibitory activity of the prenylfavonoids towards MCP-1 and IL-6 production. The α,ß-unsaturated carbonyl moiety present in chalcones such as XN was not an absolute requirement for inhibitory activity, as the saturated XN derivative, tetrahydroxanthohumol (TX), showed inhibitory activity comparable to XN. With the aim to determine the mechanism of the observed anti-inflammatory effects, cellular protein levels of Toll-like receptor 4 (TLR4) were measured by Western blot 24 h following coexposure of THP-1 cells to LPS and either XN, TX, or IX. Only XN reduced the cellular TLR4 protein content. Therefore, an additional hypothesis was developed for an anti-inflammatory mechanism that involves the TLR4 coreceptor myeloid differentiation protein-2 (MD-2), which provides the actual binding site for LPS. Molecular docking studies showed that the complementarity of prenylated flavonoids with the hydrophobic MD-2 pocket (indicating goodness of fit) directly predicted their relative ability to inhibit MCP-1 and IL-6 production. In conclusion, prenylated flavonoids may suppress LPS-induced TLR4 activation at least partly by interfering with LPS binding to the TLR4 coreceptor MD-2, and XN (but not other prenylflavonoids) exerts an additional anti-inflammatory effect by downregulating the cellular TLR4 protein content.

Epigallocatechin gallate (EGCG) potentiates the cytotoxicity of rotenone in neuroblastoma SH-SY5Y cells.

Exposure to rotenone, a widely used pesticide, has been suggested to increase the risk of developing Parkinson's disease. Studies indicate that the neurotoxicity of rotenone may be related to its ability to generate reactive oxygen species. The present work was conducted to determine to what extent (-)-epigallocatechin-3-gallate (EGCG), a widely used dietary supplement, modulates the cytotoxicity of rotenone in human neuroblastoma SH-SY5Y cells. Our results indicate that EGCG shows concentration-dependent effects on ROS production and cytotoxicity in SH-SY5Y cells. Treatment of these dopaminergic cells with rotenone (1-50 microM) alone or EGCG (25 or 50 microM) alone caused a significant decrease in cell viability. Pretreatment of SH-SY5Y cells with 25 or 50 microM EGCG potentiated the cytotoxicity of rotenone. The exacerbating effect of EGCG on rotenone toxicity may involve an apoptotic mechanism as shown by the enhancement of caspase-3 activity and activation of other caspases in rotenone-treated SH-SY5Y cells. The potentiating effect of EGCG on rotenone toxicity may be attributed to the enhanced production of intracellular superoxide in SH-SY5Y cells. The enhanced intracellular production of ROS by rotenone-EGCG combination may also account for the increased formation of protein carbonyls in 10,000xg fraction of SH-SY5Y cells detected by anti-HNE antibody. For instance, core histones and nuclear ribonuclear proteins were identified as major putative in vivo targets of HNE. Our present findings indicate that more detailed mechanistic studies are necessary to fully understand the chemistry of EGCG and to justify its use as potentially health-promoting dietary supplement, e.g. in the prevention of neurodegenerative diseases associated with oxidative stress.