A multistage human gastrointestinal model was used to digest a polyphenol-rich potato extract containing chlorogenic acid, caffeic acid, ferulic acid, and rutin as the primary polyphenols, to assess for their microbial biotransformation and to measure changes in antioxidant capacity in up to 24 h of digestion. The biotransformation of polyphenols was assessed by liquid chromatography-mass spectrometry. Antioxidant capacity was measured by the ferric reducing antioxidant power (FRAP) assay. Among the colonic reactors, parent (poly)phenols were detected in the ascending (AC), but not the transverse (TC) or descending (DC) colons. The most abundant microbial phenolic metabolites in all colonic reactors included derivatives of propionic acid, acetic acid, and benzoic acid. As compared to the baseline, an earlier increase in antioxidant capacity (T = 8 h) was seen in the stomach and small intestine vessels as compared to the AC (T = 16 h) and TC and DC (T = 24 h). The increase in antioxidant capacity observed in the DC and TC can be linked to the accumulation of microbial smaller-molecular-weight phenolic catabolites, as the parent polyphenolics had completely degraded in those vessels. The colonic microbial digestion of potato-based polyphenols could lead to improved colonic health, as this generates phenolic metabolites with significant antioxidant potential.

This study aimed to study the biotransformation of indigenous northern Thai purple rice using β-glucosidase-producing Lactobacillus (BGPL) to increase the content of bioactive anthocyanin for colorectal chemoprevention and immunization. BGPL, namely, Lactobacillus FR 332, was first isolated from Thai fermented foods. Indigenous northern Thai purple rice, namely, Khao' Gam Leum-Phua (KGLP), was selected to study bioactive anthocyanin using biotransformation by L. plantarum FR332 according to the highest amounts of cyanidin-3-glucoside. The determination of anthocyanin quantities revealed that the highest cyanidin was detected after 12 h of biotransformation, corresponding to the highest β-glucosidase activity of L. plantarum FR332 and a decrease in cyanidin-3-glucoside. The anthocyanin extract, after 12 h of biotransformation, exhibited the most potent in vitro antioxidative activity. Additionally, it showed potent anti-inflammatory activity by inhibiting cyclooxygenase-2 (COX-2), nitric oxide, and inducible nitric oxide synthase (iNOS) production in interferon-γ-stimulated colon adenocarcinoma (HT-29) cells without exerting cytotoxicity. Moreover, it also showed a potent inhibitory effect on proinflammatory cytokine interleukin-6 (IL-6) secretion and an induction effect on anti-inflammatory cytokine IL-10 secretion. These documents highlight the potential to be used of the anthocyanin extract after 12 h of biotransformation by L. plantarum FR332 as a natural active pharmaceutical ingredient (NAPI) for colorectal chemoprevention and immunization.

Antioxidants protect the structural and functional components in organisms against oxidative stress. Most antioxidants are of plant origin as the plants are permanently exposed to oxidative stress (UV radiation, photosynthetic reactions). Both carotenoids and flavonoids are prominent antioxidant and anti-radical agents often occurring together in the plant tissues and acting in lipophilic and hydrophilic milieu, respectively. They are complementary in their anti-radical activity. This study describes the synthesis of a series of hybrid ester conjugates of retinoic acid with various flavonolignans, such as silybin, 2,3-dehydrosilybin and isosilybin. Antioxidant/anti-radical activities and bio-physical properties of novel covalent carotenoid-flavonoid hybrids, as well as various mixtures of the respective parent components, were investigated. Retinoyl conjugates with silybin-which is the most important flavonolignan in silymarin complex-(and its pure diastereomers) displayed better 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging activity than both the parent compounds and their equimolar mixtures.

Silychristin A is the second most abundant compound of silymarin. Silymarin complex was previously described as an antioxidant with multidrug resistance modulation activity. Here, the results of a classical biochemical antioxidant assay (ORAC) were compared with a cellular assay evaluating the antioxidant capacity of pure silychristin A and its derivatives (anhydrosilychristin, isosilychristin and 2,3-dehydrosilychristin A). All the tested compounds acted as antioxidants within the cells, but 2,3-dehydro- and anhydro derivatives were almost twice as potent as the other tested compounds. Similar results were obtained in LPS-stimulated macrophages, where 2,3-dehydro- and anhydrosilychristin inhibited NO production nearly twice as efficiently as silychristin A. The inhibition of P-glycoprotein (P-gp) was determined in vitro, and the respective sensitization of doxorubicin-resistant ovarian carcinoma overproducing P-gp was detected. Despite the fact that the inhibition of P-gp was demonstrated in a concentration-dependent manner for each tested compound, the sensitization of the resistant cell line was observed predominantly for silychristin A and 2,3-dehydrosilychristin A. However, anhydrosilychristin and isosilychristin affected the expression of both the P-gp (ABCB1) and ABCG2 genes. This is the first report showing that silychristin A and its 2,3-dehydro-derivative modulate multidrug resistance by the direct inhibition of P-gp, in contrast to anhydrosilychristin and isosilychristin modulating multidrug resistance by downregulating the expression of the dominant transmembrane efflux pumps.

Silybin is considered to be the main biologically active component of silymarin. Its oxidized derivative 2,3-dehydrosilybin typically occurs in silymarin in small, but non-negligible amounts (up to 3%). Here, we investigated in detail complex biological activities of silybin and 2,3-dehydrosilybin optical isomers. Antioxidant activities of pure stereomers A and B of silybin and 2,3-dehydrosilybin, as well as their racemic mixtures, were investigated by using oxygen radical absorption capacity (ORAC) and cellular antioxidant activity (CAA) assay. All substances efficiently reduced nitric oxide production and cytokines (TNF-α, IL-6) release in a dose-dependent manner. Multidrug resistance (MDR) modulating potential was evaluated as inhibition of P-glycoprotein (P-gp) ATPase activity and regulation of ATP-binding cassette (ABC) protein expression. All the tested compounds showed strong dose-dependent inhibition of P-gp pump. Moreover, 2,3-dehydrosilybin A (30 µM) displayed the strongest sensitization of doxorubicin-resistant ovarian carcinoma. Despite these significant effects, silybin B was the only compound acting directly upon P-gp in vitro and also downregulating the expression of respective MDR genes. This compound altered the expression of P-glycoprotein (P-gp, ABCB1), multidrug resistance-associated protein 1 (MRP1, ABCC1) and breast cancer resistance protein (BCRP, ABCG2). 2,3-Dehydrosilybin AB exhibited the most effective inhibition of acetylcholinesterase activity. We can clearly postulate that silybin derivatives could serve well as modulators of a cancer drug-resistant phenotype.

The antioxidant activity and the chemical composition of methanol extracts from peel and pulp belonging to two species of Tunisian prickly pears Opuntia ficus indica (spiny and thornless forms) and Opuntia stricta have been studied. The antioxidant capacity was measured by DPPH radical scavenging activity. The total phenolic compound (TPC) and the total flavonoid content were determined by the Folin-Ciocalteu method and colorimetric method, respectively. The phenolic compounds were identified and quantified by high-performance liquid chromatography (HPLC) coupled with an electrospray ionization mass spectrometry (ESI-MS). The results showed that O. stricta fruits present the best antioxidant activities than the two forms of O. ficus indica, while the TPC was more important in O. ficus indica than in the O. stricta fruits. The peels have higher flavonoids than pulp, and the thornless variety has more flavonoid than the spiny. The RP-HPLC and ESI-MS analysis detected two classes of phenolic compounds and betalain pigments. Isorhamnetin derivatives are the dominant flavonol glycoside identified in O. ficus indica (spiny: 65.25 μg·g(-1); thornless: 77.03 μg·g(-1)) and O. stricta peels (19.22 μg·g(-1)).

2,3-Dehydrosilybin (DHS) was previously shown to chelate and reduce both copper and iron ions. In this study, similar experiments with 2,3-dehydrosilychristin (DHSCH) showed that this congener of DHS also chelates and reduces both metals. Statistical analysis pointed to some differences between both compounds: in general, DHS appeared to be a more potent iron and copper chelator, and a copper reducing agent under acidic conditions, while DHSCH was a more potent copper reducing agent under neutral conditions. In the next step, both DHS and DHSCH were tested for metal-based Fenton chemistry in vitro using HPLC with coulometric detection. Neither of these compounds were able to block the iron-based Fenton reaction and, in addition, they mostly intensified hydroxyl radical production. In the copper-based Fenton reaction, the effect of DHSCH was again prooxidant or neutral, while the effect of DHS was profoundly condition-dependent. DHS was even able to attenuate the reaction under some conditions. Interestingly, both compounds were strongly protective against the copper-triggered lysis of red blood cells, with DHSCH being more potent. The results from this study indicated that, notwithstanding the prooxidative effects of both dehydroflavonolignans, their in vivo effect could be protective.

Small berries are rich in polyphenols whose first-pass metabolism may alter their ultimate physiological effects. The antioxidant capacity and polyphenol profile of three freeze-dried berries (blackberry, raspberry, Red Globe grape) were measured and their apparent permeability (Papp) and first-pass biotransformation were tracked with an ex vivo bioanalytical system [everted gut sac (rat) + three detection methods: spectrophotometry, HPLC-ESI-QTOF-MS, differential pulse voltammetry (DPV)]. Total polyphenol (ratio 0.07-0.14-1.0) and molecular diversity (anthocyanins>flavan-3-ols), antioxidant capacity (DPPH, FRAP), anodic current maxima and Papp (efflux> uptake) were in the following order: blackberry > raspberry > Red Globe grape. Epicatechin, pelargonidin & cyanin (all), callistephin (raspberry/blackberry), catechin (grape), cyanidin glycosides (blackberry) and their derived metabolites [quinic acid, epicatechin, cyanidin/malvidin glucosides, and chlorogenic/caffeic acids] were fruit-specific and concentration-dependent. Time-trend DPV kinetic data revealed concurrent epithelial permeability & biotransformation processes. Regular permeability and high-biotransformation of berry polyphenols suggest fruit-specific health effects apparently at the intestinal level.

Numerous residues, such as the coffee pulp, are generated throughout coffee processing. This by-product is a source of antioxidant phytochemicals, including phenolic compounds and caffeine. However, the antioxidant properties of the phenolic compounds from the coffee pulp are physiologically limited to their bioaccessibility, bioavailability, and biotransformation occurring during gastrointestinal digestion. Hence, this study explored the phenolic and caffeine profile in the coffee pulp flour (CPF) and extract (CPE), their intestinal bioaccessibility through in vitro digestion, and their potential bioavailability and colonic metabolism using in silico models. The CPE exhibited a higher concentration of phenolic compounds than the CPF, mainly phenolic acids (protocatechuic, chlorogenic, and gallic acids), followed by flavonoids, particularly quercetin derivatives. Caffeine was found in higher concentrations than phenolic compounds. The antioxidant capacity was increased throughout the digestive process. The coffee pulp matrix influenced phytochemicals' behavior during gastrointestinal digestion. Whereas individual phenolic compounds generally decreased during digestion, caffeine remained stable. Then, phenolic acids and caffeine were highly bioaccessible, while flavonoids were mainly degraded. As a result, caffeine and protocatechuic acid were the main compounds absorbed in the intestine after digestion. Non-absorbed phenolic compounds might undergo colonic biotransformation yielding small and potentially more adsorbable phenolic metabolites. These results contribute to establishing the coffee pulp as an antioxidant food ingredient since it contains bioaccessible and potentially bioavailable phytochemicals with potential health-promoting properties.

Numerous in vitro assays are used to characterize the antioxidant properties of natural-based matrices. However, many of them generate contradictory and non-compliant results. In our study, we focused on the characterization of traditionally used biochemical (2,2'-azino-bis-(3-ethylbenzothiazoline-6 sulfonic acid) (ABTS), Oxygen Radical Absorption Capacity (ORAC), and 2,2-diphenyl-1-picrylhydrazyl (DPPH)) and cellular (CAA) antioxidant tests on a broad set of milk thistle dietary supplements containing silymarin. In addition to 26 commercially available preparations, also the natural silymarin extract available from Sigma Aldrich, St. Louis, MI, USA, and a model mixture of pure flavonoid/flavonolignans mimicking the silymarin composition were investigated as control samples. Significant differences in the antioxidant capacity of the supplements were observed. Unlike the DPPH, the results of the ABTS and ORAC methods correlated with the silymarin components determined by U-HPLC-HRMS/MS. The responses in CAA were considerably lower than in other assays. Silymarin exhibited a significantly higher antioxidant capacity than the artificially prepared flavonoid/flavonolignans mixture in all tests, indicating possible presence of other antioxidants of natural origin. The follow-up U-HPLC-HRMS/MS screening revealed the presence of tens of non-silymarin compounds with reported antioxidant activity (not only in the silymarin extract, but also in the milk thistle preparations). The sum of the total phenolics and the sum of the simple phenolics correlated with CAA results more than silymarin.

Common food flavonoids: chrysin, apigenin, luteolin, diosmetin, pinocembrin, naringenin, eriodictyol, hesperetin, and their analogues with an additional hydroxyl group at the C-8 position obtained via biotransformation were tested for antioxidant activity using the ABTS, DPPH, and ferric ion reducing antioxidant power (FRAP) methods. They were also tested for antiproliferative activity against selected human cancer cell lines-MV-4-11 (biphenotypic B myelomonocytic leukemia), MCF7 (breast carcinoma), LoVo (colon cancer), LoVo/DX (colon cancer doxorubicin resistant), and DU 145 (prostate cancer)-and two normal human cell lines-MCF 10A (breast cells) and HLMEC (lung microvascular endothelial cells). Flavonoids with a C7-C8 catechol moiety indicated much higher antioxidant activity compared with the C7 hydroxy analogues. However, because they were unstable under the assay conditions, they did not show antiproliferative activity or it was very low.

Silymarin is the standardized extract from the fruits of Silybum marianum (L.) Gaertn., a well-known hepatoprotectant and antioxidant. Recently, bioactive compounds of silymarin, i.e., silybins and their 2,3-dehydro derivatives, have been shown to exert anticancer activities, yet with unclear mechanisms. This study combines in silico and in vitro methods to reveal the potential interactions of optically pure silybins and dehydrosilybins with novel protein targets. The shape and chemical similarity with approved drugs were evaluated in silico, and the potential for interaction with the Hedgehog pathway receptor Smoothened (SMO) and BRAF kinase was confirmed by molecular docking. In vitro studies on SMO and BRAF V600E kinase activity and in BRAF V600E A-375 human melanoma cell lines were further performed to examine their effects on these proteins and cancer cell lines and to corroborate computational predictions. Our in silico results direct to new potential targets of silymarin constituents as dual inhibitors of BRAF and SMO, two major targets in anticancer therapy. The experimental studies confirm that BRAF kinase and SMO may be involved in mechanisms of anticancer activities, demonstrating dose-dependent profiles, with dehydrosilybins showing stronger effects than silybins. The results of this work outline the dual SMO/BRAF effect of flavonolignans from Silybum marianum with potential clinical significance. Our approach can be applied to other natural products to reveal their potential targets and mechanism of action.

β-Carotene has been shown to increase the risk of developing lung cancer in smokers and asbestos workers in two large scale trails, the Beta-Carotene and Retinol Efficacy Trial (CARET) and the Alpha-Tocopherol Beta-carotene Cancer Prevention Trial (ATBC). Based on this observation, it was proposed that genotoxic oxidative breakdown products may cause this effect. In support of this assumption, increased levels of sister chromatid exchanges, micronuclei, and chromosomal aberrations were found in primary hepatocyte cultures treated with a mixture of cleavage products (CPs) and the major product apo-8'carotenal. However, because these findings cannot directly be transferred to the lung due to the exceptional biotransformation capacity of the liver, potential genotoxic and cytotoxic effects of β-carotene under oxidative stress and its CPs were investigated in primary pneumocyte type II cells. The results indicate that increased concentrations of β-carotene in the presence of the redox cycling quinone dimethoxynaphthoquinone (DMNQ) exhibit a cytotoxic potential, as evidenced by an increase of apoptotic cells and loss of cell density at concentrations > 10 µM. On the other hand, the analysis of micronucleated cells gave no clear picture due to the cytotoxicity related reduction of mitotic cells. Last, although CPs induced significant levels of DNA strand breaks even at concentrations ≥ 1 µM and 5 µM, respectively, β-carotene in the presence of DMNQ did not cause DNA damage. Instead, β-carotene appeared to act as an antioxidant. These findings are in contrast with what was demonstrated for primary hepatocytes and may reflect different sensitivities to and different metabolism of β-carotene in the two cell types.