Jinkai Zheng

Surface‐Enhanced Raman Spectroscopy for the Chemical Analysis of Food

 Surface-enhanced Raman spectroscopy (SERS) is an emerging and promising technique for the chemical analysis of food. The use of metallic nanosubstrates improves the sensitivity and capacity of conventional Raman spectroscopy greatly. This paper comprehensively reviews the development and applications of SERS in the chemical analysis of food, mainly focusing on food additives and chemical contaminants. The progress of SERS development and their applications in chemical analysis of food, from detection and characterization of target analytes in simple solvents to complex food matrices, is summarized. The advantages and limitations of different SERS substrates and methodologies are discussed. As most of the current SERS research on chemical analysis of food is still in an early stage, there are still several hurdles for further advancing SERS techniques into real-world applications for complex food products. This review includes our perspectives on the future trends of the SERS technique in the field of food analysis.

Tangeretin-loaded protein nanoparticles fabricated from zein/β-lactoglobulin: preparation, characterization, and functional performance.

The aim of this study was to design a colloidal delivery system to encapsulate poor water-soluble bioactive flavonoid tangeretin so that it could be utilized in various food products as functional ingredient. Tangeretin-loaded protein nanoparticles were produced by mixing an organic phase containing zein and tangeretin with an aqueous phase containing β-lactoglobulin and then converted into powder by freeze-drying. This powder formed a colloidal suspension when dispersed in water that is relatively stable to particle aggregation and sedimentation. The influence of temperature, ionic strength, and pH on the stability of the protein nanoparticles was tested. Extensive particle aggregation occurred at high ionic strength (>100mM) and intermediate pH (4.5-5.5) due to reduced electrostatic repulsion. Extensive aggregation also occurred at temperatures exceeding 60 °C, which was presumably due to increased hydrophobic attraction. Overall, this study shows that protein-based nanoparticles can be used to encapsulate bioactive tangeretin so that it can be readily dispersed in compatible food products.

Synergistic Anti-inflammatory Effects of Nobiletin and Sulforaphane in Lipopolysaccharide-Stimulated RAW 264.7 Cells

Inflammation plays important roles in the initiation and progress of many diseases including cancers in multiple organ sites. Herein, we investigated the anti-inflammatory effects of two dietary compounds, nobiletin (NBN) and sulforaphane (SFN), in combination. Noncytotoxic concentrations of NBN, SFN, and their combinations were studied in lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophage cells. The results showed that combined NBN and SFN treatments produced much stronger inhibitory effects on the production of nitric oxide (NO) than NBN or SFN alone at higher concentrations. These enhanced inhibitory effects were synergistic based on the isobologram analysis. Western blot analysis showed that combined NBN and SFN treatments synergistically decreased iNOS and COX-2 protein expression levels and induced heme oxygenase-1 (HO-1) protein expression. Real-time polymerase chain reaction analysis indicated that low doses of NBN and SFN in combination significantly suppressed LPS-induced upregulation of IL-1 mRNA levels and synergistically increased HO-1 mRNA levels. Overall, our results demonstrated that NBN and SFN in combination produced synergistic effects in inhibiting LPS-induced inflammation in RAW 264.7 cells.

5-Demethyltangeretin inhibits human nonsmall cell lung cancer cell growth by inducing G2/M cell cycle arrest and apoptosis

SCOPE Tangeretin (TAN) and 5-demethyltangeretin (5DT) are two closely related polymethoxyflavones found in citrus fruits. We investigated growth inhibitory effects on three human nonsmall cell lung cancer (NSCLC) cells. METHODS AND RESULTS Cell viability assay demonstrated that 5DT inhibited NSCLC cell growth in a time- and dose-dependent manner, and IC50 s of 5DT were 79-fold, 57-fold, and 56-fold lower than those of TAN in A549, H460, and H1299 cells, respectively. Flow cytometry analysis showed that 5DT induced extensive G2/M cell cycle arrest and apoptosis in NSCLC cells, while TAN at tenfold higher concentrations did not. The apoptosis induced by 5DT was further confirmed by activation of caspase-3 and cleavage of PARP. Moreover, 5DT dose-dependently upregulated p53 and p21(Cip1/Waf1), and downregulated Cdc-2 (Cdk-1) and cyclin B1. HPLC analysis revealed that the intracellular levels of 5DT in NSCLC cells were 2.7-4.9 fold higher than those of TAN after the cells were treated with 5DT or TAN at the same concentration. CONCLUSION Our results demonstrated that 5DT inhibited NSCLC cell growth by inducing G2/M cell cycle arrest and apoptosis. These effects were much stronger than those produced by TAN, which is partially due to the higher intracellular uptake of 5DT than TAN.

Identification of pinostilbene as a major colonic metabolite of pterostilbene and its inhibitory effects on colon cancer cells

SCOPE Pterostilbene (PTE) is a resveratrol derivative mainly found in blueberries, and it has been shown to inhibit colon carcinogenesis in multiple animal models. To shed light on the mechanism of PTE in inhibiting colon carcinogenesis, we investigated the PTE metabolites in the mouse colon and in the human colon cancer cells. METHODS AND RESULTS CD-1 mice were fed PTE-containing diet for 3 weeks, and colonic content and colonic mucosa were collected and subjected to LC-MS analysis. Pinostilbene (PIN) was identified as a major metabolite of PTE in the mouse colon. Importantly, the level of PIN was found to be approximately equivalent to that of PTE in the colonic mucosa. PIN significantly inhibited the growth of human colon cancer cells, i.e., HCT116 and HT29. These inhibitory effects were similar to those produced by PTE. Moreover, under physiologically relevant conditions, 20 and 40 μM of PIN caused cell cycle arrest at S phase and induced apoptosis in colon cancer cells. These effects were associated with profound modulation of signaling proteins related with cell proliferation and programmed cell death. CONCLUSION Our results demonstrated that PIN is a major metabolite of PTE in the colon of mice fed with PTE, and PIN may play important roles in the anti-colon cancer effects elicited by orally administered PTE.

Evaluation of surface-enhanced Raman scattering detection using a handheld and a bench-top Raman spectrometer: A comparative study

Surface enhanced Raman scattering (SERS) detection using a handheld Raman spectrometer and a bench-top Raman spectrometer was systemically evaluated and compared in this study. Silver dendrites were used as the SERS substrate, and two pesticides, maneb and pyrrolidine dithiocarbamate-ammonium salt (PDCA) were used as the analytes. Capacity and performance were evaluated based on spectral resolution, signal variation, quantitative capacity, sensitivity, flexibility and intelligence for SERS detection. The results showed that the handheld Raman spectrometer had better data consistency, more accurate quantification capacity, as well as the capacity of on-site and intelligence for qualitative and semi-quantitative analysis. On the other hand, the bench-top Raman spectrometer showed about 10 times higher sensitivity, as well as flexibility for optimization of the SERS measurements under different parameters such as laser power output, collective time, and objective magnification. The study on the optimization of SERS measurements on a bench-top spectrometer provides a useful guide for designing a handheld Raman spectrometer, specifically for SERS detection. This evaluation can advance the application of a handheld Raman spectrometer for the on-site measurement of trace amounts of pesticides or other chemicals.

Enhanced Anti-Inflammatory Activities by the Combination of Luteolin and Tangeretin

Dietary components in combination may act synergistically and produce enhanced biological activities. Herein, we investigated the anti-inflammatory effects of 2 flavonoids, that is luteolin (LUT) and tangeretin (TAN) in combination. Lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophages were treated with noncytotoxic concentrations of LUT, TAN, and their combinations. The results showed that LUT/TAN in combination produced synergistic inhibitory effects on LPS-stimulated production of nitric oxide (NO). ELISA results demonstrated that LUT/TAN in combination caused stronger suppression on the LPS-induced overexpression of proinflammatory mediators, such as prostaglandin E2 (PGE2 ), interleukin (IL)-1β, and IL-6 than LUT or TAN alone. Immunoblotting and Real-Time PCR analyses showed that LUT/TAN combination significantly decreased LPS-induced protein and mRNA expression of inducible nitric oxide synthase and cyclooxygenase-2. These inhibitory effects of the combination treatment were stronger than those produced by LUT or TAN alone. Overall, our results demonstrated for the first time that combination of LUT and TAN produced synergistic anti-inflammatory effects in LPS-stimulated RAW 264.7 macrophages.

Nobiletin and its colonic metabolites suppress colitis-associated colon carcinogenesis by down-regulating iNOS, inducing antioxidative enzymes and arresting cell cycle progression

Nobiletin (NOB) is a major citrus polymethoxyflavone (PMF) with various beneficial biological activities. We reported previously that dietary NOB significantly inhibited colitis-associated colon carcinogenesis in azoxymethane (AOM)/dextran sulfate sodium (DSS)-treated mice, and the chemopreventive effects were associated with NOB metabolites found in the mouse colonic tissues. In this study, to better understand the role of colonic metabolites of NOB, we determined the anti-inflammation and anticancer effects of a mixture of NOB and its major metabolites (NOB-Met) at the concentrations equivalent to those found in colonic tissues of NOB-fed mice. The results demonstrated that NOB-Met effectively decreased the expression level of inducible nitric oxide synthase (iNOS), increased the level of heme oxygenase-1 (HO-1) and NADH quinone oxidoreductase 1 (NQO1) and up-regulated nuclear factor erythroid 2-related factor (Nrf2) signaling pathway in lipopolysaccharide (LPS)-stimulated macrophages. NOB-Met also caused a significant cell cycle arrest in human colon cancer cells. Validation study confirmed that dietary NOB led to the effects similar to those described above in the colon of AOM/DSS-treated mice. Specifically, dietary NOB significantly reduced the level of iNOS, up-regulated Nrf2-dependent enzymes and profoundly modulated key signaling proteins resulting in decreased cell cycle progression in the colonic tissue of AOM/DSS-treated mice. Overall, our findings demonstrated that dietary NOB led to the presence of NOB and its metabolites in the colonic tissue, which suppressed colitis-associated colon carcinogenesis via down-regulating iNOS, inducing antioxidative enzymes and arresting cell cycle progression.

Analysis of 10 metabolites of polymethoxyflavones with high sensitivity by electrochemical detection in high-performance liquid chromatography.

Polymethoxyflavones (PMFs) have been known as a type of bioactive flavones that possess various beneficial biological functions. Accumulating evidence demonstrated that the metabolites of PMFs, that is, hydroxyl PMFs (OH-PMFs), had more potent beneficial biological effects than their corresponding parent PMFs. To facilitate the further identification and quantification of OH-PMFs in biological samples, the aim of this study was to develop a methodology for the simultaneous determination of 10 OH-PMFs using high-performance liquid chromatography (HPLC) coupled with electrochemistry detection. The HPLC profiles of these 10 OH-PMFs affected by different chromatographic parameters (different organic composition in mobile phases, the concentration of trifluoroacetic acid, and the concentration of ammonium acetate) are fully discussed in this study. The optimal condition was selected for the following validation studies. The linearity of calibration curves, accuracy, and precision (intra- and interday) at three concentration levels (low, middle, and high concentration range) were verified. The regression equations were linear (r > 0.9992) over the range of 0.005-10 μM. The limit of detection for 10 OH-PMFs was in the range of 0.8-3.7 ng/mL (S/N = 3, 10 μL injection). The recovery rates ranged from 86.6 to 108.7%. The precisions of intraday and interday analyses were less than 7.37 and 8.63% for relative standard deviation, respectively. This validated method was applied for the analysis of a variety of samples containing OH-PMFs. This paper also gives an example of analyzing the metabolites of nobiletin in mouse urine using the developed method. The transformation from nobiletin to traces of 5-hydroxyl metabolites has been discovered by this effective method, and this is the first paper to report such an association.

Synergistic chemopreventive effects of nobiletin and atorvastatin on colon carcinogenesis

Different cancer chemopreventive agents may act synergistically and their combination may produce enhanced protective effects against carcinogenesis than each individual agent alone. Herein, we investigated the chemopreventive effects of nobiletin (NBT, a citrus polymethoxyflavone) and atorvastatin (ATST, a lipid-lowering drug) in colon cancer cells/macrophages and an azoxymethane (AOM)-induced colon carcinogenesis rat model. The results demonstrated that co-treatments of NBT/ATST produced enhanced growth inhibitory and anti-inflammatory effects on the colon cancer cells and macrophages, respectively. Isobologram analysis confirmed that these interactions between NBT and ATST were synergistic. NBT/ATST co-treatment also synergistically induced extensive cell cycle arrest and apoptosis in colon cancer cells. Oral administration of NBT (0.1%, w/w in diet) or ATST (0.04%, w/w in diet) significantly decreased colonic tumor incidence and multiplicity in AOM-treated rats. Most importantly, co-treatment of NBT/ATST at their half doses (0.05% NBT + 0.02% ATST, w/w in diet) resulted in even stronger inhibitory effects on colonic tumor incidence and multiplicity than did NBT or ATST alone at higher doses. Statistical analysis confirmed that the enhanced chemopreventive activities against colon carcinogenesis in rats by the NBT/ATST combination were highly synergistic. Our results further demonstrated that NBT/ATST co-treatment profoundly modulated key cellular signaling regulators associated with inflammation, cell proliferation, cell cycle progression, apoptosis, angiogenesis and metastasis in the colon of AOM-treated rats. In conclusion, for the first time, our results demonstrated a strong synergy in inhibiting colon carcinogenesis produced by the co-treatment of NBT and ATST, which provided a scientific basis for using NBT in combination with ATST for colon cancer chemoprevention in humans.