Comparison of the Main Metabolites in Different Maturation Stages of Camelliavietnamensis Huang Seeds.

Camellia vietnamensis Huang is an important woody oil crop in China, which has attracted much attention because of its abundant nutritional components and pharmaceutical value. Its seeds undergo a complex series of physiological and biochemical changes during maturation, with consequent alterations in metabolites. In order to investigate the endogenous metabolism of C. vietnamensis on Hainan Island during seed development, in this study, ultra-high-performance liquid tandem chromatography coupled with quadrupole time-of-flight mass spectrometry (UHPLC/Q-TOF-MS) and multivariate statistical analysis (MSA) were used to analyze the differences in the chemical compounds of C. vietnamensis seeds among the four maturation stages. A total of 293 metabolites were identified from the methanol extract of the seeds of C. vietnamensis. Five metabolites, belonging to benzene and substituted derivatives, 5'-deoxyribonucleosides and linear 1,3-diarylpropanoids, were found in all three comparison groups, with consistently down-regulated trends. The Kyoto Encyclopedia of Genes and Genomes (KEGG) results showed that phloretin and 5'-methylthioadenosine were the differentially expressed metabolites when seeds were in the growth periods of S2 and S3, and indole and L-tryptophan were the differentially expressed metabolites when seeds were in the growth periods of S3 and S4. In addition, 34 flavonoid metabolites were detected, of which 4 were differentially expressed. It was indicated that flavonoids dynamically change during all the oil-tea camellia seed development stages. The findings provide data for the better understanding of endogenous metabolic pathways during C. vietnamensis seed development.

Dip-and-Read, Organic Solvent-Compatible, Paper-Based Analytical Devices Equipped with Chromatographic Separation for Indole Analysis in Shrimp.

We developed an organic solvent-compatible paper-based analytical device (PAD) for the quantitative analysis of indole, which is an indicator of shrimp freshness. Although indole is insoluble in water, ethyl acetate is a suitable solvent to dissolve and extract indole from shrimp. The PADs are fabricated using a cutting method that allows the use of an organic solvent because no hydrophobic barrier is needed to form fluidic channels. Ehrlich's reagent consists of 4-(dimethylamino)benzaldehyde and p-dimethylaminobenzaldehyde and was deposited onto the reaction zone of the PAD followed by lamination to prevent evaporation of the ethyl acetate. Samples are introduced into the PAD via immersion in organic sample solutions. When the PAD is immersed into an indole solution of ethyl acetate in a closed bottle, the sample solution penetrates the channel of the PAD and successively flows into the detection zone to form a hydrophilic colored product. The PADs provide a linear relationship between the logarithm of the indole concentration and the color intensity within a range of 1.0-20 ppm with correlation coefficients of r2 > 0.99. The limits of detection and quantification are 0.36 and 0.71 ppm, respectively. Relative standard deviations for both the intraday (n = 2) and interday (n = 3) precision were less than 2.5%. In the indole analysis of shrimp, the PADs separated the interfering orange-colored astaxanthin in the extract from the colored product of indole via the paper chromatographic principle. We used the PADs to investigate the degradation of shrimp, and the results showed a rapid increase in the indole level after 7 days. High-performance liquid chromatography verified the accuracy of the PADs by showing good agreement with the obtained indole levels.

Identification and quantification of 10 indole/indazole carboxamide synthetic cannabinoids in 36 herbal blends by gas chromatography-mass spectrometry and nuclear magnetic resonance spectroscopy.

Herbal blends containing synthetic cannabinoids have become popular alternatives to marijuana. The number of synthetic cannabinoids and speed of their emergence enable this group of compounds particularly challenging in terms of detection, monitoring, and responding. In this work, both gas chromatography-mass spectrometry (GC-MS) and nuclear magnetic resonance spectroscopy (NMR) methods were developed for the identification and quantification of synthetic cannabinoids in herbal blends. Ten types of indole/indazole carboxamide synthetic cannabinoids, which showed different types of substitutions connected to nitrogen of the indole/indazole carboxamide, were detected in 36 herbal blends. The GC-MS fragmentation routes of indole/indazole carboxamide synthetic cannabinoids were discussed in detail for structure identification purpose. The concentration range of synthetic cannabinoid in 36 herbal blends was 1.9-50.6 mg/g using GC-MS method, while 1.5-49.0 mg/g by NMR method. Nicotine in herbal blends was quantified by NMR method without using reference material, and showed a variation of 5.3-44.7 mg/g. For quantitative analysis, NMR method showed great advantage in the absence of reference material, while GC-MS method showed great merit for multiple-compound analysis when reference material was available. Therefore, for the quantitative analysis of new emerged synthetic cannabinoid in herbal blends, different methods could be chosen by considering whether reference material is available, as well as the number and types of synthetic cannabinoids detected in a single sample.

Impacts of depectinization of pear juice on alcoholic fermentation and indole formation.

BACKGROUND: Recently, a producer of fermented ciders observed 'vinyl' off-odors formed during fermentation of pear juice previously depectinized at ≥ 49 °C but not if depectinized at lower temperatures. The objective of this study was to investigate the source of this spoilage and evaluate factors that affect formation. RESULTS: Analysis of untainted and tainted samples obtained from the producer determined the causative agent to be indole, a compound sometimes produced by yeast (Saccharomyces cerevisiae) during fermentation. To mimic commercial depectinization conditions, pectinases were added to pear juices held at 35 °C for 45 min (Treatment A), 49 °C for 45 min (Treatment B), or 49 °C for 90 min (Treatment C). Juice processing conditions did not affect yeast growth nor progress of alcoholic fermentation. Although neither yeast strain (DV10 or MERIT) synthesized indole during fermentation of Treatment A juices, the compound was produced by MERIT in Treatments B (27.05 µg L-1 ) and C (469.9 µg L-1 ). Supplementation of Treatment C juice with pyridoxine (vitamin B6 ) prior to fermentation resulted in no detectable indole formed. However, juices from Treatments A, B, or C contained similar concentrations of pyridoxine and non-detectable amounts of tryptophan, a potential precursor to indole. Furthermore, indole was not detected during fermentations of a synthetic pear juice medium without pyridoxine. CONCLUSION: Supplementation of cider musts with pyridoxine prior to fermentation and choice of yeast strain can lower the risk of formation of off-odors caused by indole. However, other unidentified factors are present which affect its formation in perry. © 2019 Society of Chemical Industry.

Assessing the Bioavailability of Zinc and Indole Compounds from Mycelial Cultures of the Bay Mushroom Imleria badia (Agaricomycetes) Using In Vitro Models.

Zinc and indole compounds demonstrate anti-inflammatory, antidepressant, and antioxidant activity. Edible mushrooms are good sources of these substances. Therefore, in this study, we aimed to study the accumulation, release, and absorption of zinc and indole compounds from mycelial cultures of Imleria badia species using in vitro models. Samples were analyzed using the atomic absorption spectroscopy method and the reversed-phase high-performance liquid chromatography method. The highest quantities of zinc were detected in the material grown on zinc hydrogen aspartate-enriched media (176.01 mg/100 g dry weight [d.w.]). In addition, the quantity of zinc in the control biomass was approximately 12.13 mg/100 g d.w. After passive transport, the amount of zinc was detected to be around 1.40 mg/100 g d.w., whereas after active transport with CaCo-2 cells, the quantity of zinc ranged from 0.46 mg/100 g d.w. to 12.72 mg/100 g d.w. Among the organic compounds, four indole compounds were qualitatively identified, including 5-hydroxy-l-tryptophan, melatonin, l-tryptophan, and 5-methyltryptamine. These results indicate that mushrooms and their in vitro cultures not only synthesize and accumulate these compounds, but also potentially release them into the gastrointestinal tract where they can be absorbed by the human body, which is reflected as a specific health benefit.

Tannin-rich sainfoin pellet supplementation reduces fat volatile indoles content and delays digestive parasitism in lambs grazing alfalfa.

Lambs grazing alfalfa or white clover are prone to flavour taint which can be an impediment to consumer acceptance. Here we investigated whether condensed tannin (CT)-rich sainfoin pellet supplementation of lambs grazing alfalfa influences meat sensory quality. Using three groups of 18 male Romane weaned lambs, we compared three feeding regimes: alfalfa grazing (AF), alfalfa grazing + daily supplementation with CT-rich sainfoin pellets (15 g dry matter (DM)/kg live weight, AS) and stall feeding with concentrate and grass hay indoors (SI). We also investigated the potential interest of sainfoin pellet supplementation for controlling digestive parasitism. The sainfoin pellets contained 42 g of CT/kg of DM and they represented on average 36% of the diet in AS lambs. Skatole and indole were detected in most of the AF and AS lambs, whereas in very few SI lambs. Skatole and indole concentrations in perirenal and dorsal fat were lower in the AS lambs than the AF lambs (P < 0.025 to P < 0.001), but the intensity of 'animal' odour and 'animal' flavour of the chops did not differ between both forage-grazing groups. Longissimus thoracis et lumborum muscle lightness was lower in the AF and AS lambs than the SI lambs (P < 0.001) with the other muscle colour coordinates being unaffected by the treatment and between-treatment group differences in muscle colour coordinates remaining constant throughout the 9-day display period. Subcutaneous fat colour coordinates were not influenced by the treatment. The number of individual anthelmintic drenches necessary to keep nematode faecal egg count below a threshold of 550 eggs/g of faeces was lower in the AS than the AF lambs (0.94 per lamb v. 1.63 per lamb; P < 0.001). Faecal oocyst count was lower in the AS than the AF lambs for the first measurement made 56 days after the beginning of the experiment (P < 0.001) and was not significantly different between both forage-grazing groups thereafter. The use of CT-rich sainfoin pellets to supplement lambs that are concurrently grazing alfalfa reduced fat volatile skatole and indole concentrations and delayed the onset of both helminth and coccidian infections.

Peak AAA fatty acid homolog contaminants present in the dietary supplement l-Tryptophan associated with the onset of eosinophilia-myalgia syndrome.

The eosinophilia-myalgia syndrome (EMS) outbreak that occurred in the USA and elsewhere in 1989 was caused by the ingestion of Showa Denko K.K. (SD) L-tryptophan (L-Trp). "Six compounds" detected in the L-Trp were reported as case-associated contaminants. Recently the final and most statistically significant contaminant, "Peak AAA" was structurally characterized. The "compound" was actually shown to be two structural isomers resulting from condensation reactions of L-Trp with fatty acids derived from the bacterial cell membrane. They were identified as the indole C-2 anteiso (AAA1-343) and linear (AAA2-343) aliphatic chain isomers. Based on those findings, we utilized a combination of on-line HPLC-electrospray ionization mass spectrometry (LC-MS), as well as both precursor and product ion tandem mass spectrometry (MS/MS) to facilitate identification of a homologous family of condensation products related to AAA1-343 and AAA2-343. We structurally characterized eight new AAA1-XXX/AAA2-XXX contaminants, where XXX represents the integer molecular ions of all the related homologs, differing by aliphatic chain length and isomer configuration. The contaminants were derived from the following fatty acids of the bacterial cell membrane, 5-methylheptanoic acid (anteiso-C8:0) for AAA1-315; n-octanoic acid (n-C8:0) for AAA2-315; 6-methyloctanoic acid (anteiso-C9:0) for AAA1-329; n-nonanoic acid (n-C9:0) for AAA2-329; 10-methyldodecanoic acid (anteiso-C13:0) for AAA1-385; n-tridecanoic acid (n-C13:0) for AAA2-385; 11-methyltridecanoic acid (anteiso-C14:0) for AAA1-399; and n-tetradecanoic acid (n-C14:0) for AAA2-399. The concentration levels for these contaminants were estimated to be 0.1-7.9 µg / 500 mg of an individual SD L-Trp tablet or capsule The structural similarity of these homologs to case-related contaminants of Spanish Toxic Oil Syndrome (TOS) is discussed.

Comparison of indirubin concentrations in indigo naturalis ointment for psoriasis treatment: a randomized, double-blind, dosage-controlled trial.

BACKGROUND: Indigo naturalis and its refined formulation, Lindioil, are effective in treating psoriatic symptoms topically. Indirubin is the active ingredient in indigo naturalis. OBJECTIVES: To determine the efficacy and safety of different concentrations of indirubin in Lindioil ointment for treating psoriasis. METHODS: In this randomized, double-blind trial, adult patients presenting with chronic plaque psoriasis for > 1 year and with < 20% of the body surface area (BSA) affected were randomized to apply Lindioil ointment containing 200, 100, 50 or 10 µg g-1 of indirubin twice daily for 8 weeks followed by an additional 12-week safety/extension period. The primary end point was the mean percentage change in Psoriasis Area and Severity Index (PASI) score along with the proportion of participants achieving 75% and 90% reductions in PASI scores (PASI 75 and PASI 90, respectively) from baseline to week 8. RESULTS: The results from week 8 revealed that the 200 µg g-1 group had the greatest reduction in PASI score [69·2%, 95% confidence interval (CI) 55·5-82·8], followed by the 100 µg g-1 group (63·1%, 95% CI 52·8-73·5), the 10 µg g-1 group (53·4%, 95% CI 42·8-64·0) and the 50 µg g-1 group (50·3%, 95% CI 37·4-63·2), with a between-group comparison of P = 0·0445. The group with the highest proportion of the patients achieving PASI 75 (57%, P = 0·0474) and PASI 90 (30%, P = 0·0098) was the 200 µg g-1 group. No severe treatment-related adverse events were reported during the 20-week evaluation. CONCLUSIONS: An amount of 200 µg g-1 of indirubin in Lindioil ointment is the most effective concentration studied so far for treating psoriasis topically, and is safe.

Anti-inflammatory activities of garlic sprouts, a source of α-linolenic acid and 5-hydroxy-l-tryptophan, in RAW 264.7 cells.

The purpose of this study was to analyze the indolic, phenolic, and fatty acid content and antioxidant activity of garlic sprouts growing in the dark and in the daylight. The pro- or anti-inflammatory properties of the garlic sprout extract were investigated by evaluating the cyclooxygenase-2 (COX-2), prostaglandin E synthase (cPGES), glutathione S transferase (GSTM1), nuclear factor NF-κB, peroxisome proliferator-activated receptors (PPARs), and aryl hydrocarbon receptor (AhR) protein levels in the RAW 264.7 cells activated with lipopolysaccharide (LPS). The highest amount of total indolic (73.56 mg/100 g f.w.) and phenolic compounds (36.23 mg/100 g f.w.) was detected in garlic sprouts grown in the daylight. Studies on antioxidant activity (the FRAP and DPPH method) of garlic sprouts showed that this activity is significantly higher for sprouts grown in full access to light when compared to those grown in the dark. In garlic sprout extracts, α-linolenic acid (ALA) was found to be in greater amount. COX-2 and cPGES level was lower when compared to LPS alone activated cells. After garlic extract treatment, higher level of GSTM1, PPARΥ, cytosolic p50 and p65 protein, as well as a lower NF-ĸB p50/p65 activity was noted in the RAW 264.7 cells which suggested PPARs and AhR transrepression mechanism of NF-ĸB signalling. The obtained results indicate Allium sativum sprouts are a rich source of n-3 fatty acids, indolic and phenolic compounds characterized by anti-inflammatory and antioxidative activity, which may support their high therapeutic and dietary potential.

Qualitative Analysis and Detection of the Pyrolytic Products of JWH-018 and 11 Additional Synthetic Cannabinoids in the Presence of Common Herbal Smoking Substrates.

Synthetic cannabinoids have become a ubiquitous challenge in forensic toxicology and seized drug analysis. Thermal degradation products have yet to be identified and evaluated for toxicity in comparison to parent and metabolic compounds. An investigation into these pyrolytic products, as the major route of ingestion is inhalation, may produce additional insight to understand the toxicity of synthetic cannabinoids. The pyrolysis of JWH-018 and 11 additional synthetic cannabinoids and six herbal plant substrates were conducted using an in-house constructed smoking simulator. After pyrolysis of herbal material alone, the plant substrate was spiked with the drug compounds to 2-5% w/w concentrations. Samples were collected, filtered, evaporated under nitrogen gas, reconstituted in methanol, and analyzed via gas chromatograph-mass spectrometer. Pyrolysis of the plant material alone produced 10 consistently observed compounds between the six plant species. The pyrolysis of the synthetic cannabinoids produced a total of 52 pyrolytic compounds, where 32 were unique to a particular parent compound and the remaining 20 were common products between multiple cannabinoids. The thermal degradation followed three major pathways that are outlined to assist in producing a predictive model for new synthetic cannabinoids that may arise in case samples. The observed pyrolytic products are also viable options for analysis in post mortem samples and the evaluation of toxicity.

Identification of Eight Synthetic Cannabinoids, Including 5F-AKB48 in Seized Herbal Products Using DART-TOF-MS and LC-QTOF-MS as Nontargeted Screening Methods.

Synthetic cannabinoids are sprayed onto plant material and smoked for their marijuana-like effects. Clandestine manufacturers modify synthetic cannabinoid structures by creating closely related analogs. Forensic laboratories are tasked with detection of these analog compounds, but targeted analytical methods are often thwarted by the structural modifications. Here, direct analysis in real time coupled to accurate mass time-of-flight mass spectrometry (DART-TOF-MS) in combination with liquid chromatography quadruple time-of-flight mass spectrometry (LC-QTOF-MS) are presented as a screening and nontargeted confirmation method, respectively. Methanol extracts of herbal material were run using both methods. Spectral data from four different herbal products were evaluated by comparing fragmentation pattern, accurate mass and retention time to available reference standards. JWH-018, JWH-019, AM2201, JWH-122, 5F-AKB48, AKB48-N-(4-pentenyl) analog, UR144, and XLR11 were identified in the products. Results demonstrate that DART-TOF-MS affords a useful approach for rapid screening of herbal products for the presence and identification of synthetic cannabinoids.

Quantification of synthetic cannabinoids in herbal smoking blends using NMR.

Herbal smoking blends containing synthetic cannabinoids have become popular alternatives to marijuana. These products were previously sold in pre-packaged foil bags, but nowadays seizures usually contain synthetic cannabinoid powders together with unprepared plant materials. A question often raised by the Swedish police is how much smoking blend can be prepared from certain amounts of banned substance, in order to establish the severity of the crime. To address this question, information about the synthetic cannabinoid content in both the powder and the prepared herbal blends is necessary. In this work, an extraction procedure compatible with direct NMR quantification of synthetic cannabinoids in herbal smoking blends was developed. Extraction media, time and efficiency were tested for different carrier materials containing representative synthetic cannabinoids. The developed protocol utilizes a 30 min extraction step in d4 -methanol in presence of internal standard allowing direct quantitation of the extract using NMR. The accuracy of the developed method was tested using in-house prepared herbal smoking blends. The results showed deviations less than 0.2% from the actual content, proving that the method is sufficiently accurate for these quantifications. Using this method, ten synthetic cannabinoids present in sixty-three different herbal blends seized by the Swedish police between October 2012 and April 2015 were quantified. Obtained results showed a variation in cannabinoid contents from 1.5% (w/w) for mixtures containing MDMB-CHMICA to over 5% (w/w) for mixtures containing 5F-AKB-48. This is important information for forensic experts when making theoretical calculations of production quantities in legal cases regarding "home-made" herbal smoking blends. Copyright © 2016 John Wiley & Sons, Ltd.

LC-ESI-MS/MS evaluation of forced degradation behaviour of silodosin: In vitro anti cancer activity evaluation of silodosin and major degradation products.

Silodosin (SLD) a novel α1-adrenoceptor antagonist was subjected to forced degradation involving hydrolysis (acidic, alkaline and neutral), oxidative, photolysis and thermal stress, as per ICH specified conditions. The drug underwent significant degradation under hydrolytic (acidic, alkaline and neutral) and oxidative stress conditions whereas, it was found to be stable under other stress conditions. A rapid, precise, accurate and robust chromatographic method for the separation of the drug and its degradation products (DPs) was developed on a Fortis C18 analytical column (150×4.6mm, 5µm) using 0.1% formic acid and acetonitrile as a mobile phase in gradient elution mode at a flow rate of 1.0mL/min. A total of 5 (DP 1 to DP 5) hitherto unknown DPs were identified by LC-ESI-TOF-MS/MS experiments and accurate mass measurements. The most probable mechanisms for the formation of DPs have been proposed based on a comparison of the fragmentation of the [M+H]+ ions of silodosin and its DPs. The major DPs (DP 1 and DP 2) were isolated and evaluated for anticancer activity using PC3 (human prostate cancer) cell lines by MTT assay. The results revealed that silodosin, DP 1 and DP 2 have potential anticancer activity with IC50 values (µM) 72.74 (±4.51), 25.21 (±2.36), and, 114.07 (±11.90) respectively.

Development of Eudragit RS 100 Microparticles Loaded with Ropinirole: Optimization and In Vitro Evaluation Studies.

The current study aimed to develop novel pH independent microparticles loaded with ropinirole (ROP) for sustained drug release. Eudragit RS 100 was used as release retardant and microparticles were fabricated by oil-in-oil emulsion solvent evaporation method. A three-factor three-level Box-Behnken design using Design-Expert software was employed to optimize formulation variables. Ropinirole loaded microparticles were evaluated with respect to morphology, particle size, encapsulation efficiency, and in vitro release profile. Optical microscopy and SEM micrographs indicated spherical shape with smooth surface and well-defined boundary. The particle size was in the range of 98.86 to 236.29 µm, being significantly increased with increasing polymer concentration. Higher polymer load also increased the thickness of internal polymer network, which led to reduced drug loss and higher entrapment efficiency (89%). The cumulative in vitro release was found to be in the range of 54.96 to 99.36% during the release studies (12 h) following zero order release kinetics and non-Fickian diffusion pattern. The developed microparticles have the potential to sustain the release of ropinirole, which may lead to a reduction in its adverse effects and improved management of Parkinson's disease.

Supercritical fluid chromatography for separation and preparation of tautomeric 7-epimeric spiro oxindole alkaloids from Uncaria macrophylla.

Increasing challenge arising from configurational interconversion in aqueous solvent renders it rather difficult to isolate high-purity tautomeric reference standards and thus largely hinders the holistic quality control of traditional Chinese medicine (TCM). Spiro oxindole alkaloids (SOAs), as the markers for the medicinal Uncaria herbs, can easily isomerize in polar or aqueous solvent via a retro-Mannich reaction. In the present study, supercritical fluid chromatography (SFC) is utilized to separate and isolate two pairs of 7-epimeric SOAs, including rhynchophylline (R) and isorhynchophylline (IR), corynoxine (C) and corynoxine B (CB), from Uncaria macrophylla. Initially, the solvent that can stabilize SOA epimers was systematically screened, and acetonitrile was used to dissolve and as the modifier in SFC. Then, key parameters of ultra-high performance SFC (ultra-performance convergence chromatography, UPC2), comprising stationary phase, additive in modifier, column temperature, ABPR pressure, and flow rate, were optimized in sequence. Two isocratic UPC2 methods were developed on the achiral Torus 1-AA and Torus Diol columns, suitable for UV and MS detection, respectively. MCI gel column chromatography fractionated the U. macrophylla extract into two mixtures (R/IR and C/CB). Preparative SFC, using a Viridis Prep Silica 2-EP OBD column and acetonitrile-0.2% diethylamine in CO2 as the mobile phase, was finally employed for compound purification. As a result, the purity of four SOA compounds was all higher than 95%. Different from reversed-phase HPLC, SFC, by use of water-free mobile phase (inert CO2 and aprotic modifier), provides a solution to rapid analysis and isolation of tautomeric reference standards for quality control of TCM.

Phototheranostic nanoplatform based on a single cyanine dye for image-guided combinatorial phototherapy.

This study represents a novel phototheranostic nanoplatform based on the near-infrared (NIR) heptamethine cyanine dye, IR775, which is capable of concurrent real-time fluorescence imaging and cancer eradication with combinatorial phototherapy. To achieve water solubility and enhance tumor delivery, the hydrophobic IR775 dye was loaded into a biocompatible polymeric nanoparticle with a diameter of ~40nm and slightly negative surface charge (-2.34mV). The nanoparticle-encapsulated hydrophobic IR775 dye (IR775-NP) is characterized by an enhanced fluorescence quantum yield (16%) when compared to the water soluble analogs such as ICG (2.7%) and IR783 (8%). Furthermore, the developed IR-775-NP efficiently generates both heat and reactive oxygen species under NIR light irradiation, eradicating cancer cells in vitro. Finally, animal studies revealed that the IR775-NP accumulates in cancer tumors after systemic administration, efficiently delineates them with NIR fluorescence signal and completely eradicates chemo resistant cancer tissue after a single dose of combinatorial phototherapy.

Development of propidium iodide as a fluorescence probe for the on-line screening of non-specific DNA-intercalators in Fufang Banbianlian Injection.

Fufang Banbianlian Injection (FBI) has been widely used as an anti-inflammatory and anti-tumor prescription. To understand the relationships between its bioactive ingredients and pharmacological efficacies, our previous study has been successfully identified some DNA-binding compounds in FBI using an established on-line screening system, in which 4',6-diamidino-2-phenylindole (DAPI) was developed as a probe. However, DAPI can be only used to screen ATT-specific DNA minor groove binders, leaving the potential active intercalators unknown in FBI. As a continuation of our studies on FBI, here we present a sensitive analytical method for rapid identification and evaluation of DNA-intercalators using propidium iodide (PI) as a fluorescent probe. We have firstly established the technique of high-performance liquid chromatography-diode-array detector-multistage mass spectrometry-deoxyribonucleic acid-propidium iodide-fluorescence detector (HPLC-DAD-MS(n)-DNA-PI-FLD) system. As a result, 38 of 58 previously identified compounds in FBI were DNA-intercalation active. Interestingly, all previously reported DNA-binders also showed intercalative activities, suggesting they are dual-mode DNA-binders. Quantitative study showed that flavonoid glycosides and chlorogenic acids were the main active compounds in FBI, and displayed similar DNA-binding ability using either DAPI or PI. In addition, 13 active compounds were used to establish the structure-activity relationships. In this study, PI was developed into an on-line method for identifying DNA-intercalators for the first time, and thus it will be a useful high-throughput screening technique for other related samples.

Formation of Volatile Tea Constituent Indole During the Oolong Tea Manufacturing Process.

Indole is a characteristic volatile constituent in oolong tea. Our previous study indicated that indole was mostly accumulated at the turn over stage of oolong tea manufacturing process. However, formation of indole in tea leaves remains unknown. In this study, one tryptophan synthase α-subunit (TSA) and three tryptophan synthase ß-subunits (TSBs) from tea leaves were isolated, cloned, sequenced, and functionally characterized. Combination of CsTSA and CsTSB2 recombinant protein produced in Escherichia coli exhibited the ability of transformation from indole-3-glycerol phosphate to indole. CsTSB2 was highly expressed during the turn over process of oolong tea. Continuous mechanical damage, simulating the turn over process, significantly enhanced the expression level of CsTSB2 and amount of indole. These suggested that accumulation of indole in oolong tea was due to the activation of CsTSB2 by continuous wounding stress from the turn over process. Black teas contain much less indole, although wounding stress is also involved in the manufacturing process. Stable isotope labeling indicated that tea leaf cell disruption from the rolling process of black tea did not lead to the conversion of indole, but terminated the synthesis of indole. Our study provided evidence concerning formation of indole in tea leaves for the first time.

Determination of volatile marker compounds of common coffee roast defects.

Coffee beans from the same origin were roasted using six time-temperature profiles, in order to identify volatile aroma compounds associated with five common roast coffee defects (light, scorched, dark, baked and underdeveloped). Thirty-seven volatile aroma compounds were selected on the basis that they had previously been identified as potent odorants of coffee and were also identified in all coffee brew preparations; the relative abundance of these aroma compounds was then evaluated using gas chromatography mass spectrometry (GC-MS) with headspace solid phase micro extraction. Some of the 37 key aroma compounds were significantly changed in each coffee roast defect and changes in one marker compound was chosen for each defect type, that is, indole for light defect, 4-ethyl-2-methoxyphenol for scorched defect, phenol for dark defect, maltol for baked defect and 2,5-dimethylfuran for underdeveloped defect. The association of specific changes in aroma profiles for different roast defects has not been shown previously and could be incorporated into screening tools to enable the coffee industry quickly identify if roast defects occur during production.

A new method for rapid determination of indole-3-carbinol and its condensation products in nutraceuticals using core-shell column chromatography method.

Indole-3-carbinol is a natural glucosinolate known for prevention of human breast, prostate and other types of cancer and it started to be used in commercial preparations, as food supplements. However no analytical method has been proposed for quality control of nutraceuticals with this substance yet. In this paper a new high-performance liquid chromatography (HPLC) method using core-shell column for separation of indole-3-carbinol and its condensation/degradation products was developed and used for the quantitative determination of indole-3-carbinol in nutraceuticals. Separation of indole-3-carbinol, its condensation/degradation products and internal standard ethylparaben was performed on the core-shell column Kinetex 5µ XB-C18 100A (100×4.6mm), particle size 5.0µm, with mobile phase acetonitrile/water according to the gradient program at a flow rate of 1.25mLmin(-1) and at temperature 50°C. The detection wavelength was set at 270nm. Under the optimal chromatographic conditions good linearity of determination was achieved. Available commercial samples of nutraceuticals were extracted with 100% methanol using ultrasound bath. A 5-µL sample volume of the supernatant was directly injected into the HPLC system. The developed method provided rapid and accurate tool for quality control of nutraceuticals based on cruciferous vegetable extracts with indole-3-carbinol content. The presented study showed that the declared content of indole-3-carbinol significantly varied in the different nutraceuticals available on the market. Two analyzed preparations showed the presence of condensation/degradation products of indole-3-carbinol which were not officially declared by the manufacturer. Moreover, further two analyzed nutraceutical preparations showed absolutely no content of declared amount of indole-3-carbinol.