Liang-Yu Chen

Effect of esterification condensation on the Folin-Ciocalteu method for the quantitative measurement of total phenols

The Folin-Ciocalteu method is widely applied for the determination of the total phenolic contents in natural products. This method is significantly affected by the addition of sodium carbonate. The currently applied Folin-Ciocalteu methods may have been modified without any validation in the quantitative standards and the order of processes. In this study, serial experiments were performed to investigate the effect of phenolic calibrations based on the classic Folin-Ciocalteu method. Esterification condensations were observed in the assays with prior basification for gallic acid and catechin used as quantitative standards. The phenolic contents obtained in the samples differed depending on when basification occurred compared with the gallic acid calibration. The bias of the classic Folin-Ciocalteu method derived from cross-linkage of molecules was first defined in this study. The performance of the Folin-Ciocalteu method is optimised and validated again.

Blue light induced free radicals from riboflavin on E. coli DNA damage.

The micronutrients in many cellular processes, riboflavin (vitamin B(2)), FMN, and FAD are photo-sensitive to UV and visible light to generate reactive oxygen species (ROS). The riboflavin photochemical treatment with UV light has been applied for the inactivation of microorganisms to serve as an effective and safe technology. Ultra-violet or high-intensity radiation is, however, considered as a highly risky practice. This study was working on the application of visible LED lights to riboflavin photochemical reactions to development an effective antimicrobial treatment. The photosensitization of bacterial genome with riboflavin was investigated in vitro and in vivo by light quality and irradiation dosage. The riboflavin photochemical treatment with blue LED light was proved to be able to inactivate E. coli by damaging nucleic acids with ROS generated. Riboflavin is capable of intercalating between the bases of bacterial DNA or RNA and absorbs lights in the visible regions. LED light illumination could be a more accessible and safe practice for riboflavin photochemical treatments to achieve hygienic requirements in vitro.

Investigations of riboflavin photolysis via coloured light in the nitro blue tetrazolium assay for superoxide dismutase activity

Determination of the superoxide dismutase activity is an important issue in the fields of biochemistry and the medical sciences. In the riboflavin/nitro blue tetrazolium (B2/NBT) method, the light sources used for generating superoxide anion radicals from light-excited riboflavin are normally fluorescent lamps. However, the conditions of B2/NBT experiments vary. This study investigated the effect of the light source on the light-excitation of riboflavin. The effectiveness of the photolysis was controlled by the wavelength of the light source. The spectra of fluorescent lamps are composed of multiple colour lights, and the emission spectra of fluorescent lamps made by different manufacturers may vary. Blue light was determined to be the most efficient for the photochemical reaction of riboflavin in visible region. The quality of the blue light in fluorescent lamps is critical to the photo-decomposition of riboflavin. A blue light is better than a fluorescent lamp for the photo-decomposition of riboflavin. The performance of the B2/NBT method is thereby optimized.

Investigations of blue light-induced reactive oxygen species from flavin mononucleotide on inactivation of E. coli.

The micronutrients in many cellular processes, riboflavin, flavin mononucleotide (FMN), and flavin adenine dinucleotide (FAD) are photo-sensitive to UV and visible light for generating reactive oxygen species (ROS). Produced from phosphorylation of riboflavin, FMN is more water-soluble and rapidly transformed into free riboflavin after ingestion. This study investigated the application of visible blue light with FMN to development of an effective antimicrobial treatment. The photosensitization of bacterial viability with FMN was investigated by light quality, intensity, time, and irradiation dosage. The blue light-induced photochemical reaction with FMN could inactivate Escherichiacoli by the generated ROS in damaging nucleic acids, which was validated. This novel photodynamic technique could be a safe practice for photo-induced inactivation of environmental microorganism to achieve hygienic requirements in food processing.

Determination of Schistosoma japonicum circulating antigens in dilution serum by piezoelectric immunosensor and S/N enhancement.

A piezoelectric immunosensor assay was developed with immobilizing immunoglobulin G (IgG) as a probe to detect Schistosoma japonicum circulating antigens (SjCAg). Analytical strategy utilizes the polyclonal antibodies with broad-spectrum recognition to a complex target with high specificity. The immobilized antibodies were purified from immunized rabbits sera (im-S) and infected rabbits sera (inf-S) by S. japonicum. The detection capacities of antibodies were compared between the sera of different phenotypes and purified fractions. The sample dilution ratios were also evaluated and optimized. Additionally, the sera with a variety of infection degrees for validation could be discriminated quantitatively. The linear dose-response relationship indicates that the systematic sensitivity of this method is below 150 Hz and the lowest limit of detectable range is above 500 cercariae of S. japonicum infection for 2 weeks. The novel immunosensor technique is well potential to determine the SjCAg in serum samples for clinical diagnosis of parasitosis in early stage.

Effect of colour LEDs on mycelia growth of Aspergillus ficuum and phytase production in photo-fermentations.

Aspergillus ficuum grown on plates and in liquid cultures were illuminated by a white fluorescent light and four different colour LED lights (white, blue, green and red) to evaluate the regulation of LED lights on fungal growth. Biomass conversion, pellet size and phytase activity were examined. In liquid culture, luminous intensity was highly correlated with the rate of biomass conversion but did not affect pellet size. The white fluorescent light contained several different wavelengths, and therefore, its effect on A. ficuum represents the cooperative effect of these wavelengths. Strong luminance of a white fluorescent light inhibited growth of A. ficuum mycelia on plates, whereas white LED light enhanced growth. The development of mycelia was also inhibited by blue LED light and enhanced by red LED light illumination. Investigating the effect of LED lights on the growth of A. ficuum could provide evidence on the luminous intensity that is sufficient for regulating fermentation by light.

The Degradation Mechanism of Toxic Atractyloside in Herbal Medicines by Decoction

Atractyloside (ATR) is found in many Asteraceae plants that are commonly used as medicinal herbs in China and other eastern Asian countries. ATR binds specifically to the adenine nucleotide translocator in the inner mitochondrial membrane and competitively inhibits ADP and ATP transport. The toxicity of ATR in medical herbs can be reduced by hydrothermal processing, but the mechanisms of ATR degradation are not well understood. In this study, GC-MS coupled with SPE and TMS derivatisation was used to detect ATR levels in traditional Chinese medicinal herbs. Our results suggest that ATR molecules were disrupted by decomposition, hydrolysis and saponification after heating with water (decoction) for a long period of time. Hydrothermal processing could decompose the endogenous toxic compounds and also facilitate the detoxification of raw materials used in the Chinese medicine industry.

Blue light irradiation triggers the antimicrobial potential of ZnO nanoparticles on drug-resistant Acinetobacter baumannii

Photodynamic inactivation (PDI) is a non-invasive and safe therapeutic method for microbial infections. Bacterial antibiotic resistance is caused by antibiotics abuse. Drug-resistant Acinetobacter spp. is a serious problem in hospitals around the world. These pathogens from nosocomial infections have high mortality rates in frailer people, and Acinetobacter spp. is commonly found in immunocompromised patients. Visible light is safer than ultraviolet light (UV) for PDI of nosocomial pathogens with mammalian cells. Zinc oxide nanoparticles (ZnO-NPs) were used in this study as an antimicrobial agent and a photosensitizer. ZnO is recognized as safe and has extensive usage in food additives, medical and cosmetic products. In this study, we used 0.125 mg/ml ZnO-NPs combined with 10.8 J/cm2 blue light (BL) on Acinetobacter baumannii (A. baumannii) that could significantly reduce microbial survival. However, individual exposure to ZnO-NPs does not affect the viability of A. baumannii. BL irradiation could trigger the antimicrobial ability of ZnO nanoparticles on A. baumannii. The mechanism of photocatalytic ZnO-NPs treatment for sterilization occurs through bacterial membrane disruptions. Otherwise, the photocatalytic ZnO-NPs treatment showed high microbial eradication in nosocomial pathogens, including colistin-resistant and imipenem-resistant A. baumannii and Klebsiella pneumoniae. Based on our results, the photocatalytic ZnO-NPs treatment could support hygiene control and clinical therapies without antibiotics to nosocomial bacterial infections.

Photo-catalytic polymerization of catechin molecules in alkaline aqueous

Polyphenols are associated with a wide range of physiological properties. Catechin is a flavan-3-ol with five phenolic hydroxyl groups. After blue light illumination, the transparent solution of catechin became yellowish. The effects of visible light illumination (400-800nm) were investigated on molecular structures and antioxidant capacities of catechin. Under the neutral or alkaline aqueous with the illumination of blue light, the photolysis and polymerization of catechin were observed in this study. A chromogenic catechin dimer was separated and identified as a proanthocyanidin by the chromatographic technique and mass spectrometry. For quantitative evaluation, the signal intensities of the catechin and the photochemical product show a negative correlation in the liquid chromatograms. The oligomer of flavan-3-ols (catechin dimer) is suggested as a dimeric B type proanthocyanidin, which has the molecular formula C30H26O12 and 578.14g/mol in exact mass. The mass spectrum of catechin dimer had characteristic ion signals in m/z 577, 560, 439Da. However, the total phenolic contents and scavenging O2- activity of catechin treated by blue light illumination are not changed significantly at the neutral or alkaline aqueous. Our results of photocatalytic oligomers of catechin provide a novel way to explain the sensory changes of green tea and a biochemical mechanism under the irradiation environments.

Blue light induced reactive oxygen species from flavin mononucleotide and flavin adenine dinucleotide on lethality of HeLa cells

Photodynamic therapy (PDT) is a safe and non-invasive treatment for cancers and microbial infections. Various photosensitizers and light sources have been developed for clinical cancer therapies. Flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD) are the cofactor of enzymes and are used as photosensitizers in this study. Targeting hypoxia and light-triggering reactive oxygen species (ROS) are experimental strategies for poisoning tumor cells in vitro. HeLa cells are committed to apoptosis when treated with FMN or FAD and exposed to visible blue light (the maximum emitted wavelength of blue light is 462nm). Under blue light irradiation at 3.744J/cm2 (=0.52mW/cm2 irradiated for 2h), the minimal lethal dose is 3.125μM and the median lethal doses (LD50) for FMN and FAD are 6.5μM and 7.2μM, respectively. Individual exposure to visible blue light irradiation or riboflavin photosensitizers does not produce cytotoxicity and no side effects are observed in this study. The western blotting results also show that an intrinsic apoptosis pathway is activated by the ROS during photolysis of riboflavin analogues. Blue light triggers the cytotoxicity of riboflavins on HeLa cells in vitro. Based on these results, this is a feasible and efficient of PDT with an intrinsic photosensitizer for cancer research.