Resolution of isoborneol and its isomers by GC/MS to identify "synthetic" and "semi-synthetic" borneol products.

Borneol is a plant terpene commonly used in traditional Chinese medicine. Optically pure (+)-borneol and (-)-borneol can be obtained by extraction from the plants Dipterocarpaceae and Blumea balsamifera, respectively. "Synthetic borneol" is obtained from the reduction of (±)-camphor to lead to four different stereoisomers: (+)-isoborneol, (-)-isoborneol, (+)-borneol, and (-)-borneol. In contrast, "semi-synthetic borneol" is produced from the reduction of natural camphor, (+)-camphor, to afford two isomers: (-)-isoborneol and (+)-borneol. We established a convenient method to identify them by treating the four stereoisomers with two chiral reagents, (R)-(+)-α-methoxy-α-trifluoromethylphenylacetyl chloride ((R)-(+)-MTPA-Cl) and (1S)-(-)- camphanic chloride. The resulting derivatives from the above mentioned method were analyzed by gas chromatography. The enantiomers of (+)- and (-)-isoborneol were successfully separated from (+)- and (-)-borneol isomers in this study to make this a useful method in the identification of "synthetic" and "semi-synthetic" borneols. Furthermore, we also examined five different commercial borneols. During this course, a novel and unprecedented partial epimerization from isoborneol-camphanic ester to borneol-camphanic ester was observed. However, this phenomenon did not occur in isoborneol-MTPA esters epimerization to borneol-MTPA case under the same conditions. The DFT calculation of activation energies for both reactions was in a good agreement with the results obtained from GC analysis.

Efficient pH dependent drug delivery to target cancer cells by gold nanoparticles capped with carboxymethyl chitosan.

Doxorubicin (DOX) was immobilized on gold nanoparticles (AuNPs) capped with carboxymethyl chitosan (CMC) for effective delivery to cancer cells. The carboxylic group of carboxymethyl chitosan interacts with the amino group of the doxorubicin (DOX) forming stable, non-covalent interactions on the surface of AuNPs. The carboxylic group ionizes at acidic pH, thereby releasing the drug effectively at acidic pH suitable to target cancer cells. The DOX loaded gold nanoparticles were effectively absorbed by cervical cancer cells compared to free DOX and their uptake was further increased at acidic conditions induced by nigericin, an ionophore that causes intracellular acidification. These results suggest that DOX loaded AuNPs with pH-triggered drug releasing properties is a novel nanotheraputic approach to overcome drug resistance in cancer.

Kalanchoe tubiflora extract inhibits cell proliferation by affecting the mitotic apparatus.

BACKGROUND: Kalanchoe tubiflora (KT) is a succulent plant native to Madagascar, and is commonly used as a medicinal agent in Southern Brazil. The underlying mechanisms of tumor suppression are largely unexplored. METHODS: Cell viability and wound-healing were analyzed by MTT assay and scratch assay respectively. Cell cycle profiles were analyzed by FACS. Mitotic defects were analyzed by indirect immunofluoresence images. RESULTS: An n-Butanol-soluble fraction of KT (KT-NB) was able to inhibit cell proliferation. After a 48 h treatment with 6.75 µg/ml of KT, the cell viability was less than 50% of controls, and was further reduced to less than 10% at higher concentrations. KT-NB also induced an accumulation of cells in the G2/M phase of the cell cycle as well as an increased level of cells in the subG1 phase. Instead of disrupting the microtubule network of interphase cells, KT-NB reduced cell viability by inducing multipolar spindles and defects in chromosome alignment. KT-NB inhibits cell proliferation and reduces cell viability by two mechanisms that are exclusively involved with cell division: first by inducing multipolarity; second by disrupting chromosome alignment during metaphase. CONCLUSION: KT-NB reduced cell viability by exclusively affecting formation of the proper structure of the mitotic apparatus. This is the main idea of the new generation of anti-mitotic agents. All together, KT-NB has sufficient potential to warrant further investigation as a potential new anticancer agent candidate.

Production of optically pure (-)-borneol by Pseudomonas monteilii TCU-CK1 and characterization of borneol dehydrogenase involved.

(-)-Borneol is a bicyclic plant secondary metabolite. Optically pure (-)-borneol can only be obtained from plants, and demand exceeds supply in China. In contrast, chemically synthesized borneol contains four different stereoisomers. A strain of Pseudomonas monteilii TCU-CK1, isolated in Hualien, Taiwan, can accumulate (-)-borneol in growth culture and selectively degrades the other three isomers when chemically synthesized borneol is used as sole carbon source. This (-)-borneol production method can be scaled-up for production of large quantities in the future. More importantly, laborious plant cultivation and harvest is no longer required. The main enzyme that appears in this degradation pathway, borneol dehydrogenase (BDH), and the genome sequence of TCU-CK1 are reported. The kcat/Km values of TCU-CK1 BDH on (+)- and (-)-borneol are 538.4 ± 38.4 and 17.7 ± 1.1 (s-1 mM-1), respectively. About ∼30 fold difference in the kcat/Km value between (+)-borneol and (-)-borneol was observed, in good agreement with the fact that TCU-CK1 prefers to degrade (+)-borneol, rather than (-)-borneol. A BDH isozyme was identified in a strain in which the primary BDH gene had been knocked out. (-)-Camphor can work as an inhibitor of BDH with a Ki of 1.03 ± 0.11 mM at pH 7.0, leading to the accumulation of (-)-borneol in culture. (Patent pending).

Using gas chromatography and mass spectrometry to determine 25-hydroxyvitamin D levels for clinical assessment of vitamin D deficiency.

Vitamin D is responsible for multiple metabolic functions in humans. Rickets are the most common disease caused by vitamin D deficiency. It is caused by poor calcium intake resulting in poor serum-ionized calcium. The purpose of this study is to develop a rapid, sensitive, and feasible method to determine the 25-hydroxy-vitamin D3 (25(OH)D3) levels in blood samples for clinical assessment. In this study, gas chromatography coupled mass spectrometry with trimethylsilyl derivatization (TMS-GC-MS) is the most suitable protocol for quantitative analyses of 25(OH)D3. Performance of method was evaluated and compared with liquid chromatography and immunoassay. Method validation has been carried out with plasma specimens. The limit of quantitation of TMS-GC-MS method is 1.5 ppb with good linear correlation. Furthermore, the dietary intake and nutritional status of vegetarian and non-vegetarians in Taiwan were assessed by our validated method. As a result, this vitamin D nutrition survey demonstrates that most Taiwanese people have insufficient vitamin D. Due to dietary habits; the male vegans may have the highest risk of vitamin D deficiency.

Low-dose blue light irradiation enhances the antimicrobial activities of curcumin against Propionibacterium acnes.

Propionibacterium acnes (P. acnes) is an opportunistic infection in human skin that causes acne vulgaris. Antibiotic agents provide the effective eradication of microbes until the development of drug-resistant microbes. Photodynamic inactivation (PDI) is a non-antibiotic therapy for microbial eradication. In this study, the visible blue light (BL, λmax = 462 nm) was used to enhance the antimicrobial activities of curcumin, a natural phenolic compound. Individual exposure to curcumin or BL irradiation does not generate cytotoxicity on P. acnes. The viability of P. acnes was decreased significantly in 0.09 J/cm2 BL with 1.52 µM of curcumin. Furthermore, the low-dose blue light irradiation triggers a series of cytotoxic actions of curcumin on P. acnes. The lethal factors of photolytic curcumin were investigated based on the morphology of P. acnes by SEM and fluorescent images. The membrane disruption of microbes was observed on the PDI against P. acnes. Chromatography and mass spectrometry techniques were also used to identify the photolytic metabolites. Curcumin could be photolysed into vanillin through BL irradiation, which presents a strong linear relationship in quantitation. Because the safety of blue light in mammalian cell has been proven, the photolytic curcumin treatment could support non-antibiotic therapy to eradicate P. acnes on clinical dermatology.

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.

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.

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.