Developing new green solvents is one of the key subjects in Green Chemistry. Ionic liquids (ILs) and deep eutectic solvents, thus, have been paid great attention to replace current harsh organic solvents and have been applied to many chemical processing such as extraction and synthesis. However, current ionic liquids and deep eutectic solvents have still limitations to be applied to a real chemical industry due to toxicity against human and environment and high cost of ILs and solid state of most deep eutectic solvents at room temperature. Recently we discovered that many plant abundant primary metabolites changed their state from solid to liquid when they were mixed in proper ratio. This finding made us hypothesize that natural deep eutectic solvents (NADES) play a role as alternative media to water in living organisms and tested a wide range of natural products, which resulted in discovery of over 100 NADES from nature. In order to prove deep eutectic feature the interaction between the molecules was investigated by nuclear magnetic resonance spectroscopy. All the tested NADES show clear hydrogen bonding between components. As next step physical properties of NADES such as water activity, density, viscosity, polarity and thermal properties were measured as well as the effect of water on the physical properties. In the last stage the novel NADES were applied to the solubilization of wide range of biomolecules such as non-water soluble bioactive natural products, gluten, starch, and DNA. In most cases the solubility of the biomolecules evaluated in this study was greatly higher than water. Based on the results the novel NADES may be expected as potential green solvents at room temperature in diverse fields of chemistry.

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Nuclear magnetic resonance (NMR)-based metabolomics has many applications in plant science. Metabolomics can be used in functional genomics and to differentiate plants from different origin, or after different treatments. In this protocol, the following steps of plant metabolomics using NMR spectroscopy are described: sample preparation (freeze drying followed by extraction by ultrasonication with 1:1 CD(3)OD:KH(2)PO(4) buffer in D(2)O), NMR analysis (standard (1)H, J-resolved, (1)H-(1)H correlation spectroscopy (COSY) and heteronuclear multiple bond correlation (HMBC)) and chemometric methods. The main advantage of NMR metabolomic analysis is the possibility of identifying metabolites by comparing NMR data with references or by structure elucidation using two-dimensional NMR. This protocol is particularly suited for the analysis of secondary metabolites such as phenolic compounds (usually abundant in plants), and for primary metabolites (e.g., sugars and amino acids). This procedure is rapid; it takes not more than 30 min for sample preparation (multiple parallel) and a further 10 min for NMR spectrum acquisition.

Previously it was demonstrated that natural deep eutectic solvents (NADES) are promising green solvents for the extraction of natural products. However, despite their potential, an obvious disadvantage of NADES is the high viscosity. Here we explored the dilution effect on the structures and physicochemical properties of NADES and their improvements of applications using quercetin and carthamin. The results of FT-IR and (1)H NMR experiments demonstrated that there are intensive H-bonding interactions between the two components of NADES and dilution with water caused the interactions weaken gradually and even disappeared completely at around 50% (v/v) water addition. A small amount of water could reduce the viscosity of NADES to the range of water and increase the conductivity by up to 100 times for some NADES. This study provides the basis for modulating NADES in a controllable way for their applications in food processing, enzyme reactions, pharmaceuticals and cosmetics.

The capsaicin-sensitive vanilloid receptor (VR1) was recently shown to play an important role in inflammatory pain (hyperalgesia), but the underlying mechanism is unknown. We hypothesized that pain-producing inflammatory mediators activate capsaicin receptors by inducing the production of fatty acid agonists of VR1. This study demonstrates that bradykinin, acting at B2 bradykinin receptors, excites sensory nerve endings by activating capsaicin receptors via production of 12-lipoxygenase metabolites of arachidonic acid. This finding identifies a mechanism that might be targeted in the development of new therapeutic strategies for the treatment of inflammatory pain. V R1, a cloned capsaicin receptor, is a nonspecific cation channel expressed preferentially in small sensory neurons and activated by the vanilloids, capsaicin and resiniferatoxin (1). Because VR1 is also activated by heat and acid (1, 2), it is now considered to be a molecular sensor that detects a variety of painful stimuli. Indeed, recent experiments performed in mice that lack VR1 demonstrated that the receptor is essential for inflammation-induced heat hyperalgesia (3, 4). Therefore, understanding the cellular mechanisms by which capsaicin receptors are activated by inflammatory mediators may be a key to identifying novel therapeutic targets for pain treatment. Because of the presence of VR1 in sensory neurons and an apparent role in inflammatory hyperalgesia, endogenous activators of VR1 have been suspected. We recently demonstrated that products of the lipoxygenase pathway of arachidonic acid (AA) metabolism can activate capsaicin receptors (5). Among the eicosanoids tested, the 12-lipoxygenase product, 12-hydroperoxyeicosatetraenoic acid (12-HPETE), structurally similar to capsaicin, was the most potent VR1 agonist. Thus, metabolic products of lipoxygenases become candidates for the endogenous capsaicinlike substances. However, the upstream signals that stimulate lipoxygenase and activate VR1 are elusive.Bradykinin (BK) is a potent inflammatory mediator that causes pain and hyperalgesia. BK is known to activate as well as sensitize sensory neurons to other stimuli. Various signaling pathways have been suggested to mediate the sensitizing effect of BK on sensory neurons (6, 7). However, activation mechanism by BK is not known. BK is now known to stimulate the production of AA in sensory neurons (8), a key substrate of lipoxygenases. Therefore, on the basis of previous observations that products of lipoxygenase activate VR1 (5), we hypothesized that BK excites sensory neurons by opening the capsaicin receptor via production of 12-lipoxygenase products of AA metabolism. Materials and MethodsCell Culture. Experiments were carried out according to the Ethical Guidelines of the International Association for the Study of Pain and approved by the research ethics committee for the use of animals of the Seoul National University and the University of California, San Francisco. Thoracic and lumbar dorsal root ganglia (DRGs) were dissected from 1-to 2 day-...

Developing green solvents with low toxicity and cost is an important issue for the biochemical industry. Synthetic ionic liquids and deep eutectic solvents have received considerable attention due to their negligible volatility at room temperature, high solubilization ability, and tunable selectivity. However, the potential toxicity of the synthetic ionic liquids and the solid state at room temperature of most deep eutectic solvents hamper their application as extraction solvents. In this study, a wide range of recently discovered natural ionic liquids and deep eutectic solvents (NADES) composed of natural compounds were investigated for the extraction of phenolic compounds of diverse polarity. Safflower was selected as a case study because its aromatic pigments cover a wide range of polarities. Many advantageous features of NADES (such as their sustainability, biodegradability combined with acceptable pharmaceutical toxicity profiles, and their high solubilization power of both polar and nonpolar compounds) suggest their potential as green solvents for extraction. Experiments with different NADES and multivariate data analysis demonstrated that the extractability of both polar and less polar metabolites was greater with NADES than conventional solvents. The water content in NADES proved to have the biggest effect on the yield of phenolic compounds. Most major phenolic compounds were recovered from NADES with a yield between 75% and 97%. This study reveals the potential of NADES for applications involving the extraction of bioactive compounds from natural sources.

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