Allosteric ligands for the pharmacologically dark receptors GPR68 and GPR65.

At least 120 non-olfactory G-protein-coupled receptors in the human genome are 'orphans' for which endogenous ligands are unknown, and many have no selective ligands, hindering the determination of their biological functions and clinical relevance. Among these is GPR68, a proton receptor that lacks small molecule modulators for probing its biology. Using yeast-based screens against GPR68, here we identify the benzodiazepine drug lorazepam as a non-selective GPR68 positive allosteric modulator. More than 3,000 GPR68 homology models were refined to recognize lorazepam in a putative allosteric site. Docking 3.1 million molecules predicted new GPR68 modulators, many of which were confirmed in functional assays. One potent GPR68 modulator, ogerin, suppressed recall in fear conditioning in wild-type but not in GPR68-knockout mice. The same approach led to the discovery of allosteric agonists and negative allosteric modulators for GPR65. Combining physical and structure-based screening may be broadly useful for ligand discovery for understudied and orphan GPCRs.

Transformation Mechanisms of Chemical Ingredients in Steaming Process of Gastrodia elata Blume.

To explore the transformation mechanisms of free gastrodin and combined gastrodin before and after steaming of Gastrodia elata (G. elata), a fresh G. elata sample was processed by the traditional steaming method prescribed by Chinese Pharmacopoeia (2015 version), and HPLC-ESI-TOF/MS method was used to identify the chemical composition in steamed and fresh G. elata. Finally, 25 components were identified in G. elata based on the characteristic fragments of the compounds and the changes of the 25 components of fresh and steamed G. elata were compared by the relative content. Hydrolysis experiments and enzymatic hydrolysis experiments of 10 monomer compounds simulating the G. elata steaming process were carried out for the first time. As a result, hydrolysis experiments proved that free gastrodin or p-hydroxybenzyl alcohol could be obtained by breaking ester bond or ether bond during the steaming process of G. elata. Enzymatic experiments showed that steaming played an important role in the protection of gastrodin, confirming the hypothesis that steaming can promote the conversion of chemical constituents of G. elata-inhibiting enzymatic degradation. This experiment clarified the scientific mechanism of the traditional steaming method of G. elata and provided reference for how to apply G. elata decoction to some extent.

Quality Evaluation of Gastrodia Elata Tubers Based on HPLC Fingerprint Analyses and Quantitative Analysis of Multi-Components by Single Marker.

Gastrodia elata (G. elata) tuber is a valuable herbal medicine used to treat many diseases. The procedure of establishing a reasonable and feasible quality assessment method for G. elata tuber is important to ensure its clinical safety and efficacy. In this research, an effective and comprehensive evaluation method for assessing the quality of G. elata has been developed, based on the analysis of high performance liquid chromatography (HPLC) fingerprint, combined with the quantitative analysis of multi-components by single marker (QAMS) method. The contents of the seven components, including gastrodin, p-hydroxybenzyl alcohol, p-hydroxy benzaldehyde, parishin A, parishin B, parishin C, and parishin E were determined, simultaneously, using gastrodin as the reference standard. The results demonstrated that there was no significant difference between the QAMS method and the traditional external standard method (ESM) (p > 0.05, RSD < 4.79%), suggesting that QAMS was a reliable and convenient method for the content determination of multiple components, especially when there is a shortage of reference substances. In conclusion, this strategy could be beneficial for simplifying the processes in the quality control of G. elata tuber and giving references to promote the quality standards of herbal medicines.

Novel Strategies Using Total Gastrodin and Gastrodigenin, or Total Gastrodigenin for Quality Control of Gastrodia elata.

Gastrodia elata Blume (G. elata), a traditional Chinese medicine, is widely used for treatment of various neuro dysfunctions. However, its quality control is still limited to the determination of gastrodin. In the present study, two novel strategies based on quantitative evaluation of total gastrodin and gastrodigenin with base hydrolysis and total gastrodigenin with base-enzymatic hydrolysis followed by HPLC-FLD were put forward and successfully applied to evaluate the quality of 47 batches of G. elata from eight localities. Meanwhile, a systematic comparison of the novel strategy with the multiple markers and the Pharmacopeia method was performed. The results showed that the parishins category could be completely hydrolyzed to gastrodin by sodium hydroxide solution, and gastrodin could further utterly hydrolyze to gastrodigenin with ß-d-glucosidase buffer solution. The contents of total gastrodin and gastrodigenin ranged from 1.311% to 2.034%, and total gastrodigenin from 0.748% to 1.120% at the eight localities. From the comparison, we can conclude that the two novel strategies can comprehensively reveal the characteristics of overall active ingredients in G. elata for quality control. The present study provides a feasible and credible strategy for the quality control of G. elata, suggesting a revision of the latest Chinese Pharmacopoeia or European Pharmacopoeia methods for the modernization of G. elata use.

Enhanced Agronomic Traits and Medicinal Constituents of Autotetraploids in Anoectochilus formosanus Hayata, a Top-Grade Medicinal Orchid.

This study developed an efficient and reliable system for inducing polyploidy in Anoectochilus formosanus Hayata, a top-grade medicinal orchid. The resulting tetraploid gave a significant enhancement on various agronomic traits, including dry weight, fresh weight, shoot length, root length, leaf width, the size of stoma, and number of chloroplasts per stoma. A reduction of the ratio of length to width was observed in stomata and leaves of the tetraploid, and consequently, an alteration of organ shape was found. The major bioactive compounds, total flavonoid and gastrodin, were determined by the aluminum chloride colorimetric method and ultra performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS), respectively. The tetraploid produced significantly higher contents of total flavonoid and gastrodin in the leaf, the stem, and the whole plant when compared with the diploid. The resulting tetraploids in this study are proposed to be suitable raw materials in the pharmaceutical industry for enhancing productivity and reducing cost.

[Study on metabolites from a sponge-derived fungus].

We have carried out the investigation on a sponge-derived fungus,which was identified as Emericella variecolor from the south sea of China. Two new chemical constituents,(+)-2-acetyl-dihydroterrein (1) and (+)-3-acetyl-dihydroterrein (2),with four known compounds,anditomin (3),andilesin A (4),andilesin C (5) and andilesin B (6),were isolated from this fungus by column chromatography over silica gel, Sephadex LH-20, and ODS. The structures of 1 and 2 were elucidated by spectroscopic methods including NMR,HR-ESI-MS,and CD.

Phenotypic plasticity in a willow leaf beetle depends on host plant species: release and recognition of beetle odors.

Aggregation behavior of herbivorous insects is mediated by a wide range of biotic and abiotic factors. It has been suggested that aggregation behavior of the blue willow leaf beetle Phratora vulgatissima is mediated by both host plant odor and by odor released by the beetles. Previous studies show that the beetles respond to plant odors according to their prior host plant experiences. Here, we analyzed the effect of the host plant species on odor released and perceived by adult P. vulgatissima. The major difference between the odor of beetles feeding on salicin-rich and salicin-poor host plants was the presence of salicylaldehyde in the odor of the former, where both males and females released this compound. Electrophysiological studies showed that the intensity of responses to single components of odor released by beetles was sex specific and dependent on the host plant species with which the beetles were fed. Finally, behavioral studies revealed that males feeding on salicin-rich willows were attracted by salicylaldehyde, whereas females did not respond behaviorally to this compound, despite showing clear antennal responses to it. Finally, the ecological relevance of the influence of a host plant species on the plasticity of beetle odor chemistry, perception, and behavior is discussed.

[Exploring the best dried processing condition of fresh Gastrodia elata].

OBJECTIVE: To study the processing conditions that influenced the contents of gastrodine in dried processing of fresh Gastrodia elata. METHODS: The optimized processing condition were established by using single-factor and orthogonal experimental design, which was guided by the contents of gastrodine. Fresh Gastrodia elata were harvested in the day, cleaned sediment, cooked in atmospheric pressure for 25 min, cut into 0.5 - 1.0 cm thick slices and dried in 105 degrees C. The gastrodine content was measured by HPLC. RESULTS: The gastrodine content was 0.532%, which was higher than that of traditional processing method. CONCLUSION: Dried processing condition can influence the content of gastrodine in Gastrodia elata significantly.

[Simultaneous determination of gastrodin and eight nucleosides and nucleobases in Tibet cultured gastrodia elata by HPLC method].

This study aims to establish an HPLC method for simultaneous determination of gastrodin and eight nucleosides and nucleobases components in Gastrodia elata. The separation was carried out on an Agilent Zorbax Bonus-RP (4.6 mm x 250 mm, 5 µm) column with a methanol-(0.04% acetic acid) water solution gradient elution program at a flow rate of 1.0 mL x min(-1). The column temperature was 36 degrees C, and the detection wavelength was 254 nm. The volume of injection was 20 µL. The nine components including gastrodin, cytosine, uracil, cytosine, adenine, thymine, uridine, guanosine and adenosine were well separated. The calibration curve was well linear in the range of 2.04-262.00 mg x L(-1), 0.20-24.67 mg x L(-1), 0.18-23.75 mg x L(-1), 0.20-25.83 mg x L(-1), 0.20-26.67 mg x L(-1), 0.16-20.00 mg x L(-1), 0.22-27.71 mg x L(-1), 0.20-24.29 mg x L(-1), 0.24-30.58 mg x L(-1), respectively, and the correlation coefficient was between 0.998 9-0.999 9. The average recovery of gastrodin and eight nucleosides and nucleobases were 96.4%-99.6%, RSD less than 2.7% (n = 6). The contents of gastrodin in all the seven Tibet cultured Gastrodia elata samples were over 2 mg x g(-1). Further, all samples contain higher contents of adenosine, guanosine, uridine and cytidine compared to low contents of cytosine, uracil, adenine and thymine. The established method is accurate, reproducible and suitable for the determination of gastrodin and eight nucleosides and nucleobases comppnents in Gastrodia elata.

[Research the extraction and detection method of gastrodin content in Gastrodia elata].

OBJECTIVE: To improve an extraction and determination method for gastrodin in Gastrodia elata. METHODS: On the basis of single factor experiment, used the method of hot water reflux extraction and applied the response surface methodology to determine the best conditions for extracting gastrodin and using HPLC test. RESULTS: Under the boiling water condition, pure water as solvent, the solid-liquid ratio was 1:37,extraction 2 times and extraction time 126 min per times,the content of gastrodin was 0.377%. Comparison with the 2010 edition of the China pharmacopoeia of gastrodin extracted in Gastrodia elata, the result showed the gastrodin content was increased by 2. 12 times. CONCLUSION: This extraction method of gastrodin dissolution rate is high and can reflect the real content of gastrodin of Gastrodia elata.

Exposure to tri-o-cresyl phosphate detected in jet airplane passengers.

The aircraft cabin and flight deck ventilation are supplied from partially compressed unfiltered bleed air directly from the engine. Worn or defective engine seals can result in the release of engine oil into the cabin air supply. Aircrew and passengers have complained of illness following such "fume events". Adverse health effects are hypothesized to result from exposure to tricresyl phosphate mixed esters, a chemical added to jet engine oil and hydraulic fluid for its anti-wear properties. Our goal was to develop a laboratory test for exposure to tricresyl phosphate. The assay was based on the fact that the active-site serine of butyrylcholinesterase reacts with the active metabolite of tri-o-cresyl phosphate, cresyl saligenin phosphate, to make a stable phosphorylated adduct with an added mass of 80 Da. No other organophosphorus agent makes this adduct in vivo on butyrylcholinesterase. Blood samples from jet airplane passengers were obtained 24-48 h after completing a flight. Butyrylcholinesterase was partially purified from 25 ml serum or plasma, digested with pepsin, enriched for phosphorylated peptides by binding to titanium oxide, and analyzed by mass spectrometry. Of 12 jet airplane passengers tested, 6 were positive for exposure to tri-o-cresyl phosphate that is, they had detectable amounts of the phosphorylated peptide FGEpSAGAAS. The level of exposure was very low. No more than 0.05 to 3% of plasma butyrylcholinesterase was modified. None of the subjects had toxic symptoms. Four of the positive subjects were retested 3 to 7 months following their last airplane trip and were found to be negative for phosphorylated butyrylcholinesterase. In conclusion, this is the first report of an assay that detects exposure to tri-o-cresyl phosphate in jet airplane travelers.

Functional loss of pAMT results in biosynthesis of capsinoids, capsaicinoid analogs, in Capsicum annuum cv. CH-19 Sweet.

Capsaicinoids are responsible for the spicy flavor of pungent peppers (Capsicum). The cultivar CH-19 Sweet is a non-pungent pepper mutant derived from a pungent pepper strain, Capsicum annuum CH-19. CH-19 Sweet biosynthesizes capsaicinoid analogs, capsinoids. We determined the genetic and metabolic mechanisms of capsinoid biosynthesis in this cultivar. We analyzed the putative aminotransferase (pAMT) that is thought to catalyze the formation of vanillylamine from vanillin in the capsaicinoid biosynthetic pathway. Enzyme assays revealed that pAMT activity catalyzing vanillylamine formation was completely lost in CH-19 Sweet placenta tissue. RT-PCR analysis showed normal mRNA transcription of the pAMT gene; however, SNP analysis of the cDNA sequence showed a T nucleotide insertion at 1291 bp in the pAMT gene of CH-19 Sweet. This insertion formed a new stop codon, TGA, that prevented normal translation of the gene, and the pAMT protein did not accumulate in CH-19 Sweet as determined using Western blot analysis. We developed a dCAPS marker based on the T insertion in the pAMT gene of CH-19 Sweet, and showed that the pAMT genotype co-segregated with the capsinoid or capsaicinoid fruit phenotype in the F(2) population. The T insertion was not found in other pungent and non-pungent Capsicum lines, suggesting that it is specific to CH-19 Sweet. CH-19 Sweet's pAMT gene mutation is an example of a nonsense mutation in a single gene that alters a secondary metabolite biosynthetic pathway, resulting in the biosynthesis of analogs. The dCAPS marker will be useful in selecting lines with capsinoid-containing fruits in pepper-breeding programs.

Exploiting adduct formation through an auxiliary spray in liquid chromatography-electrospray ionization mass spectrometry to improve charge-carrier identification.

We describe a simple and effective approach to probe adduct formation in liquid chromatography - electrospray ionization mass spectrometry (LC-ESI-MS) and help designate and/or confirm which particular analyte is leading to which particular charged species that is detected. A compound tends to form similar adducts with adduct-forming analogs, at various abundance levels, of course. It is based on this understanding that in this work we probed adduct formation by modulating the adduct-forming analogs and observing the adducts formed with these analogs to lend experimental evidence to adduct annotation. The approach was implemented through an auxiliary spray and made possible thanks to the interaction between the plumes of the sample spray or main spray and the auxiliary spray. Changing adduct-forming analogs by switching the auxiliary spray solution, or simply turning on and off the auxiliary spray facilitated the observation of the adducts corresponding to these analogs or lack thereof, giving rise to a simple and effective approach to probe adduct formation and thus help annotate the analyte ions.

Relationships among genetic makeup, active ingredient content, and place of origin of the medicinal plant Gastrodia tuber.

Gastrodia tuber and its component gastrodin have many pharmacological effects. The chemical fingerprints and gastrodin contents of eight Gastrodia populations were determined, and the genomic DNA polymorphism of the populations was investigated. Genetic distance coefficients among the populations were calculated using the DNA polymorphism data. A dendrogram of the genetic similarities between the populations was constructed using the genetic distance coefficients. The results indicated that the genomic DNA of Gastrodia tubers was highly polymorphic; the eight populations clustered into three major groups, and the gastrodin content varied greatly among these groups. There were obvious correlations among genetic makeup, gastrodin content, and place of origin. The ecological environments in Guizhou and Shanxi may be conducive to evolution and to gastrodin biosynthesis, and more suitable for cultivation of Gastrodia tubers. These findings may provide a scientific basis for overall genetic resource management and for the selection of locations for cultivating Gastrodia tubers.

[The determination of three effective constituents in wild and cultivated Gastrodia elata from Bomi].

OBJECTIVE: To determine the contents of gastrodin, amino acids and total flavonoids in wild and cultivated Gastrodia elata, in order to choose the best method for its cultivation. The Gastrodia elata was picked at Guxiang town Bomi county Linzhi Region in Tibet. METHODS: HPLC was used to determine the content of gastrodin. The autoanalyzer was used to determine the content of amino acid. The ultraviolet spectrophotometer was adopted to measure the content of total flavonoids. RESULTS: The Gastrodin in wild Gastrodia elata was the highest. The contents of amino acids and total flavonoids in organic cultivated Gastrodia elata were higher than those in common cultivated and wild Gastrodia elata. CONCLUSION: The organic cultivated Gastrodia elata has better quality.

[Study on the preparation of sustained-release pellets containing active components from Gastrodia elata by fluid-bed coating].

OBJECTIVE: To study the formulation of sustained-release pellets containing active components from Gastrodia elata by coating in the fluid-bed. METHODS: The sustained-release pellets were prepared with Eudragit RS 100. The formulation was optimized by the direct comparison. Then their properties were evaluated. RESULTS: The pellets presented the perfect sphericity and narrow diameter distribution and the curve of their cumulative drug release was in accord with Higuchi equation. CONCLUSION: The pellets have sustained-release effect in 12 hours.

[Determination of seven indicative ingredients in oral liquids of Chinese medicine by liquid chromatography-electrospray ionization-ion mobility spectrometry].

A two-dimensional separation and analysis method, based on liquid chromatography-electrospray ionization-ion mobility spectrometry (LC-ESI-IMS), is developed for the determination of seven indicative ingredients (danshensu, glycyrrhizic acid, gastrodin, chlorogenic acid, puerarin, baicalin, and rutin) in oral liquids of Chinese medicine. The sample was first separated on an ACQUITY UPLC BEH C18 column (50 mm×1 mm, 1.7 µm). The post-column effluent was directed to an adjustable flow splitter with a split ratio of 50:1. The low-flow and high-flow outlets were connected to an ion mobility spectrometer and a triple quadrupole mass spectrometer, respectively. The experimental conditions for LC, spray voltage, drift tube temperature, gas pre-heating temperature, and drift gas velocity were systematically optimized. The limits of detection (LODs) and quantitation (LOQs) for the seven analytes were 2-10 µg/mL and 5-25 µg/mL, respectively. The proposed method was applied for the analysis of real oral liquids of Chinese medicine samples. By coupling LC and IMS, two-dimensional separation could be achieved based on hydrophobicity difference and ionic mobility disparity, thus providing more comprehensive measurement information than LC or IMS used alone.

Integrated olfaction, gustation and toxicity detection by a versatile bioengineered cell-based biomimetic sensor.

The biological olfactory and gustation system can discriminate thousands of odor and taste substances with high sensitivity and specificity, specific receptor proteins play an important role in this process. This study used the human neuroblastoma SH-SY5Y cell line endogenously expressing the human bitter receptor, T2R16. Meanwhile, an olfactory receptor, ODR-10, was transfected on the plasma membrane of SH-SY5Y cells. T2R16 could specifically respond to bitter compounds with the structure of ß-glucopyranosides by activation of G protein coupled receptors (GPCRs) causing cell morphologic changes, which could be monitored using a cell-impedance sensor. ODR-10 could specifically respond to diacetyl by changing the extracellular potential of the cells, the resopnse was recorded by a microelectrode array (MEA). The cell index (CI) value and firing rates were extracted from the signals as the biosensor response characteristics. The results with the sensors indicated a dose-dependent response within a defined concentration range. Moreover, this cell-impedance biosensor enabled quick toxicity detection of salicin when the concentration was ≥6 mM. In conclusion, the biomimetic sensors integrated olfaction, gustation and toxicity detection using the same cell, and has showed great potential for use in both basic research and practical applications.

Protective effect of vanilloids against tert-butyl hydroperoxide-induced oxidative stress in vero cells culture.

This study investigated the effect of synthetic capsiate, a simplified analogue of capsiate, and vanillyl alcohol on the oxidative stress induced by tert-butyl hydroperoxide (TBH) in a line of fibroblasts derived from monkey kidney (Vero cells). In response to the TBH-mediated oxidative stress, a reduction of the levels of total unsaturated fatty acids and cholesterol was observed, and a rise in the concentrations of conjugated dienes fatty acids hydroperoxides and 7-ketocholesterol. Pretreatment with both synthetic capsiate and vanillyl alcohol preserved Vero cells from oxidative damage and showed a remarkable protective effect on the reduction of the levels of total unsaturated fatty acids and cholesterol, inhibiting the increase of MDA, conjugated dienes fatty acids hydroperoxides, and 7-ketocholesterol. Both compounds were effective against peroxidation of cell membrane lipids induced by TBH, with synthetic capsiate essentially acting as a pro-drug of vanillyl alcohol, its hydrophilic hydrolytic derivative.