Chemical profiling of Zilong Jin tablets using ultra high performance liquid chromatography-quadrupole/orbitrap high resolution mass spectrometry.

Zilongjin tablets as a traditional Chinese medicine are widely used for primary lung cancer patients with deficiency of "qi " and "blood " syndrome undergoing chemotherapy. It is a compound preparation that consists of eight herbs. To clarify the chemical profiling of Zilong Jin tablets rapidly, a feasible and accurate strategy was developed by the ultra high performance liquid chromatography-quadrupole/orbitrap high resolution mass spectrometry. According to the accurate mass and fragment ion information provided by high resolution mass spectrometry, the compounds were reasonably identified. In total, 74 compounds were characterized, including 20 flavonoids, 14 quinones, 15 organic acids, 6 phthalide compounds, and 19 other compounds. Among them, 34 major compounds were unambiguously confirmed by comparing with reference standards. This study could provide an important scientific basis for further research on quality control, pharmacokinetics and pharmacodynamics, and clinical application of Zilong Jin tablets.

Implications of Fagopyrin Formation In Vitro by UV Spectroscopic Analysis.

The present work aims at studying the possible biosynthesis of fagopyrin in buckwheat plants with an attempt to address the existing gaps. The developed method of differential spectrophotometry can be used for identification of naphthodianthrones fagopyrins. It was found that in the vegetative mass of buckwheat plants, fagopyrin precursor-2-(piperidine-2-yl)-emodindianthron could be present. As fagopyrin can be produced by light effect, the temperature factor may influence the formation of protofagopyrin in vitro. An optimum temperature range was estimated for protofagopyrin formation. A possible fagopyrin biosynthesis under in vitro conditions was suggested.

A rapid analysis method of safflower (Carthamus tinctorius L.) using combination of computer vision and near-infrared.

The quality of safflower (Carthamus tinctorius L.) in the market is uneven due to the problems of dyeing and adulteration of safflower, and there is no perfect standard for the classification of quality grade of safflower at present. In this study, computer vision (CV) and near-infrared (NIR) were combined to realize the rapid and nondestructive analysis of safflower. First, the partial least squares discrimination analysis (PLS-DA) model was used to qualitatively identify the dyed safflower from 150 samples. Then the partial least squares (PLS) model was used for quantitative analysis of the hydroxy safflower yellow pigment A (HSYA) and water extract of undyed safflower. Herein, the discrimination rate of PLS-DA model reached 100%, and the residual predictive deviation (RPD) values of the PLS models for HSYA and water extract were 2.5046 and 5.6195, respectively. It indicated that the rapid analysis method combining CV and NIR was reliable, and its results can provide important reference for the formulation of safflower quality classification standards in the market.

Analysis of Chemical Composition of Extractives by Acetone and the Chromatic Aberration of Teak (Tectona Grandis L.F.) from China.

In order to clarify the chemical color change of teak (Tectona grandis L.F.), the difference of chemical composition between the heartwood and sapwood of teak was investigated by gas chromatography-mass spectrometry (GC-MS) based on the acetone extractive compounds. The results showed that the difference in content of the main components between heartwood and sapwood was not obvious. However, the amount of extractives in heartwood was higher than that in sapwood, especially for phenols, quinones, and ketones. The most obvious different substances in the acetone extractive between heartwood and sapwood were 4-tert-butyl-2-phenyl-phenol,2-methyl-anthraquinone, and 2,3-dimethyl-1,4,4a,9a-tetrahydro-9,10-anthracenedione, which might be the main composition for the chromatic aberration of teak. This paper focuses on a preliminary study and further work such as high-performance liquid chromatography (HPLC) with ultraviolet photometric detector (UV)/mass spectrometry (MS) will be carried out.

Screening of blood-activating active components from Danshen-Honghua herbal pair by spectrum-effect relationship analysis.

BACKGROUND: Danshen (Salvia miltiorrhiza, DS) and Honghua (Carthamus tinctorius, HH) are commonly used traditional Chinese medicines for activating blood and removing stasis, and DS-HH (DH) herbal pair had potential synergistic effects on promoting blood circulation. Therefore, it is essential to make clear the active components of this herbal pair for better understanding their potential synergistic effects. PURPOSE: To comprehensively evaluate the activity of DH herbal pair on physiological coagulation system of rats, and seek their potential active components by spectrum-effect relationship analysis. METHODS: The water extracts of DH herbal pair with different proportions (DS: HH = 1:1, 2:1, 3:1, 5:1, 1:5 and 1:3) were prepared. Male Sprague-Dawley rats were randomly divided into eight groups: blank group, model group, model + 1:1 (DH) group, model + 2:1 group, model + 3:1 group, model + 5:1 group, model + 1:5 group and model + 1:3 group. The intragastric administration was performed for eight times with 12 h intervals. SC40 semi-automatic coagulation analyzer was employed to determine coagulation indices. Meanwhile, HPLC and LC-MS were applied for chemical analyses of DH extracts. Finally, the active ingredients were screened by spectrum-effect relationship analysis and the activities of major predicted compounds were validated in vitro. RESULTS: Different proportions of DH extracts could significantly prolong thrombin time (TT) and activated partial thromboplastin time (APTT), increase prothrombin time (PT) and decrease fibrinogen (FIB) content, reduced whole blood viscosity (WBV) and plasma viscosity (PV), decreased erythrocyte sedimentation rate blood (ESR) compared with model group. Furthermore, fifteen highly related components were screened out by the spectrum-effect relationship and LC-MS analysis, of which caffeic acid, salvianolic acid B, hydroxysafflor yellow A and lithospermate acid had significant blood-activing effect by prolong APTT and decrease FIB content at high (0.6 mM), medium (0.3 mM) and low (0.15 mM) (except lithospermate acid) concentrations in vitro. CONCLUSIONS: DH herbal pair showed strong blood-activating effect on blood stasis rat through regulating the parameters involved in haemorheology and plasma coagulation system. Four active compounds, caffeic acid, salvianolic acid B, hydroxysafflor yellow A and lithospermate acid predicted by spectrum-effect relationship analysis had good blood-activating effect. Therefore, spectrum-effect relationship analysis is an effective approach for seeking active components in herbal pairs.

Tissue distribution profiles of multiple major bioactive components in rats after intravenous administration of Xuebijing injection by UHPLC-Q-Orbitrap HRMS.

Xuebijing injection (XBJI) is a traditional Chinese medicine prescription extracted from five Chinese herbs. Hydroxysafflor yellow A, oxypaeoniflorin, ferulic acid and benzoylpaeoniflorin are the main bioactive ingredients of XBJI. This paper presents an application of ultra-high-performance liquid chromatography-Q-exactive hybrid quadrupole-Orbitrap high-resolution mass spectrometry (UHPLC-Q-Orbitrap HRMS) to quantify four compounds of XBJI in rats various tissues for tissue distribution studies. The analytes were separated on a Waters Acquity UHPLC® BEH C18 column with a gradient mobile phase consisting of acetonitrile-water (containing 0.1% formic acid) at a flow rate of 0.2 mL/min. Mass spectrometric detection was performed by parallel reaction monitoring via a heated electrospray ionization source under the negative ionization mode. The method was validated in various tissue samples, and has demonstrated great performance for rapidity, accuracy, high sensitivity and selectivity. It was successfully applied to the tissue distribution studies of XBJI after intravenous administration to rats. It was also the first study to investigate the tissue distribution of XBJI in rats and we found that the concentrations of four compounds were high in kidney, liver, stomach and intestine. The clinical use of XBJI should focus on its pharmacodynamics and safety studies in these tissues.

Tectona grandis (teak) - A review on its phytochemical and therapeutic potential.

Tectona grandis Linn (Teak), is locally known as Sagwan, belongs to Lamiaceae family. It is one of the most valuable timber in the world, due to its beautiful surface and its resistance to termite and fungal damage. The main active ingredient compounds that are responsible for these action are tectoquinone, lapachol and deoxylapachol. Naphthoquinones, anthraquinones and isoprenoid quinones are abundant metabolites in teak. In addition to these, teak contains several other phytochemicals such as triterpenoids, steroids, lignans, fatty esters and phenolic compounds. Pharmacologically, the plant has been investigated for antioxidant, anti-inflammatory, anti-pyretic, cytotoxic, analgesic, hypoglycemic, wound healing and antiplasmodial activities. The present review highlights the phytochemical and pharmacological aspects of teak.

PQQ ameliorates D-galactose induced cognitive impairments by reducing glutamate neurotoxicity via the GSK-3ß/Akt signaling pathway in mouse.

Oxidative stress is known to be associated with various age-related diseases. D-galactose (D-gal) has been considered a senescent model which induces oxidative stress response resulting in memory dysfunction. Pyrroloquinoline quinone (PQQ) is a redox cofactor which is found in various foods. In our previous study, we found that PQQ may be converted into a derivative by binding with amino acid, which is beneficial to several pathological processes. In this study, we found a beneficial glutamate mixture which may diminish neurotoxicity by oxidative stress in D-gal induced mouse. Our results showed that PQQ may influence the generation of proinflammatory mediators, including cytokines and prostaglandins during aging process. D-gal-induced mouse showed increased MDA and ROS levels, and decreased T-AOC activities in the hippocampus, these changes were reversed by PQQ supplementation. Furthermore, PQQ statistically enhanced Superoxide Dismutase SOD2 mRNA expression. PQQ could ameliorate the memory deficits and neurotoxicity induced by D-gal via binding with excess glutamate, which provide a link between glutamate-mediated neurotoxicity, inflammation and oxidative stress. In addition, PQQ reduced the up-regulated expression of p-Akt by D-gal and maintained the activity of GSK-3ß, resulting in a down-regulation of p-Tau level in hippocampus. PQQ modulated memory ability partly via Akt/GSK-3ß pathway.

Enhanced production of perylenequinones in the endophytic fungus Shiraia sp. Slf14 by calcium/calmodulin signal transduction.

Perylenequinones (PQ) that notably produce reactive oxygen species upon exposure to visible light are a class of photoactivated polyketide mycotoxins produced by fungal plant pathogens such as Shiraia sp. The involvement of Ca2+/calmodulin (CaM) signalling in PQ biosynthesis was investigated by submerged culturing of Shiraia sp. Slf14, a species that produces hypocrellins HA and HB and elsinochromes EA, EB, and EC. Our results showed that the total content of PQ reached 1894.66 ± 21.93 mg/L under optimal conditions of Ca2+ addition, which represents a 5.8-fold improvement over controls. The addition of pharmacological Ca2+ sensor inhibitors strongly inhibited PQ production, which indicates that Ca2+/CaM signalling regulates PQ biosynthesis. The expression levels of Ca2+ sensor and PQ biosynthetic genes were downregulated following addition of inhibitors but were upregulated upon addition of Ca2+. Inhibition was partially released by external Ca2+ supplementation. Fluo-3/AM experiments revealed that similar cytosolic Ca2+ variation occurred under these conditions. These results demonstrated that Ca2+ signalling via the CaM transduction pathway plays a pivotal role in PQ biosynthesis.

Chryseobacterium ginsengiterrae sp. nov., with Beta-Glucosidase Activity Isolated from Soil of a Ginseng Field.

The isolated Chryseobacterium ginsengiterrae sp. nov DCY68T was found to be Gram-negative, aerobic, non-motile, non-flagellate and rod-shaped. Their size was approximately 0.40-0.46 × 1.0-1.27 µm. The colonies were yellow-pigmented, convex, circular and 0.5-1.3 mm in diameter when grown on R2A agar for 2 days. DNA, esculin, skim milk, gelatine, starch, Tween 20, and Tween 80 were hydrolyzed, but not cellulose. The cells grew on R2A, TSA, and NA but not on MacConkey agars. Growth occured at 4-33 °C (optimum, 30 °C), at pH 5.0-8.0 (optimum, pH 6.5), and 0-2.5% NaCl. Nitrate was not reduced to nitrite. Oxidase and catalase activity were positive. Strain DCY68T contained ß-glucosidase activity in which ginsenoside Rb1 was enzymatically converted to ginsenoside F2. Analysis of the16S rRNA gene sequence revealed that strain C. ginsengiterrae sp. nov DCY68T belonged to the family Flavobacteriaceae and was most closely related to C. limigenitum SUR2T (97.4%). The genomic DNA G+C content was 42.0 mol%. The predominant quinones were MK-6 (74.5%) and MK-7 (25.5%). The major fatty acids were iso-C15:0, summed feature 3 (containing C16:1 ω7c and/or C16:1 ω6c) and iso-C17:0 3-OH. On the basis of these phenotypic, genotypic and chemotaxonomic studies, strain DCY68T represents a novel species of the genus Chryseobacterium, for which name C. ginsengiterrae sp. nov. is proposed. The type strain is DCY68T (=KCTC 32089T = JCM 18517T).

Mucilaginibacter ginsenosidivorans sp. nov., Isolated from Soil of Ginseng Field.

A Gram-reaction-negative, aerobic, nonmotile, nonspore-forming, and rod-shaped bacterial strain designated Gsoil 3017T was isolated from soil of ginseng field and investigated by phenotypic and phylogenetic analyses. Strain Gsoil 3017T grew at 10-37 °C (optimal growth at 30 °C) and at pH 5.5-8.0 (optimal growth at pH 7) on R2A and nutrient agar without additional NaCl as a supplement. Strain Gsoil 3017T possessed ß-glucosidase activity, which was responsible for its ability to transform ginsenosides Rb1, Rc, and Rd (the three dominant active components of ginseng) to F2 and C-K, respectively. Based on 16S rRNA gene phylogeny, the novel strain represents a new branch within the genus Mucilaginibacter family Sphingobacteriaceae, and clusters with Mucilaginibacter frigoritolerans FT22T (95.6%) and Mucilaginibacter gotjawali SA3-7T (95.6%). The G+C content of the genomic DNA was 48.7%. The predominant respiratory quinone was MK-7, and the major fatty acids were iso-C15:0, iso-C17:0 3-OH, and summed feature 3 (comprising C16:1 ω6c and/or C16:1 ω7c). The major polar lipid was phosphatidylethanolamine. Strain Gsoil 3017T could be differentiated genotypically and phenotypically from other type strains of the genus Mucilaginibacter. The isolate therefore represents a novel species, for which the name Mucilaginibacter ginsenosidivorans sp. nov. is proposed, with the type strain Gsoil 3017T (=KACC 14954T = JCM 17081T).

Simultaneous analysis of tert-butylhydroquinone, tert-butylquinone, butylated hydroxytoluene, 2-tert-butyl-4-hydroxyanisole, 3-tert-butyl-4-hydroxyanisole, α-tocopherol, γ-tocopherol, and δ-tocopherol in edible oils by normal-phase high performance liquid chromatography.

A normal-phase high performance liquid chromatography method for the simultaneous determination of tert-butylhydroquinone, tert-butylquinone, butylated hydroxytoluene, 2-tert-butyl-4-hydroxyanisole, 3-tert-butyl-4-hydroxyanisole, α-tocopherol, γ-tocopherol, and δ-tocopherol in edible oils was investigated. A silica column was used to separate the analytes with the gradient elution. An ultraviolet-visible detector was set at dual wavelengths mode (280 and 310nm). The column temperature was 30°C. The analytes were directly extracted with methanol. Results showed that the normal-phase high performance liquid chromatography method performed well with wide liner ranges (0.10∼500.00µg/mL, R2>0.9998), low limits of detection and quantitation (below 0.40 and 1.21µg/mL, respectively), and good recoveries (81.38∼102.34% in soybean oils and 83.03∼100.79% in lard, respectively). The reduction of tert-butylquinone caused by the reverse-phase high performance liquid chromatography during the injection was avoided with the current normal-phase method. The two isomers of butylated hydroxyanisole can also be separated with good resolution.

Comparative Analysis of the Effects of Hydroxysafflor Yellow A and Anhydrosafflor Yellow B in Safflower Series of Herb Pairs Using Prep-HPLC and a Selective Knock-Out Approach.

The flower of Carthamus tinctorius L. (Carthami Flos, safflower), important in traditional Chinese medicine (TCM), is known for treating blood stasis, coronary heart disease, hypertension, and cerebrovascular disease in clinical and experimental studies. It is widely accepted that hydroxysafflor yellow A (HSYA) and anhydrosafflor yellow B (ASYB) are the major bioactive components of many formulae comprised of safflower. In this study, selective knock-out of target components such as HSYA and ASYB by using preparative high performance liquid chromatography (prep-HPLC) followed by antiplatelet and anticoagulation activities evaluation was used to investigate the roles of bioactive ingredients in safflower series of herb pairs. The results showed that both HSYA and ASYB not only played a direct role in activating blood circulation, but also indirectly made a contribution to the total bioactivity of safflower series of herb pairs. The degree of contribution of HSYA in the safflower and its series herb pairs was as follows: Carthami Flos-Ginseng Radix et Rhizoma Rubra (CF-GR) > Carthami Flos-Sappan Lignum (CF-SL) > Carthami Flos-Angelicae Sinensis Radix (CF-AS) > Carthami Flos-Astragali Radix (CF-AR) > Carthami Flos-Angelicae Sinensis Radix (CF-AS) > Carthami Flos-Glycyrrhizae Radix et Rhizoma (CF-GL) > Carthami Flos-Salviae Miltiorrhizae Radix et Rhizoma (CF-SM) > Carthami Flos (CF), and the contribution degree of ASYB in the safflower and its series herb pairs: CF-GL > CF-PS > CF-AS > CF-SL > CF-SM > CF-AR > CF-GR > CF. So, this study provided a significant and effective approach to elucidate the contribution of different herbal components to the bioactivity of the herb pair, and clarification of the variation of herb-pair compatibilities. In addition, this study provides guidance for investigating the relationship between herbal compounds and the bioactivities of herb pairs. It also provides a scientific basis for reasonable clinical applications and new drug development on the basis of the safflower series of herb pairs.

Online-storage recycling counter-current chromatography for preparative isolation of naphthaquinones from Arnebia euchroma (Royle) Johnst.

Counter-current Chromatography (CCC) has gradually become a popular method for preparative separation, especially in natural product isolation. As an effective separation method, one-dimensional (1D) CCC often results in insufficiently resolved peaks, due to limitations in the separation efficiency and peak capacity in an equipment. Therefore, two dimensional (2D)/multi-dimensional (multi-D) CCC strategies with recycling elution mode were developed to achieve successful separation of target compounds. However, the reported 2D or multi-D CCC approaches lead to experimental costs, complicated procedures, higher requirements for equipment, and increased time consumption. In this study, an online-storage recycling (OSR) CCC strategy was designed to achieve sequential recycling elution for multi-fractions of effluent in non-stop separation with single instrument using three 6-port valves and two storage loops, which would be realized by introducing 2D or multi-D CCC method before. In this non-stop separation system, the fraction C of effluent was subjected to recycling separation while the other fractions (A and B) were storing online, following which these two fractions were subjected to subsequent recycling separations in order, after the completion of the previous recycling elution. Then, six natural occurring naphthaquinone analogues, namely, shikonin (1), propionylshikonin (2), deoxyshikonin (3), isobutyrylshikonin (4), ß, ß-dimethylacrylshikonin (5) and isovalerylshikonin (6), were isolated from the crude extract of Arnebia euchroma in single run. The purities of all compounds were > 95.0% as determined by HPLC, and their structures were determined by means of UV, MS, (1)H NMR, (13)C NMR, and optical rotatory dispersion (ORD).

UFLC-Q-TOF/MS based screening and identification of the metabolites in plasma, bile, urine and feces of normal and blood stasis rats after oral administration of hydroxysafflor yellow A.

The dried flower of Carthamus tinctorius L. (honghua) is a widely used traditional Chinese medicine in clinics to treat coronary heart disease, hypertension, and cerebrovascular disease due to its functions of ameliorating circulation and removing blood stasis. Hydroxysafflor yellow A (HSYA) is an active marker component of honghua. In this paper, ultra-flow liquid chromatography coupled with quadrupole-time-of-flight mass-spectrometry (UFLC-Q-TOF/MS) was established and successfully applied to the detection and identification of the metabolites in bile, urine, plasma and feces samples of normal and model rats with orally administrated HSYA. A total of 8 metabolites were observed in normal rats, while 7 metabolites were detected in model rats. The distribution of metabolites in the plasma, bile, urine and feces of normal and model rats had obvious differences. The major in vivo metabolic pathways for HSYA included hydroxylation, hydroxylation+methylation, acetylation and glucuronidation, and there were also dehydration, hydrogenation, hydration, and hydroxylation+glucuronidation. All of these metabolites were reported for the first time, and these results are valuable and important for the understanding of the metabolic process and therapeutic mechanism of HSYA and some other pigments in honghua.

Sphingomonas panacis sp. nov., isolated from rhizosphere of rusty ginseng.

The type strain DCY99(T) was isolated from soil collected from a ginseng field in Hwacheon, Republic of Korea. Strain DCY99(T) is Gram-negative, non-spore forming, motile, rod-shaped, and strictly aerobic. The bacteria grow optimally at 25-30 °C and pH 6.0-6.5. Phylogenetically, strain DCY99(T) is most closely related to Sphingomonas oligophenolica JCM 12082(T), followed by Sphingomonas asaccharolytica KCTC 2825(T), Sphingomonas mali KCTC 2826(T), Sphingomonas cynarae JCM17498(T), Sphingomonas pruni KCTC 2824(T), and Sphingomonas glacialis DSM 22294(T). The DNA-DNA relatedness between strain DCY99(T) and S. oligophenolica JCM 12082(T) was 15.6 ± 0.4 %, and the DNA G+C content of strain DCY99(T) was 64.4 mol%. An isoprenoid quinone was detected and identified as ubiquinone Q-10, and sym-homospermidine was identified as the major polyamine of DCY99(T). The major polar lipids were identified as sphingoglycolipid, diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylglycerol, and phosphatidylcholine. C14:02OH, C16:0, and summed feature 8 (C18:1 ω7c:/C18:1 ω6c) were identified as the major fatty acids present in DCY99(T). The results of physiological and biochemical tests allowed strain DCY99(T) to be differentiated phenotypically from other recognized species belonging to the genus Sphingomonas. Therefore, it is suggested that the newly isolated organism represents a novel species, for which the name Sphingomonas panacis sp. nov. is proposed with the type strain designated as DCY99(T) (=JCM 30806(T) =KCTC 42347(T)).

Paralcaligenes ginsengisoli sp. nov., isolated from ginseng cultivated soil.

A novel bacterial strain DCY104(T) isolated from soil of a ginseng field in Yeoncheon County, Republic of Korea is described in this study. Cells were short rod-shaped, motile by mean of one polar flagellum, strictly aerobic, Gramreaction negative, oxidase and catalase-positive. 16S rRNA gene sequence analysis showed that strain DCY104(T) shared highest similarity 98.2 % to Paralcaligenes ureilyticus GR24-5(T), and from 97.7 to 97.1 % with other type strains belong to the genera Candidimonas, Pusillimonas and Parapusillimonas Otherwise, phylogenetic trees analyses indicated that strain DCY104(T) belongs to a single group with P. ureilyticus GR24-5(T) that was distinct to other genera. The major polar lipids were phosphatidylmonomethylethanolamine, phosphatidylethanolamine, phosphatidylglycerol, and diphosphatidylglycerol. The major cellular fatty acids consisted of C16:0, cyclo-C17:0, and summed feature 8 (comprising C18:1 ω7c and/or C18:1 ω6c). The predominant polyamine was putrescine. The ubiquinone was Q-8. The genomic DNA G+C content was 55.9 mol%. These data in combination with the presence of one polar flagellum and positive activity of urease confirmed the placement of strain DCY104(T) in the genus Paralcaligenes. DNA-DNA relatedness between strain DCY104(T) and P. ureilyticus KACC 13888(T) was 40 %. The differences in the profiles of polar lipids, fatty acids and phenotypic characteristics in combination with DNA-DNA relatedness delineated strain DCY104(T) and P. ureilyticus KACC 13888(T). In summary, taxonomic analyses in this study demonstrated that strain DCY104(T) represents a novel species within the genus Paralcaligenes, for which we propose the name Paralcaligenes ginsengisoli. The type strain is DCY104(T) (= KCTC 42406(T) = JCM 30746(T)).

Fagopyrins and Protofagopyrins: Detection, Analysis, and Potential Phototoxicity in Buckwheat.

Buckwheat contains many healthy nutrients, and its consumption is therefore increasing. Buckwheat also contains fluorescent phototoxic fagopyrins. A systematic review of fagopyrins and the phototoxicity of buckwheat found that reliable quantitative data on fagopyrin toxicity are not yet available. Generally, buckwheat seeds, flour, and teas are safe in normal amounts. Diets extensively composed of buckwheat sprouts, herbs, and particularly flowers or of fagopyrin-rich buckwheat extracts may cause fagopyrism. A reference standard is needed, as it would enable the accurate evaluation of fagopyrin content in buckwheat products and would allow proper testing of their as yet unknown physical, chemical, and biological characteristics.

Cupriavidus yeoncheonense sp. nov., isolated from soil of ginseng.

A novel bacterial strain, DCY86(T) (=KCTC 42053(T) = JCM 19890(T)) was isolated from soil of a ginseng field in Yeoncheon province (38°04'00″N 126°57'00″E), Republic of Korea using a serial dilution method. Strain DCY86(T) was observed to be Gram-stain negative, strictly aerobic, to grow optimally at 25-30 °C, at pH 7-7.5 and on tryptic soya agar medium. The cells were found to be sensitive to ceftazidine and tetracycline. Based on 16S rRNA gene sequence comparisons, strain DCY86(T) was found to be most closely related to Cupriavidus basilensis LMG 18990(T) (98.48 %), Cupriavidus numazensis LMG 26411(T) (98.34 %), Cupriavidus pinatabonesis KCTC 22125(T) (98.34 %) and Cupriavidus laharis KCTC 22126(T) (98.00 %). The G+C content was determined to be 64.23 mol %. The only isoprenoid quinone detected in strain DCY86(T) was ubiquinone Q-8. The major polar lipids were identified as diphosphatidylglycerol, phosphtidylethanolamine, phosphatidylglycerol, unidentified aminophosphoglycolipids and unidentified phospholipids. The major fatty acids were identified as C16:0 summed feature 3 (C16:1 ω7c/ω6c and/or iso-C15 : 0 2-OH) and summed feature 8 (C18:1 ω7c and/or C18:1 ω6c). These data support the affiliation of strain DCY86(T) to the genus Cupriavidus. Strain DCY86(T) was also found to be able to solubilize phosphate and produce siderophores. The results of physiological and biochemical tests enabled strain DCY86(T) to be differentiated genotypically and phenotypically from the recognized species of the genus Cupriaividus. Therefore, the novel isolate can be considered to represent a novel species, for which the name Cupriavidus yeoncheonense sp. nov. is proposed here. The type strain is DCY86(T) (=KCTC 42053(T) = JCM 19890(T)).

Negadavirga shengliensis gen. nov., sp. nov., a novel member of the family Cyclobacteriaceae isolated from oil-contaminated saline soil.

Four novel Gram-stain-negative, rod-shaped, and non-motile bacterial strains, SLG210-21(T), SLG210-4, SLG210-5 and SLG210-14, were isolated from oil-contaminated saline soil in Shengli Oilfield, China. Growth were observed at 25-42 °C (optimum 37 °C), in the presence of 0-10 % (w/v) NaCl (optimum 0-1 %) and at pH 4.0-10.0 (optimum pH 7.6-8.6). All the strains were positive for catalase and α, ß-galactosidase activities and nitrogen reduction, and negative for oxidase activity, glucose fermentation and hydrolysis of agar, starch, gelatin, Tween 40, 60 and 80. The DNA G+C contents of the four strains were 41.3-43.0 mol% and the predominant respiratory quinones were all menaquinone-7. The major fatty acids were iso-C15:0, anteiso-C15:0, summed feature 3 (C16:1 ω7c and/or C16:1 ω6c), C16:1 ω5c and summed feature 9 (iso-C17:1 ω9c and/or 10-methyl C16:0), while the polar lipids consisted of phosphatidylcholine, phosphatidylethanolamine, glycolipid, two unidentified phospholipids and two unidentified amino lipids. Phylogenetic analysis based on 16S rRNA gene sequences indicated that the four strains clustered together to form a stable branch in the family Cyclobacteriaceae, and were most closely related to the genera Cyclobacterium and Echinicola with the 16S rRNA gene sequence similarities being 88.6-90.3 and 89.6-91.4 %, respectively. DNA-DNA hybridization between SLG210-21(T) and the other three strains showed the relatedness of 93.8 ± 4.5, 96.2 ± 4.2 and 82.3 ± 4.8 %, respectively. Based on the polyphasic analysis, a novel species in a new genus, Negadavirga Shengliensis gen. nov., sp. nov., is proposed with SLG210-21(T) (=LMG 27737(T) = CGMCC1.12768(T)) [corrected] as the type strain.