Pedobacter helvus sp. nov., isolated from farmland soil.

A strictly aerobic, Gram-stain-negative, rod-shaped, yellow, non-spore-forming bacterial strain, designated P-25T, was isolated from soil collected in Yantai, Shandong Province, PR China. The temperature, pH and NaCl concentration ranges for the growth of strain P-25T were 10-37 °C (optimum, 28-30 °C), pH 6.0-9.0 (optimum, pH 7.5-8.0) and 0-4 % (w/v) (optimum, 1 % w/v), respectively. Phylogenetic analysis based on 16S rRNA gene sequences showed that strain P-25T was most closely related to Pedobacter xixiisoli S27T (98.1 % 16S rRNA gene sequence similarity), followed by Pedobacter chitinilyticus CM134L-2T (97.2 %) and Pedobacter ureilyticus THG-T11T (97.1 %). The genomic DNA G+C content of strain P-25T based on its draft genome sequence was 38.1 %. MK-7 was the major respiratory quinone, and iso-C15 : 0, C16 : 1ω7c and/or C16 : 1ω6c (summed feature 3) and iso-C17 : 0 3-OH were the major fatty acids. The major polar lipids were phosphatidylethanolamine, one unidentified aminophospholipid, one unidentified phospholipid, two unidentified lipids, five unidentified aminolipids and two unidentified glycolipids. Average nucleotide identity values for the draft genomes between strain P-25T and strains S27T, CM134L-2T and THG-T11T were 81.8, 77.6 and 81.2 %, respectively, and the digital DNA-DNA hybridization (dDDH) values were 30.0, 19.2 and 27.6 %, respectively. Based on their phylogenetic and phenotypic characteristics, chemotaxonomic data, and dDDH results, strain P-25T is considered to represent a novel species of the genus Pedobacter, for which the name Pedobacter helvus sp. nov. is proposed; the type strain is strain P-25T (KCTC 62821T=CCTCC AB 2018185T).

Steroidobacter soli sp. nov., isolated from farmland soil.

A Gram-stain-negative bacterial strain, designated JW-3T, was isolated from a soil sample collected from farmland in Yantai, Shandong Province, PR China. Cells of strain JW-3T are motile rods and strictly aerobic, showing catalase- and oxidase-positive reactions. Strain JW-3T could grow at 16-37 °C (optimum, 30 °C), at pH 6.0-9.0 (pH 7.0) and in the presence of 0-1 % (w/v) NaCl (0.5 %, in Luria-Bertani broth). The major fatty acids were summed feature 3 (C16 : 1 ω7c and/or C16 : 1 ω6c; 35.5 %), iso-C16 : 0 (16.7 %) and C12 : 0 (10.8 %). The major respiratory quinone was ubiquinone-8 (Q8). The polar lipids of strain JW-3T consisted of diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylglycerol, four unidentified phospholipids, two unidentified lipids, two unidentified glycolipids and a partial unidentified aminophospholipid. Strain JW-3T was most closely related to Steroidobacter agariperforans KA5-BT with 97.67 % 16S rRNA gene sequence similarity. Results of phylogenetic analyses, based on 16S rRNA gene sequencing, showed that strain JW-3T forms a distinct phylogenic lineage within the genus Steroidobacter of the family Sinobacteraceae. The DNA G+C content of strain JW-3T was 62.57 mol%, based on its draft genome sequence. Average nucleotide identity values and digital DNA-DNA hybridization values for draft genomes, between strain JW-3T and strain KA5-BT, were 84.54 and 30.80 %, respectively. Based on its phenotypic, chemotaxonomic and molecular features, and DNA-DNA hybridization results, strain JW-3T represents a novel species of the genus Steroidobacter, for which the name Steroidobactersoli sp. nov. is proposed. The type strain is JW-3T (=CCTCC AB 2018184T=KCTC 62820T).

Determination of geniposide in adjuvant arthritis rat plasma by ultra-high performance liquid chromatography tandem mass spectrometry method and its application to oral bioavailability and plasma protein binding ability studies.

A specific, sensitive and high throughput ultra-high performance liquid chromatography-electrospray ionization tandem mass spectrometric method (UHPLC-ESI-MS/MS) was established and validated to assay geniposide (GE), a promising anti-inflammatory drug, in adjuvant arthritis rat plasma: application to pharmacokinetic and oral bioavailability studies and plasma protein binding ability. Plasma samples were processed by de-proteinised with ice-cold methanol and separated on an ACQUITY UPLC™ HSS C18 column (100 mm × 2.1mm i.d., 1.8 µm particle size) at a gradient flow rate of 0.2 mL/min using acetonitrile-0.1% formic acid in water as mobile phase, and the total run time was 9 min. Mass detection was performed in selected reaction monitoring (SRM) mode with negative electro-spray ionization includes the addition of paeoniflorin (Pae) as an internal standard (IS). The mass transition ion-pair was followed as m/z 387.4 → 122.4 for GE and m/z 479.4 → 449.0 for IS. The calibration curves were linear over the concentration range of 2-50,000 ng/mL with lower limit of quantification of 2 ng/mL. The intra-day and inter-day precisions (RSD, %) of the assay were less than 8.4%, and the accuracy was within ± 6.4% in terms of relative error (RE). Extraction recovery, matrix effect and stability were satisfactory in adjuvant arthritis rat plasma. The UHPLC-ESI-MS/MS method was successfully applied to a pharmacokinetic study of GE after oral administration of depurated GE at 33, 66, 132 mg/kg and intravenous injection at 33, 66, 132 mg/kg in adjuvant arthritis (AA) rats. In addition, it was found that GE has rapid absorption and elimination, low absolute bioavailability, high plasma protein binding ability in AA rats after oral administration within the tested dosage range. It suggested that GE showed slow distribution into the intra- and extracellular space, and the binding rate was not proportionally dependent on plasma concentration of GE when the concentration of GE was below 5.0 µg/mL.

Anti-inflammatory effects and pharmacokinetics study of geniposide on rats with adjuvant arthritis.

The aim of this study was to explore the anti-inflammatory effects of Geniposide (GE), an iridoid glycoside compound extracted from Gardenia jasminoides Ellis (GJ) fruit in adjuvant-induced arthritis (AA) rats and its pharmacokinetic (PK) basis. AA was induced by injecting with Freund's complete adjuvant (FCA). Male SD rats were subjected to treatment with GE (30, 60 and 120mg/kg) from day 17 to 24 after immunization. Fibroblast-like synoviocyte (FLS) proliferation was assessed by MTT. Interleukin (IL)-1, IL-6, TNF-α and IL-10 were determined using double-sandwich enzyme-linked immunosorbent assay (ELISA). Expression of p38 mitogen-activated protein kinases (p38MAPKs) related proteins in FLS was detected by Western blotting. PK profiles were simultaneously detected by ultra-performance liquid chromatography-electrospray ionization-tandem mass spectrometry (UPLC-ESI-MS/MS) in AA rat plasma after oral administration of GE on day 17 after immunization. As a result, GE promoted the recovery of arthritis and inhibited the colonic inflammation damage in AA rats by decreasing the expression level of TNF-α, IL-1 and IL-6, increasing the production of IL-10 and inhibiting the expression of phospho-p38 (p-p38) related proteins in FLS. PK parameters (AUC, Cmax and t1/2) tended to be associated with dosage-related decreasing of efficacy index.

Identification and distribution of four metabolites of geniposide in rats with adjuvant arthritis.

Geniposide (GE), also called Jasminoidin, is the major active ingredient of Gardenia jasminoides Ellis (GJ) fruit, which has long been used in traditional Chinese medicine (TCM). Growing evidences suggested that GE has a great potentiality for treating rheumatoid arthritis (RA). However, GE is rapidly metabolized, and we know little about its availability or metabolites in tissues. To elucidate the distribution of GE and its metabolites in tissues, three groups of adjuvant arthritis (AA) rats were given GE (33, 66 and 120 mg/kg) from days 18 to 24, and the biotransformation of GE in plasma, liver, spleen, synovium, urine and mesenteric lymph node (MLN) of rats was investigated by a novel approach named Information-Dependent Acquisition (IDA)-Mediated LC-MS/MS method. As a result, GE and its four major metabolites were detected as follows: GE, G1, G2 in plasma; GE, G2 in MLNs; only GE in liver and synovium; GE, G2, G3 and G4 in spleen; and GE, G1, G2 and G4 in urine. In total four metabolites (G1-G4) involved in the in vivo metabolism processes were identified. The results of this work have demonstrated the IDA-Mediated LC-MS/MS could screen rapidly and reliably the characterization of metabolites from iridoid compounds.

Effects and mechanisms of Geniposide on rats with adjuvant arthritis.

Geniposide (GE), an iridoid glycoside compound, is the major active ingredient of Gardenia jasminoides Ellis (GJ) fruit which has anti-inflammatory and other important therapeutic activities. The aim of this study was to investigate the effects of GE on adjuvant arthritis (AA) rats and its possible mechanisms. AA was induced by injecting with Freund's complete adjuvant (FCA). Male SD rats were subjected to treatment with GE at 30, 60 and 120mg/kg from days 18 to 24 after immunization. Lymphocyte proliferation was assessed by MTT. Interleukin (IL)-6, IL-17, IL-4 and transforming growth factor-beta 1 (TGF-ß1) were determined by ELISA. c-Jun N-terminal kinase (JNK) and phospho-JNK (p-JNK) were detected by Western blot. GE (60, 120mg/kg) significantly relieved the secondary hind paw swelling and arthritis index, along with decreased Th17-cells cytokines and increased Treg-cell cytokines in mesenteric lymph node lymphocytes (MLNL) and peripheral blood lymphocytes (PBL) of AA rats. In addition, GE decreased the expression of p-JNK in MLNL and PBL of AA rats. In vivo study, it was also observed that GE attenuated histopathologic changes of MLN in AA rats. Collectively, GE might exert its anti-inflammatory and immunoregulatory effects through inducing Th17 cell immune tolerance and enhancing Treg cell-mediated activities by down-regulating the expression of p-JNK. The mechanisms of GE on JNK signaling in MLNL and PBL may play critical roles in the pathogenesis of rheumatoid arthritis.

Comparative pharmacokinetics study after oral administration of geniposide in normal rats and adjuvant-induced arthritis rats by UPLC-MS/MS.

A simple and rapid ultra-performance liquid chromatography-electrospray ionization tandem mass spectrometry (UPLC-ESI-MS/MS) method for quantitative analysis of geniposide (GE) in rat plasma was developed, validated and applied to determine the level of GE in rat plasma after oral administration of GE in adjuvant-induced arthritis (AA) and normal rats. The investigation showed that there were significant differences in the groups between the normal rat and AA rat in pharmacokinetics parameters, such as the area under the time versus drug concentration curve (AUC(0-∞)) (3.77 ± 0.68 versus 2.27 ± 0.42, p < 0.05), the apparent volume of distribution (V) (140.41 ± 2.07 versus 136.51 ± 1.03, p < 0.05), the mean residence time (MRT) (3.98 ± 0.90 versus 3.80 ± 0.50, p < 0.05) and the clearance from the total body (CL) (16.10 ± 2.87 versus 26.44 ± 4.94, p < 0.05). The results indicated that AA could alter the pharmacokinetics of the drug and these experimental findings could be useful for the further study of the clinical applications of GE.