Metabolic profile of Phellodendron amurense Rupr. in vivo of rat and its metabolomic study on intervention in rheumatoid arthritis.

To analyze the metabolites (blood, urine and feces) in normal rats after intragastric administration of the decoction of Phellodendri Amurensis Cortex (PAC) and to map the metabolic profile of PAC in vivo of rat; meanwhile, to evaluate the anti-rheumatoid arthritis (RA) effect of PAC by blood metabolomics technique and to explore its mechanism. Performing on UPLC-Q-TOF-MS technology with a Waters ACQUITY UPLC BEH-C18 column (100 mm × 2.1 mm, 1.7 µm), the mobile phase was acetonitrile-0.1% formic acid aqueous solution (gradient elution). Prior to and following the administration of the decoction of PAC, the samples of blood, urine, and fecal were collected from the rats, in the positive ion mode, pharmacogenic metabolites in each biological sample were identified according to the accurate mass, fragment ions, retention time, metabolic reaction type, comparison of reference substance and retrieval of Pub Med database; The adjuvant-type arthritis (AA) rat model was established, and blood metabonomics method was used to study the improvement effect of rheumatoid arthritis after drug intervention with PAC, and its mechanism was preliminarily explored through analysis of metabolic pathway. A total of 72 exogenous components were identified, including 17 prototype components and 55 metabolites; 14 biomarkers were screened by blood metabolomics techniques combined with multivariate statistical analysis, and PAC significantly improved symptoms of rheumatoid arthritis in rats, and the metabolic pathway analysis mainly involves 5 metabolic pathways. The components in the aqueous decoction of PAC mainly undergo phase I metabolic reactions in rats, such as oxidation, reduction, dehydrogenation, demethylation, and phase II metabolic reactions, such as acetylation, glucuronidation, methylation; PAC has anti-rheumatoid arthritis effects, and its mechanism of action may be related to biosynthesis of aminoacyl-tRNA, metabolism of phenylalanine, metabolism of tryptophan, degradation of valine, leucine and isoleucine and biosynthesis of pantothenic acid and coenzyme A, providing a scientific basis for the study of the pharmacodynamic substances and the action mechanism of PAC against RA.

Antibacterial activity of phellodendron bark against Streptococcus mutans.

Streptococcus mutans is a major cause of tooth decay due to its promotion of biofilm formation and acid production. Several plant extracts have been reported to have multiple biological activities such as anti-inflammation and antibacterial effects. This study investigated the antibacterial activity of three plant extracts, phellodendron bark (PB), yucca, and black ginger, and found that PB had a stronger effect than the other extracts. Then, the minimum inhibitory concentration (MIC) of PB against 100 S. mutans strains was investigated. The MIC range of PB was 9.8-312.5 µg/mL. PB suppressed the growth kinetics of S. mutans in a dose-dependent manner, even at sub-MICs of PB. Then, we investigated the effect of PB on S. mutans virulence. The PB suppressed biofilm formation at high concentrations, although PB did not affect the expression of glucosyltransferase genes. Additionally, PB suppressed the decrease in pH from adding an excess of glucose. The expression of genes responsible for acid production was increased by the addition of excess glucose without PB, whereas their expression levels were not increased in the presence of 1× and 2× MIC of PB. Although PB showed a bacteriostatic effect on planktonic S. mutans cells, it was found that more than 2× MIC of PB showed a partial bactericidal effect on biofilm cells. In conclusion, PB not only showed antibacterial activity against S. mutans but also decreased the cariogenic activity in S. mutans.

Overexpression of TaLEA3 induces rapid stomatal closure under drought stress in Phellodendron amurense Rupr.

Late embryogenesis abundant (LEA) proteins participate in drought stress responses in plants. In the present study, the gene TaLEA3 from the drought-resistant plant Tamarix androssowii was transformed into Amur cork tree (Phellodendron amurense) via Agrobacterium tumefaciens to investigate the mechanism of stomatal closure in response to osmotic stress. Our results showed that P. amurense overexpressing TaLEA3 were resistant to drought stress by rapid stomatal closure. To study the stomatal movement regulated at the molecular level, a model system for stoma closure was established in in vitro P. amurense. In this work, we found that the increased Ca2+ accumulation in guard cells of transgenic plants caused stomatal closure and activated K+ efflux under polyethylene glycol (PEG) stress. Moreover, H+ changes might provide a needed pH condition for stomatal closure. Further, nitric oxide (NO) fluorescence was measured using an NO-specific fluorescent probe, diaminofluorescein-FM diacetate, which showed that guard cell NO fluorescence was stronger in transgenic plants compared with wild type plants. Additionally, five genes encoding nitrate reductase were up-regulated, indicating that TaLEA3 overexpression positively regulated NO biosynthesis and accumulation in the guard cells. This discovery will further our understanding of the LEA gene function and will help in engineering drought-resistant tree cultivars.

[Evaluation for heavy metal pollution of soil and herb from main producing area of Phellodendron amurense in China].

In order to evaluate the heavy metal potential pollution of soil and medicinal materials in main producing area of Phellodendron amurense, we collected 32 soil samples and 32 herb samples from northeast and north of China covering four provinces. In this study, the detection of heavy metal contents was conducted by ICP emission spectroscopy and atomic fluorescence spectrometry. The results showed that the soil from all areas of Ph amurense generally reached the national standard. As, Hg, Cr, Cd, Pb and Cu content of herb samples met the requirtment of the national standard except Hg content exceeding standard slight in a few samples. The reason of excessive Hg was the ability of Hg accumulation in Ph. amurense and atmospheric environment was polluted. So, national standard and Good Agricultural Practice (GAP) must be carried out severely in Ph. amurense resources production.

Phenyl-ß-D-Glucopyranoside Exhibits Anti-inflammatory Activity in Lipopolysaccharide-Activated RAW 264.7 Cells.

Phenyl-ß-D-glucopyranoside is a component of Phellodendron amurense with anti-cancer and anti-inflammatory activities. In the present study, we investigated the role of phenyl-ß-D-glucopyranoside in inflammation using lipopolysaccharide (LPS)-stimulated murine Raw 264.7 macrophages. Phenyl-ß-D-glucopyranoside not only inhibited nitric oxide (NO) production but also significantly inhibited the expression of inducible NO synthase (iNOS) and cyclooxygenase-2 (COX-2) without inducing cytotoxicity. Phenyl-ß-D-glucopyranoside also attenuated proinflammatory cytokines, including tumor necrosis factor-α (TNF-α), interleukin-1ß (IL-1ß) and other inflammation-related genes, such as IL-6 in a concentration-dependent manner. Furthermore, phenyl-ß-D-glucopyranoside abolished increased adhesion, ninjurin 1 (Ninj1) expression, and matrix metalloproteinase (MMP) activity induced by endotoxin treatment. Finally, phenyl-ß-D-glucopyranoside inhibited the nuclear translocation of nuclear factor-κB (NF-κB), which is one of the most important transcription factors involved in the inflammatory process. Taken together, phenyl-ß-D-glucopyranoside may be beneficial for the prevention and treatment of anti-inflammatory diseases.

Agrobacterium tumefaciens-mediated transformation of Phellodendron amurense Rupr. using mature-seed explants.

An efficient transformation protocol was developed for Agrobacterium-mediated transformation of Phellodendron amurense Rupr. for using explants from mature seeds. The binary vector pCAMBIA1303, which contained hygromycin phosphotransferase (hptII) as a selectable marker gene and ß-glucuronidase (GUS) as a reporter gene, was used for transformation studies. Different factors that affect survival of transformed buds, namely Agrobacterium infection method, bacterial strain, pre-culture duration, acetosyringone concentration, co-culture duration, and co-culture temperature were examined and optimized for transformation efficiency on the basis of GUS staining of hygromycin-resistant buds. Polymerase chain reaction (PCR), Southern blot and reverse transcription PCR confirmed the presence of the GUS gene. A transformation frequency of 13.1 % was achieved under optimized conditions for transformation (A. tumefaciens strain EHA105, 4 days co-cultivation at 4 °C, and infection of the pre-cultured mature-seed explants for 2 days). This is the first report of a successful genetic transformation protocol for P. amurense.

[Seasonal dynamics of carbon and nitrogen concentrations in Phellodendron amurense fine roots].

Taking a 23 years old Phellodendron amurense plantation as test object, the first five order roots of P. amurense were sampled to study the seasonal dynamics of their total carbon (TC), total nitrogen (TN), total non-structural carbohydrates (TNC), and soluble N concentrations, with the correlations among these parameters analyzed. In the first five order roots, the TNC occupied 49% of TC, and the soluble N accounted for 26% of TN. Within the growth season, the rate of TNC to TC increased from 42% in the first order roots to 52% in the fifth order roots, and the rate of soluble N to TN decreased from 28% to 21% correspondingly. All the first five order roots had the lowest concentration of TC but the highest concentration of TN in spring, and the lowest concentrations of TNC and soluble N in summer. The increase of the TC concentration in the roots decreased the concentrations of TNC and soluble N, whereas the increase of the TN concentration decreased the TNC concentration significantly and increased the concentration of soluble N. From the first to the fifth order roots, the TC and TN had an increasing correlation with TNC but a decreasing correlation with soluble N, suggesting the close correlations of TNC and soluble N with TC and TN in P. amurense fine roots.

Storage behavior and changes in concentrations of abscisic acid and gibberellins during dormancy break and germination in seeds of Phellodendron amurense var. wilsonii (Rutaceae).

The medicinal Asian plant genus Phellodendron is known to contain several very important compounds that have biological action. The main purpose of this study was to determine whether seeds of Phellodendron amurense var. wilsonii can be stored and to characterize their dormancy. Seeds of this taxon stored at -20 and -80 degrees C and in liquid nitrogen retained their high germinability, indicating that they have orthodox storage behavior. Intact seeds from freshly collected fruits were dormant and required 12 weeks of cold stratification at 4 degrees C for complete germination. Scarifying the seed coat was partially effective in breaking seed dormancy. Exogenous gibberellins (GA(3), GA(4) and GA(4+7)) promoted germination of scarified seeds, GA(4) and GA(4+7) being more effective than GA(3). Fluridone, an abscisic acid (ABA) biosynthesis inhibitor, was efficient in breaking dormancy, but it was less effective than GA(4) or GA(4+7) alone. Paclobutrazol, a GA biosynthesis inhibitor, inhibited seed germination, and the inhibitory effect was reversed completely by GA(4) and by GA(4+7). ABA content of seeds subjected to cold stratification or to incubation at 35/10 degrees C, which enhanced seed germination, was reduced about four- to sixfold compared to that of fresh seeds. Higher concentrations of GA(3), GA(4) and GA(7) were detected in nondormant seeds and in seeds with an emerged radicle than in fresh seeds. Present results seem to indicate that dormancy in P. amurense var. wilsonii seeds is imposed partially by the seed coat and partially by high ABA content. ABA content decreased and GA(3), GA(4) and GA(7) content increased during germination.

Akt-and CREB-mediated prostate cancer cell proliferation inhibition by Nexrutine, a Phellodendron amurense extract.

Evidence from epidemiological studies suggests that plant-based diets can reduce the risk of prostate cancer. However, very little information is available concerning the use of botanicals in preventing prostate cancer. As a first step toward developing botanicals as prostate cancer preventives, we examined the effect of Nexrutine on human prostate cancer cells. Nexrutine is a herbal extract developed from Phellodendron amurense. Phellodendron extracts have been used traditionally in Chinese medicine for hundreds of years as an antidiarrheal, astringent, and anti-inflammatory agent. The present study investigated its potential antitumor effect on human prostate cancer cells. Our results suggest that it inhibits tumor cell proliferation through apoptosis induction and inhibition of cell survival signaling. The results of the present study indicate that Nexrutine treatment 1) inhibits the proliferation of both androgen-responsive and androgen-independent human prostate cancer cells through induction of apoptosis; 2) reduces levels of pAkt, phosphorylated cAMP response-binding protein (pCREB) and CREB DNA-binding activity; and 3) induces apoptosis in prostate cancer cells stably overexpressing Bcl-2. Further, Akt kinase activity was reduced in cells treated with Nexrutine, and ectopic expression of myristoylated Akt protected from Nexrutine induced inhibition of proliferation, implicating a role for Akt signaling.

[Hairy roots induction of Phellodendron chinense and production of its active constituents].

OBJECTIVE: To introduce the hairy roots of Phellodendron chinense and determine the content of its active constituents. METHOD: Transformed hairy roots of P. chinense were obtained by the transformation of Agrobacterium rhizogenes A4, R1600, ATCC15834 and R1000. RESULT AND CONCLUSION: It was clearly demonstrated that T-DNA of A. rhizogenes Ri plasmid was integrated into the cells of hairy roots by PCR. The content of berberine hydrochlodride, which was determined by HPLC, was higher in hairy roots than that in the axenic plantet and callus.

[Effects of water stress on protective enzyme activities and lipid peroxidation in Phellodendron amurense seedlings].

This paper studied the effects of water stress on the dynamic variations of malondialdehyde (MDA) and free proline contents, and of superoxide dismutase (SOD), peroxidase (POD) and catalase (CAT) activities in Phellodendron amurense seedlings. The results showed that after treated with light drought, heavy drought and waterlogging for 40 days, the MDA content in corktree seedlings was significantly higher, being 2.49, 2.37 and 4.12 times of the control, respectively, but after treated for 80 days, there was no significant difference among the three treatments. The free proline content had no obvious variation under waterlogging and in the control, but increased after 40 days of light and heavy drought while declined after 80 days of drought stresses, with the increment being higher under heavy drought than under light drought. No regularity was found for the dynamic variations of SOD, POD and CAT activities, but they were significantly higher under heavy drought than under light drought, waterlogging, and the control.