Coelonin protects against PM2 .5 -induced macrophage damage via suppressing TLR4/NF-κB/COX-2 signaling pathway and NLRP3 inflammasome activation in vitro.

One of the important monitoring indicators of the air pollution is atmospheric fine particulate matter (PM2.5 ), which can induce lung inflammation after inhalation. Coelonin can alleviate PM2.5 -induced macrophage damage through anti-inflammation. However, its molecular mechanism remains unclear. We hypothesized that macrophage damage may involve the release of inflammatory cytokines, activation of inflammatory pathways, and pyrosis induced by inflammasome. In this study, we evaluated the anti-inflammation activity of coelonin in PM2.5 -induced macrophage and its mechanism of action. Nitric oxide (NO) and reactive oxygen species (ROS) production were measured by NO Assay kit and dichlorofluorescein-diacetate (DCFH-DA), and apoptosis were measured by Flow cytometry and TUNEL staining. The concentration of inflammatory cytokines production was measured with cytometric bead arrays and ELISA kits. The activation of NF-κB signaling pathway and NLRP3 inflammasome were measured by immunofluorescence, quantitative reverse transcription-polymerase chain reaction and western blot. As expected, coelonin pretreatment reduced NO production significantly as well as alleviated cell damage by decreasing ROS and apoptosis. It decreased generation of interleukin (IL)-6 and tumor necrosis factor (TNF)-α in PM2.5 -induced RAW264.7 and J774A.1 cells. Moreover, coelonin markedly inhibited upregulating the expression of toll-like receptor (TLR)4 and cyclo-oxygenase (COX)-2, blocked activation of p-nuclear factor-kappa B (NF-κB) signaling pathway, and suppressed expression of NLRP3 inflammasome, ASC, GSDMD, IL-18 and IL-1ß. In conclusion, the results showed that coelonin could protect against PM2.5 -induced macrophage damage via suppressing TLR4/NF-κB/COX-2 signaling pathway and NLRP3 inflammasome activation in vitro.

Polysaccharides from Tetrastigma hemsleyanum Diels et Gilg: optimum extraction, monosaccharide compositions, and antioxidant activity.

The optimization of extraction of Tetrastigma hemsleyanum Diels et Gilg polysaccharides (THP) using ultrasonic with enzyme method and its monosaccharide compositions and antioxidant activity were investigated in this work. Single-factor experiments and response surface methodology (RSM) were performed to optimize conditions for extraction, and the independent variables were (XA) dosage of cellulase, (XB) extraction time, (XC) ultrasonic power, and (XD) ratio of water to the material. The extraction rate of THP was increased effectively under the optimum conditions, and the maximum (4.692 ± 0.059%) was well-matched the predicted value from RSM. THP was consisted of mannose, glucuronic acid, rhamnose, galacturonic acid, glucose, galactose, and arabinose, while glucose was the dominant (26.749 ± 0.634%). According to the total antioxidant capacity assay with the FRAP method, DPPH, and hydroxyl radical scavenging assay, THP showed strong antioxidant activity with a dose-dependent behavior. The results indicated that THP has the potential to be a novel antioxidant and could expand its application in food and medicine.

Role of Militarine in PM2.5-Induced BV-2 Cell Damage.

A growing number of studies have shown that air fine particulate matter (PM2.5) pollution is closely associated with neuroinflammation in humans. Militarine, a glucosyloxybenzyl 2-isobutylmalate compound isolated from Bletilla striata, has been found to exert significant neuroprotective effects. However, the anti-inflammatory, antioxidant and antiapoptotic effects of militarine on PM2.5-stimulated BV-2 microglial cells have not been reported. This study aimed to investigate the protective effects of militarine against PM2.5-induced cytotoxicity and its mechanism in BV-2 microglial cells. Our results revealed that pretreatment with 0.31-1.25 µg/mL militarine reversed the morphological changes caused by PM2.5 and decreased proinflammatory cytokine generation and gene expression in PM2.5-treated BV-2 cells. In particular, tumor necrosis factor-α and interleukin-6 expression was inhibited in a dose-dependent manner. Notably, militarine markedly inhibited the upregulation of Toll-like receptor 4, Toll-like receptor 2, and cyclo-oxygenase-2 expression at both the mRNA and protein levels and reduced NF-κB pathway-associated protein expression. Immunofluorescence analysis showed that militarine suppressed NF-κB activity through inhibiting p65 nuclear translocation. Our data suggested that militarine alleviated neuroinflammation in BV-2 microglial cells, possibly by inhibiting the expression of neuroinflammatory cytokines through the TLR/NF-κB signaling pathway. Additionally, militarine significantly reduced PM2.5-mediated reactive oxygen species (ROS) generation and cell apoptosis and restored the mitochondrial membrane potential (MMP; ΔΨm). Collectively, these findings demonstrate that militarine played a protective role against PM2.5-induced damage in BV-2 cells by exerting anti-inflammatory, antioxidant, and antiapoptotic effects.

The Antibacterial Properties of 4, 8, 4', 8'-Tetramethoxy (1,1'-biphenanthrene) -2,7,2',7'-Tetrol from Fibrous Roots of Bletilla striata.

Biphenanthrene compound, 4, 8, 4', 8'-tetramethoxy (1, 1'-biphenanthrene)-2, 7, 2', 7'-tetrol (LF05), recently isolated from fibrous roots of Bletilla striata, exhibits antibacterial activity against several Gram-positive bacteria. In this study, we investigated the antibacterial properties, potential mode of action and cytotoxicity. Minimum inhibitory concentrations (MICs) tests showed LF05 was active against all tested Gram-positive strains, including methicillin-resistant Staphylococcus aureus (MRSA) and staphylococcal clinical isolates. Minimum bactericidal concentration (MBC) tests demonstrated LF05 was bactericidal against S. aureus ATCC 29213 and Bacillus subtilis 168 whereas bacteriostatic against S. aureus ATCC 43300, WX 0002, and other strains of S. aureus. Time-kill assays further confirmed these observations. The flow cytometric assay indicated that LF05 damaged the cell membrane of S. aureus ATCC 29213 and B. subtilis 168. Consistent with this finding, 4 × MIC of LF05 caused release of ATP in B. subtilis 168 within 10 min. Checkerboard test demonstrated LF05 exhibited additive effect when combined with vancomycin, erythromycin and berberine. The addition of rat plasma or bovine serum albumin to bacterial cultures caused significantly loss in antibacterial activity of LF05. Interestingly, LF05 was highly toxic to several tumor cells. Results of these studies indicate that LF05 is bactericidal against some Gram-positive bacteria and acts as a membrane structure disruptor. The application of biphenanthrene in the treatment of S. aureus infection, especially local infection, deserves further study.

Effective fraction of Bletilla striata reduces the inflammatory cytokine production induced by water and organic extracts of airborne fine particulate matter (PM2.5) in vitro.

BACKGROUND: Bletilla striata is a traditional Chinese medicine used to treat hemorrhage, scald, gastric ulcer, pulmonary diseases and inflammations. In this study, we investigated bioactivity of the effective fraction of B. striata (EFB) in reducing the inflammatory cytokine production induced by water or organic extracts of PM2.5. METHODS: PM2.5 extracts were collected and analyzed by chromatographic system and inductively coupled plasma mass spectrometer. Cell viability was measured using MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) assay, and cell supernatant was analyzed by flow cytometry, ELISA, and qRT-PCR in cultured mouse macrophage cell line RAW264.7 treated with EFB and PM2.5 extracts. Expressions of nuclear factor-kappa B (NF-κB) and mitogen-activated protein kinase (MAPK) signaling pathway were measured by Western blot. RESULTS: PM2.5 composition is complex and the toxicity of PM2.5 extracts were not noticeable. The treatment of EFB at a wide dose-range of 0-40 µg/mL did not cause significant change of RAW264.7 cell proliferation. EFB pretreatment decreased the inflammatory cytokines in the macrophage. Further analysis showed that EFB significantly attenuated PM2.5-induced proinflammatory protein expression and downregulated the levels of phosphorylated NF-κBp65, inhibitor of kappa B (IκB)-α, c-Jun N-terminal kinase (JNK), extracellular signal-regulated kinase (ERK), and p38. CONCLUSIONS: Our study demonstrated the potential effectiveness of B. striata extracts for treating PM2.5-triggered pulmonary inflammation.

[Comparison of contents of three active ingredients in Bletilla striata from different sources].

In order to understand the difference of contents of coelonin,batatasin Ⅲ and 3'-O-methylbatatasin Ⅲ in 60 different sources of Bletilla striata planted under the same conditions. UPLC method was used and the analysis was performed on a Waters ACQUITY UPLC BEH C18 column( 2. 1 mm×100 mm,1. 7 µm),eluted with acetonitril-0. 1% formic acid solution by gradient. The flow rate was 0. 208 m L·min-1,the detection wavelength was 270 nm,the column temperature was 35 ℃ and the injection volume was 4µL. Under the above chromatographic conditions,the three components can be separated well with good linearity in the range of 0. 156-5. 000 mg·L-1. The average contents of coelonin,batatasin Ⅲ and 3'-O-methylbatatasin Ⅲ were( 0. 116 ± 0. 071) %,( 0. 386 ±0. 185) % and( 0. 086±0. 034) %,respectively. After planting for two years under the same conditions,there was no significant difference in chemical composition among different sources and varieties,but the contents of the three components had some regional differences,which indicated that the western region was higher than the eastern region,while the contents of coelonin and batatasin Ⅲ in B.sinensis were slightly higher than those in B. striata. The chromatographic method above is simple,stable and reproducible,and can be used for quantitative analysis of three components. The content analysis of different sources of B. striata can provide reference for future B. striata breeding and quality control.

[Effect of pinostrobin chalcone on adipogenic differentiation of mesenchymal stem cell C3H10T1/2].

Chalcones is a flavonoid wildly presented in many herbs. It has the effect to inhibit cells adipogenic differentiation. In order to study the effect of pinostrobin chalcone extracted and isolated from leaves of hickoryes on the adipogenic differentiation of murine embryonic mesenchymal stem cell (C3H10T1/2), MTS [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)- 2H-tetrazolium] method was used to detect the cell proliferation; adipogenic differentiation was characterized by oil red O staining and isopropanol extraction; the triglyceride content was detected by GAP-PAP enzyme method; and the C3H10T1/2 cell differentiation into adipocytes was also examined by the mRNA and protein expression of PPARγ, C/EBPα and FABP4 by RT-PCR and Western blot respectively. Results indicated that pinostrobin chalcone almost had no effect on cell proliferation activity when the concentration was less than or equal to 50 µmol•L⁻¹; the oil red O staining, isopropanol extraction and GAP-PAP enzyme method showed that pinostrobin chalcone significantly decreased the C3H10T1/2 adipogenic differentiation and triglyceride content in the cytoplasm of adipocytes; the RT-PCR and Western blot analysis showed that pinostrobin chalcone can down-regulate the mRNA and protein levels of FABP4, PPARγ and C/EBPα in C3H10T1/2 cells(P<0.05 or P<0.01). The experiment results suggest that pinostrobin chalcone can inhibit C3H10T1/2 adipogenic differentiation.

The anti-Staphylococcus aureus activity of the phenanthrene fraction from fibrous roots of Bletilla striata.

BACKGROUND: Bletillae Rhizoma, the tuber of Bletilla striata, has been used in Chinese traditional medicine to treat infectious diseases. Chemical studies indicated that phenanthrene was one of the most important components of the herb, with a broad spectrum of antibiotic activity against Gram-positive bacteria. The objective of this study was to further characterize the antibacterial activity of the phenanthrene fraction from the fibrous root of the pseudobulb of B. striata. METHODS: The phenanthrene fraction (EF60) from the ethanol extract of fibrous roots of Bletilla striata pseudobulbs was isolated using polyamide column chromatography. The antibacterial activity of the fraction was evaluated in vitro using a 96-well microtiter plate and microbroth dilution method. The cytotoxicity of EF60 against mammalian cells was tested by hemolysis and MTT assays. RESULTS: EF60 was obtained using alcohol extraction and polyamide column chromatography, with a yield of 14.9 g per 1 kg of the fibrous roots of B. striata. In vitro tests indicated that EF60 was active against all tested strains of Staphylococcus aureus, including clinical isolates and methicillin-resistant S. aureus (MRSA). The minimum inhibitory concentration (MIC) values of EF60 against these pathogens ranged from 8 to 64 µg/mL. Minimum bactericidal concentration tests demonstrated that EF60 was bactericidal against S. aureus 3304 and ATCC 29213 and was bacteriostatic against S. aureus 3211, ATCC 25923, and ATCC 43300. Consistently, the time-kill assay indicated that EF60 could completely kill S. aureus ATCC 29213 at 2× the MIC within 3 h but could kill less than two logarithmic units of ATCC 43300, even at 4× the MIC within 24 h. The postantibiotic effects (PAE) of EF60 (4× MIC) against strains 29213 and 43300 were 2.0 and 0.38 h, respectively. Further studies indicated that EF60 (160 µg/mL) showed no cytotoxicity against human erythrocytes, and was minimally toxic to Human Umbilical Vein Endothelial Cells with an IC50 of 75 µg/mL. CONCLUSIONS: Our studies indicated that EF60 is worthy of further investigation as a potential phytotherapeutic agent for treating infections caused by S. aureus and MRSA.

[Protective Effect of Total Flavonoids from Carya cathayensis Leaf on Cultured H9C2 Cardiomyocytes During Hypoxia / Reoxygenation Injury].

Objective: To study the protective effect of total flavonoids from Carya cathayensis leaf on cultured H9C2 cardimyocytes during hypoxia / reoxygenation( H / R) injury. Methods: Co Cl2 was used to induce the H / R injury model in H9C2 cells at different concentrations,different H / R time. Total flavonoids from Carya cathayensis leaf was added into culture medium with final concentration of2. 5,5 and 10 µg / m L before H / R. Lactate dehydrogenase( LDH), Malondialdehyde( MDA) content, Superoxidedimutase( SOD) activity in the bathing medium were assayed for the evaluation of myocardial cell injury. Myocardial cell viability was detected by the MTS assay kit. Apoptotic changes in H9C2 cells were observed by using Hoechst-PI staining and flow cytometry analysis. The expression of HIF-1αprotein was detected by Western blot. Results: Using 1 200 µmol / L Co Cl2 to hypoxia the cells for 18 h and then reoxygenation for 2 h were the best condition of H / R injury model. Compared with model group,the cells treated with total flavonoids from Carya cathayensis leaf could decrease the activity of LDH and the content of MDA, while increased the activity of SOD. Moreover, they could regulate the expression of HIF-1α protein to normal level. Conclusion: It is suggested that total flavonoids from Carya cathayensis leaf has a protective effect on H9C2 cardiomyocyte during H / R injury. The mechanism may be relate to enhancing the capability of the cell clearing the oxygen free radial, decreasing the production of lipid peroxidation and reducing apoptosis.

[Chrysin inhibits adipogenic differentiation of mouse embryonic fibroblasts].

Chrysin is an active flavonoid wildly presented in many herbs. It has the effect to reduce serum lipid. To investigate the effect of chrysin on the adipogenic differentiation of mouse embryonic fibroblasts, methyl thiazolyl tetrazolium (MTT) and crystal violet were used to detect the cytotoxic effect of chrysin on Immortalized mouse embryonic fibroblasts (iMEFs). Propidium iodide (PI) staining combined with flow cytometry (FCM) was employed to detect the effects of different concentrations of chrysin on iMEFs cell cycle. The effect of chrysin on adipogenic differentiation ability of iMEFs was determined by oil red O staining. Semi-quantitative PCR was employed to detect the effect of chrysin on mRNA transcriptional levels of adipogenic differentiation markers, including perilipin 2, adiponectin (adipoq), Fabp4, LPL, MCP-1 and adipogenic differentiation key transcription factor peroxisome proliferators-actiated receptor-gamma 2(PPAR-γ2). Results indicated that chrysin had certain cytotoxic effect for iMEFs in a dose-dependent manner, and the IC50 was identified nearly to 30 µmol•L⁻¹. FCM analysis showed that chrysin could affect the cell-cycle distribution of iMEFs, increasing the ratio of cells in G1 phase. Adipogenic differentiation inducing experiment showed that 30 µmol•L⁻¹ chrysin significantly reduced lipid drops accumulation induced by insulin and dexamethasone. In addition, the mRNA transcriptional levels of PPAR-γ2 and LPL were significantly decreased and mRNA levels of fabp 4, MCP-1, adipoq were also affected after chrysin treatment. The experiment results suggest that chrysin attenuates the adipogenic differentiation capacity of mesenchymal stem cells.

[Antibacterial Activity of Chemical Constituents Isolated from Fibrous Roots of Bletilla striata].

Objective: To investigate the chemical constituents isolated from the fibrous roots of Bletilla striata, and to research their antibacterial activities. Methods: The native products were isolated and purified by silica gel, Sephadex LH-20 column chromatography and preparative HPLC. Their structures were elucidated on the basis of various spectroscopic analysis, and their antibacterial activities were tested by microbroth dilution method in a 96-well microtiter plate. Results: Seven compounds were isolated from the ethanol extract of the fibrous roots of Bletilla striata, and identified as p-hydroxybenzaldehyde( 1),2,7-dihydroxy-4-methoxy-9,10-dihydrophenanthrene( 2),4,5-dihydroxy-2-methoxy-9,10-dihydrohenanthrpene( 3),2-dihydroxy-4,7-dimethoxyphenan-threne( 4), militarine( 5), dactylorhin A( 6) and gastrodin( 7). Among them, compounds 2 ~ 4 showed moderate antibacterial activities against several Gram-positive bacterial strains( MIC 8 ~ 128 µg / m L),such as Staphylococcus aureus, Staphylococcus epidermidis, Enterococcus faecalis and Bacillus subtilis. Conclusion: The fibrous roots and tubers of Bletilla striata contain similar compounds, including glucosyloxybenzyl 2-isobutylmalates,and phenanthrene compounds, which showed antimicrobial activities against Gram-positive bacterial strains. And compounds 3,4 are isolated from Bletilla genus for the first time.

[Research on the Anti-Pulmonary Fibrosis Effect of the Bletilla striata Polysaccharide in Rat Silicosis Model].

Objective: To investigate the anti-pulmonary fibrosis effect and the possible molecular mechanism of the Bletilla striata polysaccharide. Methods: Polysaccharide was prepared by water reflux extraction plus ethanol precipitation method, and following deproteinization process by Sevage method. Rat silicosis model was established by invasive intratracheal instillation method. The effect and molecular mechanism of the polysaccharide was evaluated by lung indexes, lung pathological change, serum levels of SOD,MDA,NF-κB,IL-1ß,PDGF,TGF-ß1,TNF-α,HYP were detected, and the contents of CD3~+,CD4~+,CD8~+T lymph cells and CD4~+/ CD8~+ratio were detected by flow cytometry. Results: Both low( 100 mg / kg) and high( 400 mg / kg) dosage polysaccharide treatment could remarkably elevate the serum SOD level and reduce the MDA,NO level, and effectively reverse the CD4~+/ CD8~+ratio comparing with the model group( P < 0. 01). Except the TNF-α level was significantly lower in the high dosage treatment group, there was no other effect in inflammatory cytokines and HYP content in serum. HE pathological section confirmed that the Bletilla striata polysaccharide treatment group can not effectively prevent lung fibrosis. Conclusion: The Bletilla striata polysaccharide has remarkable regulation effect on antioxidation system and immune system, but can not effectively prevent lung fibrosis, more effort should be made to study the active antipulmonary fibrosis components of Bletilla striata.

Antibacterial Biphenanthrenes from the Fibrous Roots of Bletilla striata.

Four new 9',10'-dihydro-biphenanthrenes, including an unprecedented 1,2'-linked biphenanthrene, 4,7,3',5'-tetramethoxy-9',10'-dihydro(1,2'-biphenanthrene)-2,7'-diol (1), a new 1,3'-linked biphenanthrene, 4,7,7'-trimethoxy-9',10'-dihydro(1,3'-biphenanthrene)-2,2',5'-triol (2), and two new 1,1'-linked biphenanthrenes, 4,7,4'-trimethoxy-9',10'-dihydro(1,1'-biphenanthrene)-2,2',7'-triol (3) and 4,7,3',5'-tetramethoxy-9',10'-dihydro(1,1'-biphenanthrene)-2,2',7'-triol (4), as well as two known biphenanthrenes (5, 6), were isolated from a 95% ethanol extract of the fibrous roots of Bletilla striata. Their structures were determined by spectroscopic and spectrometric methods. Atropisomerism of these compounds was considered based on their chiral optical properties and potential energy surface scans at the ab initio HF/3-21G level, which revealed their racemic mixture form. Compounds 2-6 showed potent antibacterial activities against six Gram-positive bacterial strains.

[Study on anti-angiogenesis effect of three curcumin pigments and expression of their relevant factors].

To study the in vitro anti-angiogenesis effect of three curcumin pigments (curcumin, demethoxycurcumin, bisdemethoxycurcumin). In the study, the inhibitory effect of the three curcumin pigments on proliferation of HUVEC cells induced by OX-LDL and the effect on migration of HUVEC cells were detected. The effect on neovascularization was observed by chorioallantoic membrane (CAM) test. The effect on cell adhesion factors ICAM-1 and VCAM-1 of HUVECs were tested by Real-time RT-PCR. It was found that the three curcumins could inhibit the proliferation of HUVEC cells induced by OX-LDL within the dosage range 4, 8, 16 mg x L(-1), with a dose-dependence. The proliferative effect of curcumins on HUVECs was greater than the other two derivatives (P < 0.01). All of the three curcumin pigments inhibited the migration of HUVEC cells and the angiogenesis of chick chorioallantoic membrane (CAM). The migration inhibition rate of curcumins at middle and high concentrations was greater than the other two (P < 0.01). All of the three curcumin could down-regulate the expression of VEGF and ICAM-1, and curcumins showed more obvious effect in down-regulating VEGF than demethoxycurcumin and bisdemethoxycurcumin(P < 0.01); Bisdemethoxycurcumin showed the most significant effect in down-regulating ICAM-1 (P < 0.01). All of the three showed no remarkable effect on expression of VCAM-1, and only bisdemethoxycurcumin showed the down-regulating effect (P < 0.05). According to the findings, all of the three curcumin pigments could resist angiogenesis by inhibiting proliferation and migration of endothelial cells and down-regulating the expression of VEGF and adhesion molecules ICAM-1.

Lasiodiplodia sp. ME4-2, an endophytic fungus from the floral parts of Viscum coloratum, produces indole-3-carboxylic acid and other aromatic metabolites.

BACKGROUND: Studies on endophytes, a relatively under-explored group of microorganisms, are currently popular amongst biologists and natural product researchers. A fungal strain (ME4-2) was isolated from flower samples of mistletoe (Viscum coloratum) during a screening program for endophytes. As limited information on floral endophytes is available, the aim of the present study is to characterise fungal endophytes using their secondary metabolites. RESULTS: ME4-2 grew well in both natural and basic synthetic media but produced no conidia. Sequence analysis of its internal transcribed spacer rDNA demonstrated that ME4-2 forms a distinct branch within the genus Lasiodiplodia and is closely related to L. pseudotheobromae. This floral endophyte was thus identified as Lasiodiplodia sp. based on its molecular biological characteristics. Five aromatic compounds, including cyclo-(Trp-Ala), indole-3-carboxylic acid (ICA), indole-3-carbaldehyde, mellein and 2-phenylethanol, were found in the culture. The structures of these compounds were determined using spectroscopic methods combined with gas chromatography. To the best of our knowledge, our work is the first to report isolation of these aromatic metabolites from a floral endophyte. Interestingly, ICA, a major secondary metabolite produced by ME4-2, seemed to be biosynthesized via an unusual pathway. Furthermore, our results indicate that the fungus ME4-2 is a potent producer of 2-phenylethanol, which is a common component of floral essential oils. CONCLUSIONS: This study introduces a fungal strain producing several important aromatic metabolites with pharmaceutical or food applications and suggests that endophytic fungi isolated from plant flowers are promising natural sources of aromatic compounds.

[Differences of fungal diversity and structure in rhizosphere of Fritillaria thunbergii from different provenances].

To explore the mechanism of soil microbial ecology, the differences of fungal diversities in rhizosphere of different provenances of Fritillaria thunbergii were analyzed. The diversities and compositions of rhizo-fungi of the samples were analyzed by using DGGE and 454 pyrosequencing. DGGE results showed the Shannon index of Ninbo provenance planted in Ninbo was the highest one. And its dominant fungi were Ascomycota, Deuteromycota and Zygomycota. Except the same fungi, every provenance planted in Ninbo had its own special ones. From the 454 pyrosequencing, the fungal diversity in Panan producing was the highest which was similar with DGGE result. Among the ten phylum detected in its rhizosoil, Fungi_incertae_sedis, Ascomycota, Mucoromycotina, Basidiomycota and Chytridiomycota almost amounted to 90% of the whole community. The fungal types and amounts in Panan were more than those in Ninbo indicating the differences between producing areas and the advantage of macro genome sequencing. There were 10 phyla, 29 families, 28 genus and 159 species of fungi in Panan provenance, 6 phyla, 20 families, 19 genus, 136 species in Ninbo provenance, 8 phyla, 37 families, 47 genus, 289 species in Nantong provenance and 7 phyla, 25 families, 24 genus, 102 species in the bulk soil. Some genus such as Dothidea, Capnobotryella and Conidiobolus were only existed in Nantong provenance, while Pyrenochae- ta, Glomus and Pseudonectria were only in Panan provenance, which implied these species could grew because F. thunbergii influenced the existence of fungi. Experiments of provenance and producing area of F. thunbergii showed that the fungal diversity of indigenous provenance was higher than that of exotic provenance and each provenance had unique fungal species in the rhizosphere, which indicated that the diversity and structure was shaped cooperatively by the species and soil type. These fungal species are interacted with the soil-rhizhosphere-microbe microecological system, which in turn influence the growth of F. thunbergii.

[Effects of growth years of Paeonia lactiflora on bacterial community in rhizosphere soil and paeoniflorin content].

To explore the relationship between microecological environment and Paeonia lactiflora the effects of growth years of P. lactillora on rhizosphere bacterial communities were studied by PCR-DGGE and the paeoniflorin content determined by HPLC. Results showed that the soil pH increased with growing years of P. lactillora. In the fourth year, soil pH and enzyme activity reached the highest level, while organic matter content was the lowest. The bacterial diversity had a positive correlation with growing years varied from 3.38 to 3.61. Sequencing results demonstrated that Gammaproteobacteria, llphaproteobacteria, Actinobacteria, Acidobacte- ria and Firmicutes were predominant bacteria kinds in the soil of P. lactillora. Gammaproteobacteria was only detected in the bulk soil, while llphaproteobacteria, Acidobacteria G1l, Actinobacteria were only in the rhizosphere soil and the bacterial community among different growing years were similar except few species. HLPC results showed that paeoniflorin content was 3.26%, 3.30%, 3.36%, 3.41% separately from one to four-year-old P. lactiflora with an upward trend. The correlation analysis indicated that the paeoniflorin content had a positive correlation with soil pH and bacterial diversity, conversely, had a negative correlation with organic matter con- tent. During the growth years the rhizosphere bacterial diversity increased without changes of predominant bacteria and the paeoniflorin content increased without significant differences while its production increased significantly, which was different from the plants showing replanting diseases. This is in line with the farming practice choosing 4-year-old P. lactllora, but not the 1-3 year old one. In addition, the accumulation of paeoniflorin is closely related to soil pH, organic matter content and bacteria diversity, confirming that the geoherblism of P. lactiflora is closely related with microbial environment in the soil.

[Effect of different treatment on endophytic bacterial communities in continuous cropping of Chrysanthemum morifoliu].

To reveal the effect of rotation cropping and bacterial manure on the growth of Chrysanthemum morifolium and screen the beneficial endophytic, the diversity of endophytic and dominant genera of different treatment groups were analyzed. Four different treatments were continuous cropping, rotation, self-made organic fertilizer and commercially available fertilizer, respectively. Endophytic bacterial diversity and dominant genera in different organs were examined using Terminal Restriction Fragment Length Polymorphism (T-RFLP). The results showed that enzyme Hae III was more appropriate than enzyme Hinfl because the number of TRFs digested by enzyme Hae III was more than that of enzyme Hinfl. In comparison of diversity, the endophytic bacterial communities' diversity index in group of cropping rotation and fertilizer was higher than that of continuous cropping which indicated that the addition of exogenous microorganism in soil could increase the diversity of plant endophyte. 18 dominant species were selected, including 3 kinds of Firmicutes, 4 kinds of Actinomycetes and 11 kinds of Proteobacteria. The results of dominant species comparison showed that the number of dominant species in continuous cropping of Ch. morifolium was significantly less than that of the rotation group. Some dominant bacteria in rotation group and fertilizer group such as Arthrobacter, Streptomyces, Streptomyces, Flavobacterium and Mycobacterium were not found in the continuous cropping of Ch. mortfolium group. Dominant species of fertilizer treatment group was similar with the rotation group, and the continuous cropping group's dominant species was more abundant. It indicates that these bacteria may be able to mitigate hindrance in continuous cropping, especially the Flavobacterium which can decompose the pathogenic fungi is worthy of further attention. Compared with leaves, there are more dominant species in roots and stems. The diversity of edophytic bacterial communities in continuous cropping of Ch. morifolium stays below than that in the rotation of Ch. morifolium, and fertilizer treatment can increase the diversity of continuous cropping so that it could mitigate hindrance in continuous cropping.

[Protoplasts isolation, purification and plant regeneration of Pinellia cordata].

The main factors which affected the isolation, purification and cultivation of Pinellia cordata protoplasts from leaves were studied. The results indicated that the optimum enzyme solution for P. cordata leaves was 13% CPW + 1.0% Cellulose +0.1% Pectolase, at pH 6.0, temperature (25-28 degrees C ) for 4 h. The sucrose density gradient centrifugation was adopted to purificate the protoplasts collected, when 25% sucrose was used as mediator, centrifugating at 500 rpm for 10 min. When the protoplasts were shallow liquid and liquid-solid double layer cultured on the medium of MS + 0.5 mg x L(-1) 6-BA + 0.25 mg x L(-1) NAA + 13% mannitol at the density of 2.5 x 104 protoplasts/mL, or fed and nursed cultured at the density of 100-500 protoplasts/mL, cell division could be observed for 3 days; granular calli appeared for 30 days. Calli was proliferated on the medium of MS + 0.5 mg x L(-1) 6-BA + 0.25 mg x L(-1) NAA solidified by 0.55% agar, and differentiated and regenerated after 5-6 months. Plant generation of P. cordata is successfully established.

The induced and intrinsic resistance of Escherichia coli to sanguinarine is mediated by AcrB efflux pump.

IMPORTANCE: The use of plant extracts is increasing as an alternative to synthetic compounds, especially antibiotics. However, there is no sufficient knowledge on the mechanisms and potential risks of antibiotic resistance induced by these phytochemicals. In the present study, we found that stable drug resistant mutants of E. coli emerged after repetitive exposure to sanguinarine and demonstrated that the AcrB efflux pump contributed to the emerging of induced and intrinsic resistance of E. coli to this phytochemical. Our results offered some insights into comprehending and preventing the onset of drug-resistant strains when utilizing products containing sanguinarine.