Effects of plant essential oils and their constituents on Helicobacter pylori : A Review

Essential oils (EOs) obtained from different medicinal and aromatic plant families by steam distillation have been used in the pharmaceutical, food, and fragrance industries. The plant EOs and their broad diversity of chemical components have attracted researchers worldwide due to their human health benefits and antibacterial properties, especially their treatment of Helicobacter pylori infection. Since H. pylori has been known to be responsible for various gastric and duodenal diseases such as atrophic gastritis, peptic ulcer, gastric adenocarcinoma, and mucosa-associated lymphoid tissue lymphoma, several combination antibiotic therapies have been increasingly used to enhance the eradication rate of the bacterial infection. However, in the last decades, the efficacy of the therapies has decreased significantly due to widespread emergence of multidrug resistant strains of H. pylori. In addition, side-effects from commonly used antibiotics and recurrence of the bacterial infection have drawn public health concern globally.Therefore, this review focuses on in vitro effects of plant EOs and their bioactive constituents on the growth, cell morphology and integrity, biofilm formation, motility, adhesion, and urease activity of H. pylori. Their inhibitory effects on expression of genes necessary for growth and virulence factor productions of the bacterial pathogen are also discussed. Further in vivo and clinical evaluations are required so that plant EOs and their bioactive constituents can be possibly applicable in pharmacy or as adjuvants to the current therapies of H. pylori infection

Current advances in seagrass research: A review from Viet Nam

Seagrass meadows provide valuable ecosystem services but are fragile and threatened ecosystems all over the world. This review highlights the current advances in seagrass research from Viet Nam. One goal is to support decision makers in developing science-based conservation strategies. In recent years, several techniques were applied to estimate the size of seagrass meadows. Independent from the method used, there is an alarming decline in the seagrass area in almost all parts of Viet Nam. Since 1990, a decline of 46.5% or 13,549 ha was found. Only in a few protected and difficult-to-reach areas was an increase observed. Conditions at those sites could be investigated in more detail to make suggestions for conservation and recovery of seagrass meadows. Due to their lifestyle and morphology, seagrasses take up compounds from their environment easily. Phytoremediation processes of Thalassia hemprichii and Enhalus acoroides are described exemplarily. High accumulation of heavy metals dependent on their concentration in the environment in different organs can be observed. On the one hand, seagrasses play a role in phytoremediation processes in polluted areas; on the other hand, they might suffer at high concentrations, and pollution will contribute to their overall decline. Compared with the neighboring countries, the total Corg stock from seagrass beds in Viet Nam was much lower than in the Philippines and Indonesia but higher than that of Malaysia and Myanmar. Due to an exceptionally long latitudinal coastline of 3,260 km covering cool to warm water environments, the seagrass species composition in Viet Nam shows a high diversity and a high plasticity within species boundaries. This leads to challenges in taxonomic issues, especially with the Halophila genus, which can be better deduced from genetic diversity/population structures of members of Hydrocharitaceae. Finally, the current seagrass conservation and management efforts in Viet Nam are presented and discussed. Only decisions based on the interdisciplinary cooperation of scientists from all disciplines mentioned will finally lead to conserve this valuable ecosystem for mankind and biodiversity

Safety and efficacy of fluoxetine on functional outcome after acute stroke (AFFINITY): a randomised, double-blind, placebo-controlled trial

Background Trials of fluoxetine for recovery after stroke report conflicting results. The Assessment oF FluoxetINe In sTroke recoverY (AFFINITY) trial aimed to show if daily oral fluoxetine for 6 months after stroke improves functional outcome in an ethnically diverse population. Methods AFFINITY was a randomised, parallel-group, double-blind, placebo-controlled trial done in 43 hospital stroke units in Australia (n=29), New Zealand (four), and Vietnam (ten). Eligible patients were adults (aged ≥18 years) with a clinical diagnosis of acute stroke in the previous 2–15 days, brain imaging consistent with ischaemic or haemorrhagic stroke, and a persisting neurological deficit that produced a modified Rankin Scale (mRS) score of 1 or more. Patients were randomly assigned 1:1 via a web-based system using a minimisation algorithm to once daily, oral fluoxetine 20 mg capsules or matching placebo for 6 months. Patients, carers, investigators, and outcome assessors were masked to the treatment allocation. The primary outcome was functional status, measured by the mRS, at 6 months. The primary analysis was an ordinal logistic regression of the mRS at 6 months, adjusted for minimisation variables. Primary and safety analyses were done according to the patient's treatment allocation. The trial is registered with the Australian New Zealand Clinical Trials Registry, ACTRN12611000774921. Findings Between Jan 11, 2013, and June 30, 2019, 1280 patients were recruited in Australia (n=532), New Zealand (n=42), and Vietnam (n=706), of whom 642 were randomly assigned to fluoxetine and 638 were randomly assigned to placebo. Mean duration of trial treatment was 167 days (SD 48·1). At 6 months, mRS data were available in 624 (97%) patients in the fluoxetine group and 632 (99%) in the placebo group. The distribution of mRS categories was similar in the fluoxetine and placebo groups (adjusted common odds ratio 0·94, 95% CI 0·76–1·15; p=0·53). Compared with patients in the placebo group, patients in the fluoxetine group had more falls (20 [3%] vs seven [1%]; p=0·018), bone fractures (19 [3%] vs six [1%]; p=0·014), and epileptic seizures (ten [2%] vs two [<1%]; p=0·038) at 6 months. Interpretation Oral fluoxetine 20 mg daily for 6 months after acute stroke did not improve functional outcome and increased the risk of falls, bone fractures, and epileptic seizures. These results do not support the use of fluoxetine to improve functional outcome after stroke

적작약 뿌리 성분 및 구조적 유사 화합물의 human rhinovirus에 대한 항바이러스 활성 및 장내세균에 대한 항균활성

학위논문 (박사)-- 서울대학교 대학원 : 협동과정 농업생물공학전공, 2013. 2. 안영준.An assessment was made of the antiviral and antibacterial activities of paeony, Paeonia lactiflora Pallas (Paeoniaceae), root constituents and structurally related compounds against two serotypes of human rhinovirus, HRV-2 and HRV-4, in HeLa ATCC CCL–2 (a human cervix epithelial cell line) and MRC5 CCl-171 (a normal human lung fibroblast cell line) and antibiotic-susceptible and -resistant strains of Helicobacter pylori and human intestinal bacteria (nine harmful bacteria and eight lactic acid-producing bacteria). Modes of antiviral and antibacterial action also were examined using molecular and biochemical techniques. The antiviral activities of 1,2,3,4,6-penta-O-galloyl-beta-D-glucopyranose (PGG), paeonol (PA), and gallic acid (GA) identified in paeony root against HRV-2 and HRV-4 were evaluated using a tetrazolium assay. Results were compared with those of ribavirin, a broad spectrum antiviral agent. PA and ribavirin did not cause cytotoxicity to both cell lines (CC50, >500 μg/mL). PGG exhibited low toxicity to both cell lines (CC50, ~102 μg/mL). GA caused high toxicity to MRC5 (CC50, 37.0 μg/mL), whereas it caused very low toxicity to HeLa cells (CC50, 255.2 μg/mL). Based on fifty percent inhibitory concentration (IC50) values, PGG was the most active compound against the HRVs (10.9 and 13.53 μg/mL in HeLa cells16.83 and 16.31 μg/mL in MRC5 cells to HRV-2 and HRV-4, respectively), followed by GA (72.6 and 76.23 μg/mL in HeLa cells to HRV-2 and HRV-4, respectively). GA did not inhibit the viruses in the MRC5 cells. Ribavirin and PA exhibited high and moderate activity, respectively against the HRVs in both cell lines (IC50, 58‒79 μg/mL and 82‒101 μg/mL, respectively). PGG and PA inhibited RNA replication of HRV-2 and HRV-4 and also reduced expression of rhinovirus receptors and inflammatory cytokines induced by the two HRV serotypes. Quantitative structure–activity relationship (QSAR) of paeonol and 10 structurally related compounds in MRC5 cells indicates that structural characteristics, such as types of functional groups and carbon skeleton appear to play a role in determining the anti-rhinovirus activity. The introduction of methoxy group(s) into acetophenone may be required for rhinovirus inhibition. The growth-inhibiting, bactercidial, and urease inhibitory activities of PA, benzoic acid (BA), methyl gallate (MG), and PGG identified in paeony root, structurally related compounds, root steam distillate constituents, and four antibiotics toward three reference strains and four clinical isolates of H. pylori were assessed using broth dilution bioassay and Western blot. BA and PA showed strong bactericidal effect at pH 4, while MG and PGG were effective at pH 7. These constituents exhibited strong growth-inhibiting and bactericidal activity toward the five strains resistant to amoxicillin (minimal inhibitory concentration (MIC), 12.5 mg/L), clarithromycin (64 mg/L), metronidazole (64 mg/L), or tetracycline (15 mg/L), indicating that these constituents and the antibiotics do not share a common mode of action or elicit cross-resistance. QSAR of paeonol and 10 structurally related compounds indicates that structural characteristics, such as types of functional groups and carbon skeleton, and hydrophobicity appear to play a role in determining the anti-H. pylori activity. H. pylori urease inhibitory activity of PGG was comparable to that of acetohydroxamic acid, while MG was less potent at inhibiting urease than thiourea. The UreB band disappeared at 250 mg/L PGG on Western blot, while the UreA bands were fainted visible at 1,000 mg/L PGG. These constituents showed no significant cytotoxicity to three human cell lines, HeLa, MRC5, and A549 (a human lung carcinoma cell line). High potent anti-H. pylori activity was also produced by steam distillate and its constituents. Thymol, α-terpinolene, (1R)-(‒)-myrtenol, (1S,2S,5S)-(‒)-myrtanol, (‒)-perilla alcohol, (‒)-borneol, (1R)-(‒)-myrtenal and paeonol showed high activity (MIC, 40–160 mg/L). The other constituents of the SD showed moderate or weak activity. The steam distillate exhibited strong growth-inhibiting and bactericidal activity against the five resistant strains indicating that these strains were lack of mechanisms of resistance to the steam distillate. In addition, the steam distillate inhibited H. pylori colonization in mouse stomach. The growth inhibitory activities of paeony root steam distillate constituents and structurally related compounds against nine harmful intestinal bacteria and eight lactic acid-producing bacteria were compared with those of two antibiotics, amoxicillin and tetracycline. Thymol, α-terpinolene, (‒)-perilla alcohol, and (1R)-(‒)-myrtenol exhibited high to extremely high levels of growth inhibition of all the harmful bacteria, whereas thymol and α-terpinolene (except for Lactobacillus casei ATCC 393) inhibited the growth of all the beneficial bacteria (MIC, both 0.08–0.62 mg/mL). Tetracycline and amoxicillin exhibited extremely high level of growth inhibition of all the test bacteria (MIC, <0.00002–0.001 mg/mL). 1,8-Cineole, geraniol, (‒)-borneol, (1S,2S,5S)-(‒)-myrtanol, nerol, (S)-(‒)-β-citronellol, and (+/-)-lavandulol also exhibited inhibitory activity but with differing specificity and levels of activity. Structure–activity relationship indicates that structural characteristics, such as geometric isomerism, degrees of saturation, types of functional group and types of carbon skeleton, appear to play a role in determining the growth-inhibiting activity of monoterpenoids. In conclusion, global efforts to reduce the level of antibiotics justify further studies on P. lactiflora root-derived materials as potential antiviral or antibacterial products, or lead molecules for the prevention or eradication of human diseases caused by rhinovirus, H. pylori, and harmful intestinal bacteria such as clostridia. Keywords: Paeonia lactiflora root, natural growth inhibitor, natural bactericide, gatrointestinal bacteria, Helicobacter pylori, human rhinovius, antibiotic resistance, urease, terpenoids, structure– activity relationship Student number: 2007 – 30696ABSTRACT ………….………........………………………………… i ABBREVIATIONS …………….........…….…………………………… x LIST OF TABLES ………….…….........………………………………… xii LIST OF FIGURES ……….……….........……………………………… xvi INTRODUCTION ……….………........………………………………… 1 LITERATURE REVIEW ……………........……………………………… 4 Chapter 1. Antiviral activity of constituents identified in Paeonia lactiflora root against rhinovirus Abstract 35 1.1. Introduction ………………………..…….….........………….………………… 37 1.2. Materials and methods ……..…………….........…………….……………… 38 1.2.1. Instrumental analyses 38 1.2.2. Materials 38 1.2.3. Plant 39 1.2.4. Cell lines and human rhinovirus serotypes 39 1.2.5. Cytotoxicity assay 41 1.2.6. HRV production 41 1.2.7. Virus titration 42 1.2.8. Cytopathic effect inhibition assay 43 1.2.9. Extraction and isolation 43 1.2.10. Effect of active constituents on the infectivity of human rhinovirus particles 48 1.2.11. Time course of compound addition 48 1.2.12. Real-time quantitative RT-PCR analysis 49 1.2.13. Measurement of ICAM-1 and LDLR expression 50 1.2.14. Western Blotting 52 1.2.15. Measurement of cytokine production 53 1.2.16. Data analysis 53 1.3. Results …………………..………………………………………………………… 55 1.3.1. Bioassay-guided fractionation and isolation of active principles 55 1.3.2. Cytotoxicity of test compounds 65 1.3.3. Antiviral activity of test compounds 66 1.3.4. Effects of PA and PGG on the infectivity of HRV particles 72 1.3.5. Time course of test compound addition 73 1.3.6. Effects of PA and PGG on the level of HRV replication 74 1.3.7 Effects of PA and PGG on ICAM-1 and LDLR expressions 75 1.3.8. Effects of PA and PGG on expression of cytokines IL-6 and IL-8 80 1.3.9. Effects of PA and PGG on expressions of other cytokines 84 1.3.10. Effects of PA and PGG on TLR3 mRNA expression 87 1.4. Discussion …..…………………………………………………………….............. 88 Chapter 2. Growth-inhibiting, bactericidal, and urease inhibitory effects of Paeonia lactiflora root constituents on antibiotic-susceptible and -resistant strains of Helicobacter pylori Abstract 96 2.1. Introduction …………………………...………………………………...... 98 2.2. Materials and Methods ………………………………..…………........... 101 2.2.1. Chemicals 101 2.2.2. Bacterial strains and culture conditions 101 2.2.3. Steam distillation 102 2.2.4. Gas chromatography 102 2.2.5. Gas chromatography-mass spectrometry 102 2.2.6. Extraction and isolation 103 2.2.7. Microbiological assay 106 2.2.8. Measurement of bactericidal activity at various pH values 107 2.2.9. Microscopic observation 108 2.2.10. Scanning and transmission electron microscopic analysis 108 2.2.11. Cytotoxicity assay 109 2.2.12. Inhibition of urease in vitro 109 2.2.13. SDS-PAGE and Western blot analysis 110 2.2.14. Native PAGE and zymogram analysis 111 2.2.15. In vivo study 111 2.2.16. Data analysis 113 2.3. Results …………………………………...………………………................ 115 2.3.1. Antibiotic resistance 115 2.3.2. Paeonia lactiflora root steam distillate 117 2.3.2.1. Chemical composition of P. lactiflora root steam distillate 117 2.3.2.2. Growth-inhibiting activities of P. lactiflora root steam distillate constituents 119 2.3.2.3. Bactericidal activities of P. lactiflora root steam distillate constituents 119 2.3.2.4. Effect on morphology of H. pylori 123 2.3.2.5. Effects of P. lactiflora root steam distillate on H. pylori colonization in mouse stomach 124 2.3.3. Paeonia lactiflora root extract 125 2.3.3.1. Bioassay-guided fractionation and identification 125 2.3.3.2. Growth inhibitory activity of test compounds 133 2.3.3.3. Bactericidal activity of test compounds 134 2.3.3.4. Effect of test compounds on the viability of H. pylori at varying pH values 136 2.3.3.5. Structure–activity relationship 142 2.3.3.6. Cytotoxicity 145 2.3.3.7. Effect on morphology of H. pylori 145 2.3.3.8. Urease inhibitory activity 147 2.3.3.9. Effect on urease production 148 2.4. Discussion ………………………………...…………………………… 151 Chapter 3. Growth-inhibiting effects of Paeonia lactiflora root steam distillate constituents and structurally related compounds on human intestinal bacteria Abstract 156 3.1. Introduction ……………………………………………………..........… 157 3.2. Materials and Methods …………...……………………………….......... 159 3.2.1. Steam distillation 159 3.2.2. Chemicals 159 3.2.3. Bacterial strains and culture conditions 159 3.2.4 Growth-inhibiting assay 161 3.3. Results …………………………………………………………………… 162 3.4. Discussion ………………...……………………………………………… 170 Conclusion .................................................................................................. 175 References ................................................................................................... 180 Abstract in Korean ..................................................................................... 219 Acknowledgement .................................................................................. 224Docto

Antimicrobial Effect of Platelet-Rich Plasma against Porphyromonas gingivalis

Aim. The aim of our study was to evaluate whether there was a difference in antimicrobial effect between the PRP of healthy volunteers and that of patients with chronic periodontitis against P. gingivalis, which was fresh cultured from subgingival plaque. Methods. Subgingival plaque of patients with moderate and severe chronic periodontitis was collected to isolate P. gingivalis. The PRP of four individuals with healthy periodontium and four patients with moderate and severe periodontitis were collected with a specific kit using a two-centrifuge procedure, and then, the antibacterial properties against P. gingivalis were tested, through their minimum inhibitory concentration (MIC), adhesion resistance assay, and biofilm susceptibility assay. Results. P. gingivalis was successfully isolated from the subgingival plaque of the 21st patient. The round, smooth, and black colony appeared in the agar disk after 7–10 days of incubation under anaerobic conditions. Bacterial identification was performed by MALDI-TOF and confirmed by PCR. All PRP samples tested showed the ability to inhibit P. gingivalis growth. The MIC value (expressed as fraction of PRP) was 1/2, and PRP prevented P. gingivalis attachment on the disk surface. However, PRP did not have a strong effect on the suppression of P. gingivalis biofilm. Conclusion. PRP of individuals with healthy periodontium and chronic periodontitis patients showed antibacterial properties against P. gingivalis. This material can become an adjunct to periodontal treatment

Antiviral activity and possible mechanism of action of constituents identified in Paeonia lactiflora root toward human rhinoviruses.

Human rhinoviruses (HRVs) are responsible for more than half of all cases of the common cold and cost billions of USD annually in medical visits and missed school and work. An assessment was made of the antiviral activities and mechanisms of action of paeonol (PA) and 1,2,3,4,6-penta-O-galloyl-β-D-glucopyranose (PGG) from Paeonia lactiflora root toward HRV-2 and HRV-4 in MRC5 cells using a tetrazolium method and real-time quantitative reverse transcription polymerase chain reaction and enzyme-linked immunosorbent assay. Results were compared with those of a reference control ribavirin. Based on 50% inhibitory concentration values, PGG was 13.4 and 18.0 times more active toward HRV-2 (17.89 μM) and HRV-4 (17.33 μM) in MRC5 cells, respectively, than ribavirin. The constituents had relatively high selective index values (3.3->8.5). The 100 μg/mL PA and 20 μg/mL PGG did not interact with the HRV-4 particles. These constituents inhibited HRV-4 infection only when they were added during the virus inoculation (0 h), the adsorption period of HRVs, but not after 1 h or later. Moreover, the RNA replication levels of HRVs were remarkably reduced in the MRC5 cultures treated with these constituents. These findings suggest that PGG and PA may block or reduce the entry of the viruses into the cells to protect the cells from the virus destruction and abate virus replication, which may play an important role in interfering with expressions of rhinovirus receptors (intercellular adhesion molecule-1 and low-density lipoprotein receptor), inflammatory cytokines (interleukin (IL)-6, IL-8, tumor necrosis factor, interferon beta, and IL-1β), and Toll-like receptor, which resulted in diminishing symptoms induced by HRV. Global efforts to reduce the level of synthetic drugs justify further studies on P. lactiflora root-derived materials as potential anti-HRV products or lead molecules for the prevention or treatment of HRV

Effect on expression of TLR3 mRNA.

<p>Toll-like receptor 3 (TLR3) mRNA expression was detected by real-time quantitative reverse transcription-PCR in MRC5 2 days after infection in the presence of 100 μg/mL paeonol (PA) and 20 μg/mL 1,2,3,4,6-penta-<i>O</i>-galloyl-β-D-glucopyranose (PGG). Each bar represents the mean ± SD of triplicate samples of three independent experiments. ***p<0.001, **p<0.01, using a Bonferroni multiple comparison post-test.</p

Effect on mRNA and protein expressions of ICAM1 and LDLR.

<p>The mRNA expressions of membrane-bound intercellular adhesion molecule-1 (mICAM-1) (<b>A</b>) and low-density lipoprotein receptor (LDLR) (<b>B</b>) were detected by real-time quantitative reverse transcription-PCR in MRC5 2 days after infection in the presence of 100 μg/mL paeonol (PA) and 20 μg/mL 1,2,3,4,6-penta-<i>O</i>-galloyl-β-D-glucopyranose (PGG). Soluble ICAM-1 (sICAM-1) (<b>C</b>) was detected by ELISA. Each bar represents the mean ± SD of triplicate samples of three independent experiments. ***p<0.001, **p<0.01, *p<0.05, using a Bonferroni multiple comparison post-test.</p

Effect on protein expression of cytokines.

<p>Concentrations of interleukin (IL)-6 (<b>A</b>) and IL-8 (<b>B</b>) was detected by ELISA in MRC5 supernatants after 2 day infection in the presence of 100 μg/mL paeonol (PA) and 20 μg/mL 1,2,3,4,6-penta-<i>O</i>-galloyl-β-D-glucopyranose (PGG). Each bar represents the mean ± SD of triplicate samples of three independent experiments. ***p<0.001, **p<0.01, using a Bonferroni multiple comparison post-test.</p