Major chemotypes and antioxidative activity of the leaf essential oils of Cinnamomum osmophloeum Kaneh. from a clonal orchard

Essential oils of 92 cutting clones from a clonal orchard of Cinnamomum osmophloeum Kaneh. were obtained by hydrodistillation and characterised by gas chromatography–mass spectrometry. Our results showed that the yields of essential oils ranged between 0.09% and 2.65% (vol/fresh wt). The constituents of essential oils varied among samples. The major chemotypes classified in the individual cutting clones were cinnamaldehyde (50 plants, representing 50–95% of the total volatiles), linalool (1 plant, 73.3%), β-cubebene (2 plants, 59.4% and 78.7%), and cinnamyl acetate (1 plant, 61.8%). The antioxidant activities of the four chemotypes were determined using a 1,1-diphenyl-2-picrylhydrazyl (DPPH) assay. The antioxidant activities of the essential oil decreased in the order of cinnamyl acetate > cinnamaldehyde > β-cubebene > linalool. Indigenous cinnamon oil extract showed a good free radical-scavenging capacity at all concentrations studied, except at 2 μg/ml. The scavenging activity increased with increasing concentration of the extract. The capability of the four essential oil chemotypes to reduce the stable radical, DPPH, to DPPH-H was assayed by a decrease in the IC₅₀ values of 10.4 (cinnamyl acetate type) to 29.7 (linalool type) μg/ml. These results suggest that the leaf essential oil of C. osmophloeum possesses chemical compounds with antioxidant activity which can be used as natural preservatives in food and/or by the pharmaceutical industry. Trees in this plantation which can be used for further propagation for the production of chemotypes of interest were identified.     

Antifungal property of the essential oils and their constituents from Cinnamomum osmophloeum leaf against tree pathogenic fungi

This study compares the chemical constituents of leaf essential oils from various geographical provenances of Cinnamomum osmophloeum and investigates their antifungal activities against six tree pathogenic fungi. According to GC‐MS and cluster analyses, the leaf essential oils obtained from different geographical provenances and their relative contents were classified into six chemotypes: cinnamaldehyde type, cinnamaldehyde–cinnamyl acetate type, cinnamyl acetate type, linalool type, camphor type, and mixed type. Results from the antifungal tests show that the leaf essential oils of cinnamaldehyde type and cinnamaldehyde–cinnamyl acetate type have excellent inhibitory effect against Rhizoctonia solani, Collectotrichum gloeosporioides, Ganoderma australe and Fusarium solani. Furthermore, among the fourteen constituents of C osmophloeum leaf essential oils, Z‐cinnamaldehyde, eugenol, geraniol and citral display the best antifungal properties. Comparisons of the antifungal properties of Z‐cinnamaldehyde congeners reveal that Z‐cinnamaldehyde exhibits the best antifungal property of this group. Copyright © 2005 Society of Chemical Industry     

Cinnamon essential oil and cinnamaldehyde in the control of bacterial biofilms formed on stainless steel surfaces

The antibacterial effects of Cinnamomum cassia essential oil (EO) and cinnamaldehyde were evaluated against single- and mixed-species cultivation of enteropathogenic Escherichia coli (EPEC) and Listeria monocytogenes attached to stainless steel. A central composite rotational design with two variables and eleven assays was used to optimize the concentrations (0.00–1.00% v/v for the EO and 0.00–0.80% v/v for cinnamaldehyde) and contact times (1–21 min). The models generated were validated, and the effectiveness of C. cassia EO and cinnamaldehyde was compared with that of commercially available chemical sanitizers. Cinnamaldehyde and C. cassia EO proved to be efficient alternatives to commercial chemical sanitizers in the reduction or elimination of sessile bacterial cells. The activity of these natural compounds was, in most cases, equivalent or superior to that of the chemical sanitizers tested. However, L. monocytogenes was more resistant than EPEC to C. cassia EO and cinnamaldehyde, and the bacterial association in mixed-species biofilms made them more susceptible to these compounds.     

Oryzadine,a new alkaloid of Oryza sativa cv. Heugjinjubyeo, attenuates oxidative stress-induced cell damage via a radical scavenging effect

The antioxidant properties of oryzadine, a new alkaloid, obtained from Oryza sativa cv. Heugjinjubyeo was investigated by applying various methods based on cell-free and cell experiments. Oryzadine showed scavenging effects on the hydroxyl radical, superoxide radical, and intracellular reactive oxygen species (ROS). Oryzadine inhibited H₂O₂-induced DNA damage, which was demonstrated by DNA tail formation, lipid peroxidation which was demonstrated by the formation of thiobarbituric acid reactive substance (TBARS), and protein oxidation which was demonstrated by protein carbonyl formation. Therefore, oryzadine protected H₂O₂-induced cell damage. Our results show that the cytoprotective effects of oryzadine stem from its ability to inhibit H₂O₂-induced apoptosis, as demonstrated by a decrease in apoptotic body formation and the inhibition of mitochondrial membrane potential (ΔΨ) loss. Taken together, these findings suggest that oryzadine protected cells against H₂O₂-induced cell damage via ROS scavenging effect. Therefore, oryzadine could be considered a significant natural source of antioxidant.     

Principal phenolic phytochemicals and antioxidant activities of three Chinese medicinal plants

The principal antioxidant components and content of cinnamon (Cinnamomum cassia), turmeric (Curcuma longa) and golden thread (Coptidis rhizoma) extracts were determined using high performance liquid chromatography (HPLC) with UV detection. In general, C. cassia, C. longa and C. rhizoma extracts from domestic Taiwan were rich in cinnamaldehyde, curcumin, and berberin, respectively. The contents of cinnamaldehyde, curcumin, and berberin in the acetone extracts were 1911, 2029, and 840 mg l⁻¹, respectively. The Folin–Ciocalteu method was used to measure the total phenolic concentrations of extracts, which had the content of 9.6 (C. cassia), 2.6 (C. longa), and 4.3 (C. rhizoma) mM l⁻¹. In addition, DPPH radical-scavenging, ferric-reducing antioxidant power (FRAP), and ferric thiocyanate (FTC) assays were employed to measure antioxidant activities. The C. cassia fresh extracts had higher antioxidant activities which were 84–90% (DPPH), 17–33 μmol l⁻¹ (FRAP), and 53–82% (FTC). The activities of C. longa fresh extracts were 22–44% (DPPH), 7–11 μmol l⁻¹ (FRAP), and 53–81% (FTC) while C. rhizoma were 53–64% (DPPH), 18–26 μmol l⁻¹ (FRAP), and 59–82% (FTC).     

Kinetic of production and mode of action of the Lactobacillus paracasei subsp. paracasei anti-listerial bacteriocin, an Algerian isolate

Lactobacillus paracasei subsp. paracasei was isolated in our laboratory from breast-fed newborn faeces and identified phenotypically and genotypically. The strain was able to produce a bacteriocin-like substance active towards listerial strains (Listeria innocua CLIP 74915 and Listeria monocytogenes EGDe). The maximum production of the substance by producing strain was detected in the late logarithmic growth phase (14 h in MRS and 18 h in BHI broths). It displayed bactericidal mode of action with leakage of cellular content (K⁺ and ATP) leading to cell lysis as secondary effect. A reduction of about 5 log with 35.7 ± 0.2% of cell lysis and of about 4 log with 82.0 ± 0.3% of cell lysis were observed, respectively, in a phosphate buffer and BHI suspensions. This was further demonstrated by electron microscopy that showed severe modifications in the cell morphology with a concomitant lysis.     

Inhibition of β-amyloid peptide-induced neurotoxicity by kaempferol 3-O-(6″-acetyl)-β-glucopyranoside from butterbur (Petasites japonicus) leaves in B103 cells

One of predominant hallmarks in Alzheimer’s disease (AD) is extracellular senile plaques containing β-amyloid peptide (Aβ). Aβ is known to be directly responsible for the free radical production and lipid peroxidation, leading to apoptosis and cellular death. In this study, we investigated the possible protective effect of kaempferol 3-O-(6″-acetyl)-β-glucopyranoside (KAG) isolated from butterbur (Petasites japonicus) leaves against Aβ-induced neurotoxicity. Exposure of mouse neuroblastoma B103 cells to Aβ(25–35) at the concentration of 50 μM significantly reduced cellular viability and increased both oxidative stress and apoptotic rate. However, pretreatment of B103 cell with isolated KAG at 10 μM significantly inhibited Aβ-induced apoptotic cellular damage and reactive oxygen species (ROS) generation. Pretreatment of KAG also completely inhibited caspase-3 activity to the basal level at the concentration of 10 mM. This study therefore demonstrated that Aβ induced cellular death might be prevented by KAG from butterbur leaves by the suppression of ROS and the subsequent recovery of apoptotic cell death.     

Toxin profile, antibiotic resistance, and phenotypic and molecular characterization of Bacillus cereus in Sunsik

Sunsik, a ready-to-eat food in Korea, is comprised of various agricultural and marine products, and has been an important concern in Bacillus cereus food poisoning. The aim of this study was to investigate the toxin profiles, genotypic and phenotypic patterns as well as antibiotic resistance of B. cereus strains isolated from Sunsik. A subtyping method known as automated repetitive sequence-based PCR system (DiversiLab™) was used to assess the intraspecific biodiversity of these isolates. Thirty-five B. cereus strains were isolated from 100 commercial Sunsik samples, all of which harbored at least 1 enterotoxin gene. The detection rates of nheABC, hblCDA, cytK, and entFM enterotoxin gene among all isolates were 97%, 86%, 77%, and 100%, respectively. Most strains also produced corresponding enterotoxins such as HBL (83%) and NHE (94%). One strain (2.9%) carried the emetic toxin genes, including ces and EM1, and was positive for the HEp-2 cell emetic toxin assay. Most strains were positive for various biochemical tests such as salicin hydrolysis (86%), starch fermentation (89%), hemolysis (89%), motility test (100%) and lecithinase hydrolysis (89%). All isolates were susceptible to most antibiotics although they were highly resistant to β-lactam antibiotics. By using the automated rep-PCR system, all isolates were successfully differentiated, indicating the diversity of B. cereus strains present in Sunsik.     

Cyphomandra betacea Sendt. phenolics protect LDL from oxidation and PC12 cells from oxidative stress

Cyphomandra betacea Sendt. (tamarillo) is a subtropical fruit containing rich contents of anthocyanins and carotenoids. The antioxidant activity was investigated using a crude ethanol extract of C. betacea fruit and its partitioned fractions, i.e. the ethyl acetate, butanol and water fractions. The ethyl acetate fraction exhibited the highest DPPH scavenging activity, Trolox equivalent antioxidant capacity (TEAC) and total phenol content. C. betacea phenolics in ethyl acetate fraction inhibited copper-induced LDL oxidation equally to or more effectively than dl-α-tocopherol, as measured by decreased formation of thiobarbituric acid-reactive substances (TBARS), conjugated diene and relative electrophoretic mobility (REM). Furthermore, 3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyl tetrazolium (MTT) reduction assay showed that C. betacea phenolics in ethyl acetate fraction prevent oxidative stress-induced cell death in neuronal PC12 cells in a dose-dependent manner via attenuation of ROS production. In conclusion, C. betacea phenolics are potent antioxidants which can inhibit LDL oxidation in vitro and ROS production in PC12 cells.     

Antimicrobial effects of selected plant essential oils on the growth of a Pseudomonas putida strain isolated from meat

The inhibitory effect of 60 different essential oils was evaluated on a Pseudomonas putida strain of meat origin, associated with meat spoilage. Essential oils were tested at concentrations from 0.003 to 0.8% (wt/vol) to determine minimum inhibitory and maximal tolerated concentrations (MIC and MTC, respectively) using an agar medium culture. Of the 60 samples tested, Corydothymus capitatus essential oil was the most active showing a MIC of 0.025% and a MTC of 0.06%. Seven essential oils (Cinnamomum cassia, Origanum compactum, Origanum heracleoticum, Satureja hortensis, Satureja montana, Thymus vulgaris carvacroliferum, Thymus vulgaris thymoliferum) have shown a strong antimicrobial activity against P. putida with a MIC of 0.05% and a MTC ranging from 0.013% to 0.025%. Ten other oils (Cinnamomum verum (leaf and bark), Eugenia caryophyllus, Cymbopogon martinii var. motia, Cymbopogon nardus, Melaleuca linariifolia, Origanum majorana, Pimenta dioica, Thymus satureoides, Thymus serpyllum) showed a high antimicrobial activity showing a MIC ranging from 0.1% to 0.4%, while the remaining were less active showing a MIC ? 0.8%.     

Identification and epidemiological relationships of Aeromonas isolates from patients with diarrhea, drinking water and foods

A collection of Aeromonas isolates obtained over a three-year period in the same geographic area (León, NW of Spain) was characterized by (GTG)₅-PCR fingerprinting, amplified fragment length polymorphism (AFLP) analysis and gyrB gene sequence analysis. The isolates originated from human diarrheal stools (29 isolates), potable water (13 isolates), rabbit meat (13 isolates) and marine fish (5 isolates). The distribution of Aeromonas species varied with the strain source. Aeromonas caviae HG4 and Aeromonas media HG5 were predominant in clinical and water isolates, respectively, whereas motile Aeromonas salmonicida HG3 strains were most frequently found in fish and meat. Molecular typing revealed several genotypic relationships among specific isolate subsets: (i) two clones of A. media HG5 persisted in drinking water over the study period, (ii) different patients harbored identical or closely related clones during several months, and (iii) clonal relatedness was observed in two sets of water and human isolates. The first of these sets comprised nine water isolates and two human A. media HG5 isolates, whereas the other one included a water isolate and a human isolate of A. caviae HG4. The latter finding suggests that Aeromonas transmission in the studied region followed a waterborne route. Interestingly, the three human isolates closely related to water isolates were recovered in a period of four days in June 2006 from non-related patients without underlying medical conditions that tested negative for other enteric pathogens. The data imply the transmission through contaminated water of strains of the A. caviae group that can produce disease in humans.     

Intragastric administration of a superoxide dismutase-producing recombinant Lactobacillus casei BL23 strain attenuates DSS colitis in mice

Human immune cells release large amounts of reactive oxygen species (ROS) such as superoxide radical and hydrogen peroxide via respiratory burst. In inflammatory bowel diseases, a sustained and abnormal activation of the immune response results in oxidative stress of the digestive tract and in a loss of intestinal homeostasis. We previously reported that heterologous production of the Lactobacillus plantarum manganese catalase (MnKat) enhances the survival of Lb. casei BL23 when exposed to oxidative stress. Anti-inflammatory effects were observed after Lb. casei BL23 oral administrations in moderate murine dextran sodium sulfate (DSS)-induced colitis, without added effects of the MnKat production. Here, we evaluated the protective effects obtained by an improved antioxidative strategy. The Lactococcus lactis sodA gene was expressed in Lb. casei BL23 which acquired an efficient manganese superoxide dismutase (MnSOD) activity. The effects of Lb. casei MnSOD alone or in combination with Lb. casei MnKat were compared first in eukaryotic cell PMA-induced oxidative stress model and then in severe murine DSS-induced colitis. Based on ROS production assays as well as colonic histological scores, a significant reduction of both oxidative stress and inflammation was observed with Lb. casei MnSOD either alone or in combination with Lb. casei MnKat. No added effect of the presence of Lb. casei MnKat was observed. These results suggest that Lb. casei BL23 MnSOD could have anti-inflammatory effects on gut inflammation.     

Properties of xylanolytic enzyme system in bifidobacteria and their effects on the utilization of xylooligosaccharides

The xylanolytic enzyme system was examined in Bifidobacterium adolescentis, B. infantis and B. bifidum to determine their ability to utilize xylooligosaccharides (XOSs). All these species produced only xylosidase and arabinosidase, and no xylanase, α-glucuronidase and acetyl xylan esterase were found. The optimal activity of β-d-xylosidase from B. adolescentis was at pH 5.6 and 45 °C, and α-l-arabinoside was at pH 5.0 and 40 °C. The degradation products of cell-free extracts and the growth rate of B. adolescentis were tested over XOSs and XOSs de-branched by recombinant α-glucuronidase. The results showed that de-branching α-glucuronidase increased the production of xylose and the total cell density by 10%, and accelerated the growth of B. adolescentis by 20%.     

Molecular identification of Anisakis type I larvae isolated from hairtail fish off the coasts of Taiwan and Japan

Anisakid nematodes are known to cause the zoonotic disease, anisakiasis, through the consumption of raw or undercooked fish. The parasites most frequently associated with the disease in humans are categorized as Anisakis type I, which comprise several species of the genus Anisakis. The larvae show primitive forms and lack the detailed morphological characteristics required for precise species identification. Thus, molecular characterization is necessary for determining the species of Anisakis type I larvae and acquiring important clinical and epidemiological information. In this study, we isolated Anisakis type I larvae from hairtail fish caught off the coasts of Taiwan and Japan. The ribosomal DNA (rDNA) internal transcribed spacer (ITS) region was sequenced, and restriction fragment length polymorphism (RFLP) analyses using HinfI and HhaI was carried out for species identification. Most larvae isolated from hairtail caught in Taiwan were Anisakis typica (84%), while those isolated from hairtail caught in Japan were almost exclusively identified either as Anisakis simplex sensu stricto (65%) or Anisakis pegreffii (33%). This is the first report of A. typica in fish obtained from Taiwan. Our results shed the light on the epidemiology of Anisakis type I larvae, which is a potential cause of human anisakiasis in Taiwan and Japan.     

Selected plant essential oils and their main active components,a promising approach to inhibit aflatoxigenic fungi and aflatoxin production in food

Recent research has showed that Aspergillus flavus and Aspergillus parasiticus are aflatoxigenic species that can become very competitive in the framework of climate change. Aflatoxins show carcinogenic, mutagenic, immunotoxic and teratogenic effects on human and animals. Effective and sustainable measures to inhibit these species and aflatoxins in food are required. Origanum vulgare and Cinnamomum zeylanicum essential oils (EOs) and their major active constituents, carvacrol and cinnamaldehyde, respectively, were assayed for inhibiting these species and aflatoxin production in maize extract medium under different environmental conditions. Doses of 10–1000 mg l^?1 were assayed and the effective doses for 50 (ED₅₀) and 90% (ED₉₀) growth inhibition were determined. The ED₅₀ of cinnamaldehyde, carvacrol, oregano EO, and cinnamon EO against A. flavus were in the ranges 49–52.6, 98–145, 152–505, 295–560 mg l^?1 and against A. parasiticus in the ranges 46–55.5, 101–175, 260–425 and 490–675 mg l^?1, respectively, depending on environmental conditions. In A. flavus treatments ED₉₀ were in the ranges 89.7–90.5, 770–860 and 820–>1000 mg l^?1 for cinnamaldehyde, carvacrol and cinnamon EO, and in A. parasiticus treatments in the ranges 89–91, 855–>1000 and 900–>1000 mg l^?1, respectively. ED₉₀ values for oregano EO against both species were >1000 mg l^?1. Growth rates of both species were higher at 37 than at 25°C and at 0.99 than at 0.96 a_w. Aflatoxin production was higher at 25 than at 37°C. Stimulation of aflatoxin production was observed at low doses except for cinnamaldehyde treatments. The effectiveness of EOs and their main constituents to inhibit fungal growth and aflatoxin production in contact assays was lower than in vapour phase assays using bioactive EVOH-EO films previously reported.     

Identification and safety evaluation of Bacillus species occurring in high numbers during spontaneous fermentations to produce Gergoush, a traditional Sudanese bread snack

Gergoush is a naturally fermented Sudanese Bread snack produced in three fermentation steps (primary starter, adapted starter and final dough), followed by three baking steps for a half to one hour at above 200 °C. This study examines the microbiota of two sets of fermentations performed at a traditional production site in Khartoum, Sudan in 2006 and 2009, respectively. In 2006 four different milk/legume based primary starters (faba bean, chick pea, lentil and white bean) were sampled in order to enumerate and identify the Bacillus at species or group level. In 2009 specific focus was on the enumeration and safety evaluation of the dominant Bacillus cereus group species occurring during various Gergoush productions (including the three fermentations steps and after baking). In 2006, the primary starters contained Bacillus spp. in the order of between 7.7 and 8.1 log₁₀ CFU/g. Species identifications were performed by M13-PCR typing using the Escherichia coli phage M13 derived primer PM13 combined with internally transcribed 16-23S rRNA PCR, 16S rRNA gene and gyrA or gyrB gene sequencing, and selected phenotypic tests. Depending on the legume used, 40-68% of the isolates were identified as B. cereus sensu lato, 16-27% as Bacillus licheniformis, 8-32% as Bacillus subtilis and 4-20% as Bacillus sonorensis. During the second set of fermentation trials performed in 2009, the Bacillus spp. and B. cereus occurred in numbers of between 7.7-9.9 and 6.1-7.8 log₁₀ CFU/g, respectively, while no bacteria were detected after baking. A total of 180 B. cereus sensu lato isolates from four different primary starters, adapted starters and final doughs were further identified as B. cereus sensu stricto (118 isolates) and Bacillus thuringiensis (62 isolates). The safety of Gergoush was evaluated based on the counts and toxin gene profiles of the dominant B. cereus species. Considering that no bacteria survived the baking process, and that the cereulide synthetase gene cesB involved in the production of the heat stable emetic toxin cereulide was not detected in any of the investigated B. cereus isolates, the results indicate, that Gergoush produced at the traditional production site is safe for human consumption. This study is the first to identify the Bacillus of Gergoush to species level, and it is the first to perform a safety evaluation of the product, based on the dominant B. cereus species.     

Clitocybe alexandri extract induces cell cycle arrest and apoptosis in a lung cancer cell line: Identification of phenolic acids with cytotoxic potential

Mushrooms are a possible rich source of biologically active compounds with the potential for drug discovery. The aim of this work was to gain further insight into the cytotoxicity mechanism of action of Clitocybe alexandri ethanolic extract against a lung cancer cell line (NCI-H460 cells). The effects on cell cycle profile and levels of apoptosis were evaluated by flow cytometry, and the effect on the expression levels of proteins related to cellular apoptosis was also investigated by Western blot. The extract was characterised regarding its phenolic composition by HPLC-DAD, and the identified compounds were studied regarding their growth inhibitory activity, by sulforhodamine B (SRB) assay. The effect of individual or combined compounds on viable cell number was also evaluated using the Trypan blue exclusion assay. It was observed that the C. alexandri extract induced an S-phase cell cycle arrest and increased the percentage of apoptotic cells. In addition, treatment with the GI₅₀ concentration (concentration that was able to cause 50% of cell growth inhibition; 24.8 μg/ml) for 48 h caused an increase in the levels of wt. p53, cleaved caspase-3 and cleaved poly (ADP-ribose) polymerase (PARP). The main components identified in this extract were protocatechuic, p-hydroxybenzoic and cinnamic acids. Cinnamic acid was found to be the most potent compound regarding cell growth inhibition. Nevertheless, it was verified that the concomitant use of the individual compounds provided the strongest decrease in viable cell number. Overall, evidence was found for alterations in cell cycle and apoptosis, involving p53 and caspase-3. Furthermore, our data suggests that the phenolic acids identified in the extract are at least partially responsible for the cytotoxicity induced by this mushroom extract.     

Identification of two licorice species, Glycyrrhiza uralensis and Glycyrrhiza glabra, based on separation and identification of their bioactive components

The purpose of this study was to identify two licorice species, Glycyrrhiza uralensis and Glycyrrhiza glabra. A simple method including HPLC and LC/MS was developed to simultaneously separate, identify, and quantify 15 bioactive components of Glycyrrhiza species. G. uralensis was found to contain quercetin but G. glabra did not. G. glabra provided by Brion Research Institute of Taiwan contained uralsaponin B, but G. glabra brought from herbal shops did not. The DPPH radical scavenging activity of G. uralensis and G. glabra achieved approximately 72–75% if 10 mg/mL or more licorice extract was used. G. uralensis had slightly better radical scavenging activity than G. glabra. G. uralensis also showed higher reducing ability than G. glabra.