Evaluation of the anticancer activity of Origanum Marjoram as a safe natural drink for daily use.

BACKGROUND: Chemotherapeutic agents have numerous side effects. There is a major interest in using natural and safe plants as food or drink to prevent from cancer. Origanum marjoram (OMAE) is a medicinal plant that can be used as a tea, food, and additive in traditional medicine. OBJECTIVE: This study aimed to evaluate the potential anticancer effects of OMAE as a soft drink for daily use against a model cancer, prevention and treatment. METHOD: MCF-7 cells were chosen as model cancer cells. The MTT assay was used to assess the in vitro inhibitory effects of OMAE on cell growth. Moreover, quantitative real-time PCR (qRT-PCR) was used to detect specific genes associated with cancer, such as ESR1, Bax, Bcl-2, and p53. Furthermore, the DNA damage was evaluated using the comet assay. RESULTS: OMAE has IC50 of 53.1 and IC90 of 97.5 µg/ml dependent inhibition of cell proliferation after 48 h of treatment toward MCF-7. Also, a significant decrease in the expression level of the ESR1 gene in the MCF-7 cell line. Furthermore, there was a significant increase in the comet length and comet-positive cells after treatment with OMAE (88.7%) compared with those in the untreated control cells (9.5%), suggesting a high induction of DNA damage by OMAE. Also, OMAE showed a modification in bcl-2, tumor suppressor gene (p53), and Bax levels and influenced the BAX/BCL-2 ratio via releasing the cytochrome C. CONCLUSION: The results of the study were promising, suggesting that the reduced apoptotic rate of MCF-7 breast cancer cells in this work was correlated to the potential anticancer effect of OMAE which would be a suitable preventable drink against cancer. However, further studies are needed to fully understand the potential of OMAE as a cancer treatment.

Intraspecific divergence in essential oil content, composition and genes expression patterns of monoterpene synthesis in Origanum vulgare subsp. vulgare and subsp. gracile under salinity stress.

BACKGROUND: Oregano (Origanum vulgare L.), one of the important medicinal plants in the world, has valuable pharmacological compounds with antimicrobial, antiviral, antioxidant, anti-inflammatory, antispasmodic, antiurolithic, antiproliferative and neuroprotective activities. Phenolic monoterpenes such as thymol and carvacrol with many medical importance are found in Oregano essential oil. The biosynthesis of these compounds is carried out through the methyl erythritol-4 phosphate (MEP) pathway. Environmental stresses such as salinity might improve the secondary metabolites in medicinal plants. The influence of salinity stress (0 (control), 25, 50 and 100 mM NaCl) on the essential oil content, composition and expression of 1-deoxy-D-xylulose-5-phosphate reductoisomerase (DXR), γ-terpinene synthase (Ovtps2) and cytochrome P450 monooxygenases (CYP71D180) genes involved in thymol and carvacrol biosynthesis, was investigated in two oregano subspecies (vulgare and gracile). RESULTS: Essential oil content was increased at low NaCl concentration (25 mM) compared with non-stress conditions, whereas it was decreased as salinity stress intensified (50 and 100 mM). Essential oil content was significantly higher in subsp. gracile than subsp. vulgare. The highest (0.20 mL pot-1) and lowest (0.06 mL pot-1) amount of essential oil yield was obtained in subsp. gracile at 25 and 100 mM NaCl, respectively. The content of carvacrol, as the main component of essential oil, decreased with increasing salinity level in subsp. gracile, but increased in subsp. vulgare. The highest expression of DXR, Ovtps2 and CYP71D180 genes was observed at 50 mM NaCl in subsp. vulgare. While, in subsp. gracile, the expression of the mentioned genes decreased with increasing salinity levels. A positive correlation was obtained between the expression of DXR, Ovtps2 and CYP71D180 genes with carvacrol content in both subspecies. On the other hand, a negative correlation was found between the expression of CYP71D180 and carvacrol content in subsp. gracile. CONCLUSIONS: The findings of this study demonstrated that both oregano subspecies can tolerate NaCl salinity up to 50 mM without significant reduction in essential oil yield. Also, moderate salinity stress (50 mM NaCl) in subsp. vulgare might increase the carvacrol content partly via increment the expression levels of DXR, Ovtps2 and CYP71D180 genes.

The comparison of the effect of Origanum vulgare L. extract and vitamin C on the gentamycin-induced nephrotoxicity in rats.

The gentamicin renal toxicity has been the focal point for much discussion. The objective of this study was to investigate the impacts of Origanum vulgare L. extract and vitamin C on gentamicin dose-dependent toxicity in rats' kidney. The present study was conducted on 60 male Wistar rats divided into ten experimental groups: control (untreated), G1, G2, G3 (100, 200, 300 mg/kg gentamicin), M1, M2 and M3 (500 mg/kg marjoram extract) + 100, 200 and 300 gentamicin, V1, V2 and V3 (Vitamin C 500 mg/kg) + 100, 200 and 300 of gentamicin. On the last day, the serum was separated from heart blood and the kidney tissues were extracted to measure the biochemical and oxidative stress parameters and histological changes. Kidney damage was confirmed as dose-dependent gentamicin by biochemical and pathological parameters. Urea, Blood Urea Nitrogen (BUN), and creatinine showed a significant increase in the G3 group compared to the control, M1, and V1 groups (p < 0.01). Catalase (CAT), Glutathione peroxidase (GPx), and Superoxide dismutase (SOD) showed a significant reduction in renal tissue in the G3 group compared to the other groups (p < 0.001). Malondialdehyde (MDA) concentration in the kidney tissue of the G3 group also showed a significant increase compared to other groups (p < 0.001). Furthermore, TNFα and IL-1 levels were the highest in the G3 group, and serum total antioxidant capacity (TAC) concentration had the lowest amount compared to other groups. Moreover, histopathological lesions of the kidney showed significant statistical differences among the groups that received gentamicin with the control and M1 group. Marjoram extract at the dose of 500 mg/kg had a desirable effect on controlling gentamicin damage in the kidneys compared with vitamin C. In particular, controlling gentamicin-induced oxidative stress and inflammation by the consumption of marjoram extract and vitamin C plays an important role in protecting the kidneys.

Natural Antibiotic Oregano in Hydroxyapatite-Coated Titanium Reduces Osteoclastic Bone Resorption for Orthopedic and Dental Applications.

Traditional infection prevention and treatment methods include synthetic antibiotics, which can cause severe adverse side effects. Carvacrol and thymol are biologically active monoterpenoid extractants from oregano leaves with antibiotic capabilities; however, little is known regarding their effects on bone tissue engineering. The objective of this work is to understand their effects on osteogenesis, specifically with osteoblast and osteoclast cells, from surface-modified Ti6Al4V with plasma sprayed hydroxyapatite (HA) coatings. This system is an alternative to cemented implants to aid in bone healing. Results reveal that full carvacrol release from the HA matrix is successful in aqueous environments and modulation of release kinetics can also be made using polycaprolactone (PCL) and polyethylene glycol (PEG) polymers. From HA-pressed disc samples in physiological pH, full carvacrol release is achieved in 10 days using PCL/PEG, about 95% release in 50 days using no polymer, and 60% in 50 days when using a PCL coating. Without polymer, full carvacrol release is achieved after 3 days from HA coatings in both physiological pH and acidic pH, mimicking the post-surgery environment. The release is assessed as a diffusion-based mechanism in phosphate-buffered saline but degradation-based mechanism in acetate buffer solution. Carvacrol and thymol show bacterial inhibition of Staphylococcus epidermidis and no cytotoxic effects on osteoblast proliferation in vitro. Carvacrol and thymol also induce a significant 7% reduction in osteoclast tartrate-resistant acid phosphatase (TRAP) activity, caused by poorly attached cellular morphologies, leading to an approximately 65% reduction in osteoclast resorption pit formation. Our goal is to demonstrate a natural medicinal system that can support bone healing while providing infection prevention and reducing costly revision surgeries for orthopedic and dental applications.

Potential synergistic antimicrobial efficiency of binary combinations of essential oils against Bacillus cereus and Paenibacillus amylolyticus-Part A.

The checkerboard method was used to study the potential interactions between eight essential oils (Basil, Cinnamon, Eucalyptus, Mandarin, Oregano, Peppermint, Tea tree, and Thyme) when used as antibacterial agents against Bacillus cereus LSPQ 2872 and Paenibacillus amylolyticus ATCC 9995. The minimum inhibitory concentration (MIC) of each essential oil (EO) and the fractional inhibitory concentration (FIC) index for the binary combinations of essential oils (EOs) were determined. According to FIC index values, some of the compound binary combinations showed an additive effect; however, Thyme/Tea tree and Cinnamon/Thyme EOs exhibited a synergistic effect against P. amylolyticus and B. cereus, respectively. Cinnamon/Thyme EOs mixture exhibited no interactive effect against P. amylolyticus, but a synergistic effect against B. cereus. The combination of Oregano/Thyme EOs displayed the best antibacterial activity and showed a synergistic effect against B. cereus and P. amylolyticus bacteria. The Oregano/Thyme EOs mixture has potential application in food preservation to reduce the contamination of B. cereus and P. amylolyticus.

In vitro bioaccessibility and activity of Greek oregano (Origanum vulgare L. ssp. hirtum (link) Ietswaart) compounds as affected by nitrogen fertilization.

BACKGROUND: Greek oregano is a culinary and medicinal herb native to the Mediterranean region; however nowadays it is cultivated in many regions all over the world. It is commonly used as a spice for flavoring food products and in various traditional medicine applications. This study investigated the effect of nitrogen fertilization at 0-150 kg N ha-1 on the potential bioaccessibility and activity of Greek oregano compounds. For this purpose the total phenolic content, rosmarinic acid content, and antioxidant activities of the raw material, as well as digested fractions, were determined. RESULTS: Nitrogen fertilization had a negative influence on the phenolic content and antioxidant activity of raw material; however, its effect on the potential bioaccessibility varied depending on the dose. The highest potential bioaccessibility and activity was determined for plants fertilized with 30 kg N ha-1 . For the gastric and intestinal phases of digestion, the potential bioaccessibility percentages were 39.5% and 29.6% for total phenolics, 53.1% and 11.2% for rosmarinic acid content, 45.2% and 44.4% for antiradical activity against 2,2'-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid (ABTS), 39.2% and 27.2% for antiradical activity against 2,2-diphenyl-1-picrylhydrazyl (DPPH) and 50.2% and 23.4% for reducing power, respectively. CONCLUSION: The results showed that nitrogen fertilization is an important factor determining the in vitro bioaccessibility of Greek oregano compounds. Furthermore, the nutraceutical potential of herbs, in term of the bioaccessibility of bioactive compounds, may be optimized during plant cultivation by applying an accurate nitrogen level. © 2020 Society of Chemical Industry.

Phenolic compounds, antioxidant capacity and antimutagenic activity in different growth stages of in vitro raised plants of Origanum vulgare L.

Efficient micropropagation procedure was developed for Origanum vulgare, a high-value culinary herb, and the phytochemicals, phenolic content, antioxidant and antimutagenic activity of leaf and stem, derived from different growing stages were analyzed. The agar solidified Murashige and Skoog (MS) medium supplemented with a combination of 6-benzylaminopurine and α-naphthaleneacetic acid was optimized as best shoot-multiplication-medium. Shoots were rooted best on 1/2 strength MS medium supplemented with 50 µM indole-3-butyric acid (IBA). The plantlets were successfully acclimatized ex vitro in a soil, sand and farmyard manure mixture (2:1:1 v/v/v) with 100% survival rate in greenhouse. The total anthocyanin and total phenolic content were observed significantly higher in leaves of in vitro-raised plants. However, total tannin, flavonoid and antioxidant activity remained higher in leaves of mother plant maintained under ployhouse condition. All the plant extracts have shown significant antimutagenic activity except in vitro-growing plants. A total of 13 polyphenolic compounds were detected in different extracts using high performance liquid chromatography. Among these, catechin was detected maximum in in vitro-growing cultures and chlorogenic acid in leaves of mother plant. These findings will help the farmers, medicinal plant growers, and industries for mass multiplication and effective extraction of phytochemicals from O. vulgare.

Phytochemical Profiles and Antibacterial Activities of Hydroalcoholic Extracts of Origanum vulgare and Hypericum perforatum and Carvacrol and Hypericin as a Promising Anti-Staphylococcus aureus.

OBJECTIVES: Staphylococcus aureus, a Gram-positive bacteria, is ranked second among the causes of hospital infections and is one of the three main causes of food poisoning. In recent times, the spread of antibiotic resistance in S. aureus has become very worrisome. Therefore, research for new effective drugs is important. The present study aims to investigate the phytochemical profiles and antibacterial effects of hydroalcoholic extracts of Origanum vulgare (Lamiaceae family) and Hypericum perforatum (Clusiaceae family) and their active compounds on S. aureus (ATCC 12600) in vitro. METHODS: The identification of phytochemical compounds in both plants was performed by Highperformance liquid chromatography (HPLC), headspace-solid-phase microextraction (HS-SPME) and Fourier-transform infrared spectroscopy (FTIR). To investigate microbial susceptibility, minimum inhibitory concentration (MIC), minimum bactericidal concentration (MBC) and disc diffusion method (DAD) were used. Finally, the results of the study were compared with methicillin. RESULTS: Of the 42 combinations of O. vulgare, carvacrol (48%) and of the 38 combinations of H. perforatum, hypericin (46.2%) were the most abundant. The MIC, MBC and DAD of O. vulgare and H. perforatum, carvacrol, hypericin and methicillin were 625, 625, 312.5, 78.12 and 384 µg/mL, 10000, 10000, 2500, 2500 and 384 µg/mL, and 15.66 ± 4.49, 12.66 ± 0.47 and 22 ± 0.81 mm, respectively. CONCLUSION: Due to the significant effects of O. vulgare and H. perforatum and their active components against S. aureus, it is expected that in the future, hypericin, carvacrol and their derivatives can be used as effective antibacterial agents against S. aureus.

Origanum vulgare essential oils inhibit glutamate and aspartate metabolism altering the photorespiratory pathway in Arabidopsis thaliana seedlings.

Essential oils (EOs) have been extensively studied as valuable eco-friendly compounds with herbicidal activity for weed management. Phytotoxic potential of EOs, extracted from a wild population of Origanum vulgare ssp. hirtum (Link) Ietswaart, has been here evaluated on plant model Arabidopsis, through a physiological and metabolomic approach. The EOs composition was mainly characterized by monoterpenes and sesquiterpenes, with a strong abundance of two monoterpenic phenols, namely carvacrol and thymol, and the monoterpene o-cymene. The in vitro bioassay confirmed a strong phytotoxic effect of EOs on Arabidopsis rosettes, showing by both a strong growth reduction and highly chlorotic leaves. In well-developed seedlings, EOs firstly caused growth reduction and leaf chlorosis, together with a series of interconnected metabolic alterations: i) impairing the nitrogen assimilation into amino acids, which affects in particular the glutamine metabolism; and as consequence ii) excessive accumulation of toxic ammonia into the leaves, associated with oxidative stress and damage; iii) declining the efficiency of the photosynthetic apparatus, connected to the reduced CO2 fixation and photooxidation protection; iv) impairing the photorespiratory pathway. Overall, the results highlights that EOs alters principally the ability of Arabidopsis seedlings to incorporate inorganic nitrogen into amino acids, principally glutamine, leading to a dramatic accumulation of ammonia in leaf cells. This primary effect induces, in turn, a cascade of reactions that limits the efficiency of PSII, inducing oxidative stress and finally causing a strong plant growth reduction, leaf necrosis and eventually plant death. These findings suggest that O. vulgare EOs might be proficiently exploited as a potential bioherbicide in an ecofriendly agriculture. Moreover, its multitarget activity could be advantageous in limiting weed resistance phenomenon.

Performance and methane emissions in dairy cows fed oregano and green tea extracts as feed additives.

Plant extracts have been proposed as substitutes for chemical feed additives due to their potential as rumen fermentation modifiers and because of their antimicrobial and antioxidant activities, possibly reducing methane emissions. This study aimed to evaluate the use of oregano (OR), green tea extracts (GT), and their association as feed additives on the performance and methane emissions from dairy between 28 and 87 d of lactation. Thirty-two lactating dairy cows, blocked into 2 genetic groups: 16 Holstein cows and 16 crossbred Holstein-Gir, with 522.6 ± 58.3 kg of body weight, 57.2 ± 20.9 d in lactation, producing 27.5 ± 5.0 kg/cow of milk and with 3.1 ± 1.8 lactations were evaluated (means ± standard error of the means). Cows were allocated into 4 treatments: control (CON), without plant extracts in the diet; oregano extract (OR), with the addition of 0.056% of oregano extract in the dry matter (DM) of the diet; green tea (GT), with the addition of 0.028% of green tea extract in the DM of the diet; and mixture, with the addition of 0.056% oregano extract and 0.028% green tea extract in the DM of the diet. The forage-to-concentrate ratio was 60:40. Forage was composed of corn silage (94%) and Tifton hay (6%); concentrate was based on ground corn and soybean meal. Plant extracts were supplied as powder, which was previously added and homogenized into 1 kg of concentrate in natural matter, top-dressed onto the total mixed diet. No treatment by day interaction was observed for any of the evaluated variables, but some block by treatment interactions were significant. In Holstein cows, the mixture treatment decreased gross energy and tended to decrease the total-tract apparent digestibility coefficient for crude protein and total digestible nutrients when compared with OR. During the gas measurement period, GT and OR increased the digestible fraction of the ingested DM and decreased CH4 expressed in grams per kilogram of digestible DMI compared with CON. The use of extracts did not change rumen pH, total volatile fatty acid concentration, milk yield, or most milk traits. Compared with CON, oregano addition decreased fat concentration in milk. The use of plant extracts altered some milk fatty acids but did not change milk fatty acids grouped according to chain length (short or long), saturation (unsaturated or saturated), total conjugated linoleic acids, and n-3 and n-6 contents. Green tea and oregano fed separately reduced gas emission in cows during the first third of lactation and have potential to be used as feed additives for dairy cows.

Effect of In Vitro Digestion on the Total Antioxidant Capacity and Phenolic Content of 3 Species of Oregano (Hedeoma patens, Lippia graveolens, Lippia palmeri).

Oregano phenolic compounds have been studied for their anti-inflammatory properties. Nonetheless, after ingestion, the gastrointestinal environment can affect their antioxidant stability and thus their bioactive properties. To evaluate the effect of in vitro gastrointestinal (GI) digestion on the phenolic compounds of 3 species of oregano (Hedeoma patens, Lippia graveolens, and Lippia palmeri), the total reducing capacity, total flavonoid content, and antioxidant capacity were evaluated before and after in vitro GI digestion. In addition, the phenolic compounds of the 3 oregano species were identified and quantified by UPLC-PDA before and after in vitro GI digestion. It was shown that the reducing capacity, flavonoid content and antioxidant capacity were affected by the GI digestion process. Moreover, the phenolic compounds identified were apigenin-7-glucoside, scutellarein, luteolin, luteolin-7-glucoside, phloridzin and chlorogenic acid, and their levels were affected by the in vitro GI process. Our results showed that the phenolic compounds from these 3 species of oregano are affected by the in vitro digestion process, and this effect is largely attributable to pH changes. These changes can modify the bioavailability and further anti-inflammatory activity of oregano phenolics, and thus, further research is needed. PRACTICAL APPLICATION: Oregano is a rich source of polyphenols that have shown bioactive properties like anti-inflammatory potential. However, little is known of the gastrointestinal fate of oregano polyphenols which is imperative to fully understand its bioaccessibility. Our results are important to develop new administration strategies which could help protect the antioxidant and anti-inflammatory potential and bioaccessibility of such compounds.

Possible Molecular Mechanisms by Which an Essential Oil Blend from Star Anise, Rosemary, Thyme, and Oregano and Saponins Increase the Performance and Ileal Protein Digestibility of Growing Broilers.

Phytogenic feed additives represent a potential alternative to antibiotics with attributed health and growth-promoting effects. Chickens supplemented with an essential oil blend, a Quillaja saponin blend, or a combination of both phytogenic preparations showed a comprehensively and significantly improved apparent ileal digestibility of crude protein and amino acids compared to control birds. Accordingly, holistic transcriptomic analyses of jejunum and liver samples indicated alterations of macromolecule transporters and processing pathways likely culminating in an increased uptake and metabolizing of carbohydrates and fatty acids. Complementary analyses in Caco-2 showed a significant increase in transporter recruitment to the membrane (SGLT1 and PEPT1) after addition of essential oils and saponins. Although the penetrance of effects differed for the used phytogenic feed additives, the results indicate for an overlapping mode of action including local effects at the intestinal border and systemic alterations of macronutrient metabolism resulting in an improved performance of broilers.

Changes in thermo-tolerance and survival under simulated gastrointestinal conditions of Salmonella Enteritidis PT4 and Salmonella Typhimurium PT4 in chicken breast meat after exposure to sequential stresses.

This study assessed changes in thermo-tolerance and capability to survive to simulated gastrointestinal conditions of Salmonella Enteritidis PT4 and Salmonella Typhimurium PT4 inoculated in chicken breast meat following exposure to stresses (cold, acid and osmotic) commonly imposed during food processing. The effects of the stress imposed by exposure to oregano (Origanum vulgare L.) essential oil (OVEO) on thermo-tolerance were also assessed. After exposure to cold stress (5°C for 5h) in chicken breast meat the test strains were sequentially exposed to the different stressing substances (lactic acid, NaCl or OVEO) at sub-lethal amounts, which were defined considering previously determined minimum inhibitory concentrations, and finally to thermal treatment (55°C for 30min). Resistant cells from distinct sequential treatments were exposed to simulated gastrointestinal conditions. The exposure to cold stress did not result in increased tolerance to acid stress (lactic acid: 5 and 2.5µL/g) for both strains. Cells of S. Typhimurium PT4 and S. Enteritidis PT4 previously exposed to acid stress showed higher (p<0.05) tolerance to osmotic stress (NaCl: 75 or 37.5mg/g) compared to non-acid-exposed cells. Exposure to osmotic stress without previous exposure to acid stress caused a salt-concentration dependent decrease in counts for both strains. Exposure to OVEO (1.25 and 0.62µL/g) decreased the acid and osmotic tolerance of both S. Enteritidis PT4 and S. Typhimurium PT4. Sequential exposure to acid and osmotic stress conditions after cold exposure increased (p<0.05) the thermo-tolerance in both strains. The cells that survived the sequential stress exposure (resistant) showed higher tolerance (p<0.05) to acidic conditions during continuous exposure (182min) to simulated gastrointestinal conditions. Resistant cells of S. Enteritidis PT4 and S. Typhimurium PT4 showed higher survival rates (p<0.05) than control cells at the end of the in vitro digestion. These results show that sequential exposure to multiple sub-lethal stresses may increase the thermo-tolerance and enhance the survival under gastrointestinal conditions of S. Enteritidis PT4 and S. Typhimurium PT4.

Antioxidant, Antibacterial, and Cytotoxic Activities of the Ethanolic Origanum vulgare Extract and Its Major Constituents.

Oregano is a perennial shrub that grows in the mountains of the Mediterranean and Euro/Irano-Siberian regions. This study was conducted to identify the major constituents of the ethanolic Origanum vulgare extract and examine the cytotoxic, antioxidant, and antibacterial properties of the extract but more importantly the contribution of its specific major constituent(s) or their combination to the overall extract biological activity. Gas chromatography/mass spectroscopy analysis showed that the extract contained monoterpene hydrocarbons and phenolic compounds, the major ones being carvacrol and thymol and to a lesser extent p-cymene, 1-octacosanol, creosol, and phytol. A549 epithelial cells challenged with the extract showed a concentration-dependent increase in cytotoxicity. A combination of thymol and carvacrol at equimolar concentrations to those present in the extract was less cytotoxic. The A549 cells pretreated with nonlethal extract concentrations protected against hydrogen-peroxide-induced cytotoxicity, an antioxidant effect more effective than the combination of equimolar concentrations of thymol/carvacrol. Inclusion of p-cymene and/or 1-octacosanol did not alter the synergistic antioxidant effects of the carvacrol/thymol mixture. The extract also exhibited antimicrobial properties against Gram-positive and Gram-negative bacterial strains including clinical isolates. In conclusion, the oregano extract has cytotoxic, antioxidant, and antibacterial activities mostly attributed to carvacrol and thymol.

Development of an experimental apparatus and protocol for determining antimicrobial activities of gaseous plant essential oils.

There is a growing interest in the use of naturally-occurring antimicrobial agents such as plant essential oils (EOs) to inhibit the growth of hazardous and spoilage microorganisms in foods. Gaseous EOs (EO gases) have many potential applications in the food industry, including use as antimicrobial agents in food packaging materials and sanitizing agents for foods and food-contact surfaces, and in food processing environments. Despite the potentially beneficial applications of EO gases, there is no standard method to evaluate their antimicrobial activities. Thus, the present study was aimed at developing an experimental apparatus and protocol to determine the minimal inhibitory concentration (MIC) and minimal lethal concentration (MLC) of EO gases against microorganisms. A sealed experimental apparatus was constructed for simultaneous evaluation of antimicrobial activities of EO gases at different concentrations without creating concentration gradients. A differential medium was then evaluated in which a color change allowed for the determination of growth of glucose-fermenting microorganisms. Lastly, an experimental protocol for the assessment of MIC and MLC values of EO gases was developed, and these values were determined for 31 EO gases against Escherichia coli O157:H7 as a model bacterium. Results showed that cinnamon bark EO gas had the lowest MIC (0.0391 µl/ml), followed by thyme-thymol EO gas (0.0781 µl/ml), oregano EO gas (0.3125 µl/ml), peppermint EO gas (0.6250 µl/ml), and thyme-linalool EO gas (0.6250 µl/ml). The order of the MLC values of the EO gases against the E. coli O157:H7 was thyme-thymol (0.0781 µl/ml)

LC-MS-based metabolite profiling of methanolic extracts from the medicinal and aromatic species Mentha pulegium and Origanum majorana.

INTRODUCTION: There has been increasing interest dedicated to the phenolic compounds with a view to their antioxidant and healthy properties. Recent studies have focused on plants from the Lamiaceae family with special interest in phenolic compounds antioxidant potential. OBJECTIVE: The metabolite profile of methanolic extracts from two Lamiacea medicinal plants was investigated. MATERIALS AND METHODS: Mentha pulegium and Origanum majorana methanolic extracts were analysed using reversed-phase ultra-high-performance liquid chromatography (RP-UHPLC) coupled to electrospray ionisation quadrupole time-of-flight mass spectrometry (ESI-QTOF-MS) detection in the negative ion mode. RESULTS: A total of 85 metabolites were characterised from different families, such as organic acids and derivatives, amino acids and derivatives, nucleosides, phenolic compounds as well as other polar metabolites, by using the MS and MS/MS information provided by the QTOF-MS. However, the total phenols and flavonoids were also quantified spectrophotometrically and they registered higher amounts in Mentha pulegium than in Origanum majorana extract. Gallocatechin was the major compound in M. pulegium extract whereas quercetin dimethyl ether, jaceidin and dihydrokaempferide were the major ones in O. majorana extract. CONCLUSION: The distribution of phenolic compounds in the methanolic extract showed a variation among studied plants. Mentha pulegium can be considered as a source of gallocatechin.

Effect of the icing with thyme, oregano and clove extracts on quality parameters of gutted and beheaded anchovy (Engraulis encrasicholus) during chilled storage.

The effect of ice containing thyme (0.04% w/v), oregano (0.03% w/v) and clove (0.02% w/v) extracts on the quality parameters of anchovy (Engraulis encrasicholus) was assessed through the chemical, sensory and microbiological methods. According to sensory analyses, anchovy stored in ice prepared with each plant extracts had a shelf life of 12 days, while batch stored in traditional ice had a shelf life of 9 days. pH were not significantly affected by the presence of plant extracts in the ice. However, a marked antioxidant effect (P<0.05) could be detected with fish kept under plant extract icing systems, according to peroxide (PV) and thiobarbituric acid (TBA) values. Moreover, the employment of such icing systems led to lower (P<0.05) total volatile base-nitrogen (TVB-N) value and free fatty acid (FFA) formation. Thus, TVB-N proved to be a suitable index for the spoilage of anchovies during chilled storage. The employment of such icing systems also led to significantly lower counts of aerobic mesophiles and psychrotrophic bacteria in anchovy muscle with skin, as compared with the traditional ice batch. This work demonstrates that the use of ice with plant extracts for the storage of gutted and beheaded anchovy can be recommended to improve the quality and extend the shelf life.

Traffic-related trace element fate and uptake by plants cultivated in roadside soils in Toronto, Canada.

This research examines traffic-related trace element emissions and their uptake by plants grown in urban roadside environments in Toronto, Canada. Oregano (Origanum vulgare), beets (Beta vulgaris) and eggplants (Solanum melongena) were cultivated at four locations with variable traffic-related metal inputs in 2010. The top 30 cm of soil at the medium-traffic location was first replaced with a triple mix soil (topsoil, compost and peat) to control for pre-existing contamination. The same soil was used to cultivate at the two no/low traffic locations. Soil at the heavy traffic location was not remediated. Soil, plant tissue and plant rhizosphere samples were collected for the analysis of a range of traffic-related metal(loids) using ICP-MS, including Cr, Mn, Cu, Ni, Cd, As, Sb and Pb. Samples were digested with HNO(3) and HCl using a microwave-assisted digestion procedure and then treated with HF prior to analysis. Two certified reference materials, San Joaquin soil (NIST 2709a) and trace elements in spinach leaves (NIST 1570a), were used for QA/QC purposes. Metal(loid) concentrations and accumulation over time were highly variable at the medium traffic site where the soil was replaced. Mn (p<0.10), As (p<0.10) and Sb (p<0.01) concentrations significantly increased in bulk soils from May to November 2010, while Ce (p<0.01) and Cd (p<0.10) levels decreased. For instance, median As concentrations increased from 4.39 to 8.40 mg/kg over this period. Metals were found to be more bioaccessible to O. vulgare grown in the new soil at the medium traffic volume site, compared to the aged soil at the heavy traffic location. Several elements, most notably Cd, were also found to accumulate in the root zone of sampled S. melongena. Metal concentrations in S. melongena rhizosphere were better predictors of plant tissue levels, providing evidence that soil quality guidelines based on total metal concentrations for bulk soils are inadequate.

Are leaf glandular trichomes of oregano hospitable habitats for bacterial growth?

Phyllospheric bacteria were isolated from microsites around essential-oil-containing glands of two oregano (Origanum vulgare subsp. hirtum) lines. These bacteria, 20 isolates in total, were subjected to bioassays to examine their growth potential in the presence of essential oils at different concentrations. Although there were qualitative and quantitative differences in the essential oil composition between the two oregano lines, no differences were recorded in their antibacterial activity. In disk diffusion bioassays, four of the isolated strains could grow almost unrestrained in the presence of oregano oil, another five proved very sensitive, and the remaining 11 showed intermediate sensitivity. The strain least inhibited by oregano essential oil was further identified by complete16s rRNA gene sequencing as Pseudomonas putida. It was capable of forming biofilms even in the presence of oregano oil at high concentrations. Resistance of P. putida to oregano oil was further elaborated by microwell dilution bioassays, and its topology on oregano leaves was studied by electron microscopy. When inoculated on intact oregano plants, P. putida was able not only to colonize sites adjacent to essential oil-containing glands, but even to grow intracellularly. This is the first time that such prolific bacterial growth inside the glands has been visually observed. Results of this study further revealed that several bacteria can be established on oregano leaves, suggesting that these bacteria have attributes that allow them to tolerate or benefit from oregano secondary metabolites.

Chitosan oligosaccharides promote the content of polyphenols in Greek oregano (Origanum vulgare ssp. hirtum).

Greek oregano is commonly used as a spice and in traditional medicine in Eurasia. The plant is rich in secondary metabolites, such as volatile organic compounds (VOC) and polyphenols. Chitosan oligosaccharides (COS) are used as a plant elicitor. The objectives of this study were to determine the effects of COS on the growth and content of secondary metabolites in Greek oregano. Four COS treatments (50, 200, 500, and 1000 ppm) were used in a field experiment. The 200 and 500 ppm COS treatments promoted plant height growth, whereas 50 and 200 ppm COS upregulated the content of polyphenols significantly (38 and 29%, respectively). The COS treatments induced H(2)O(2) generation in Greek oregano leaves; thus, the effect of H(2)O(2) treatment was studied to investigate the possible role of H(2)O(2) in growth and polyphenol production. A low concentration of H(2)O(2) also promoted plant height growth, but only tendencies to higher polyphenol content were seen.