Physiological and molecular mechanisms underlying salicylic acid-mitigated mercury toxicity in lemon balm (Melissa officinalis L.).

Mercury (Hg) is one of the most toxic heavy metals with strong negative effects on the plant growth and functions. Salicylic acid (SA) is an important signaling molecule which confers tolerance to metal toxicities but little is known about the mechanisms of SA-mediated alleviation of Hg stress. Here, physiochemical and molecular responses of Hg-stressed lemon balm (Melissa officinalis L.) to exogenous SA were investigated to reveal SA-induced tolerance mechanisms. The CHLG gene of lemon balm which encodes chlorophyll synthase was also partly isolated and sequenced for the first time. Hg stress markedly decreased growth, relative water content (RWC) and photosynthetic pigments of the plant. However, exogenous SA significantly mitigated the toxic effects of mercury on the growth and RWC and enabled plant to maintain chlorophylls to the similar levels of unstressed plants. Hg-induced oxidative damage was also reduced following treatment with SA and treated plants showed the lower extent of lipid peroxidation which was accompanied with the higher free proline and phenolics contents and elevation of the antioxidant capacity as evidenced by DPPH radical scavenging and FRAP assays. Moreover, SA treatment resulted in up-regulation of CHLG and phenylalanine ammonia-lyase (PAL) genes as key components of chlorophyll and phenylpropanoid routes, respectively. Our results collectively indicate the ameliorative effects of exogenous SA in mercury toxicity through coordinated alternations in plant metabolic processes which provide insights to better understand mechanisms of Hg tolerance in lemon balm plant.

Modulation of secondary metabolite profiles by biologically synthesized MgO/perlite nanocomposites in Melissa officinalis plant organ cultures.

In this work, biological synthesis of MgO/perlite nanocomposites (NCs) besides their effects on morphology and secondary metabolite profiles of Melissa officinalis plant organ cultures were evaluated. MgO NPs were immobilized on the surface of nanoperlite using M. officinalis extract as a capping agent. The as-synthesized MgO/perlite NCs were characterized by using FTIR, XRD, SEM, EDS and DLS. The average particle size of nanoperlite and MgO/perlite NCs was about 10 and 30 nm, respectively. Morphological observations showed that nanoperlite and MgO/perlite NCs had no effect on root number, as well as root and shoot length. None of the applied concentrations of perlite and MgO/perlite NCs could significantly increase the growth parameters in comparison to the control, except for 150 mg/L of nanoperlite which caused an increase in the shoot number. Although, the contents of chlorophyll and carotenoids were not affected, the maximum content of volatile compounds obtained at 100 of MgO/perlite NCs. Rosmarinic acid was detected in shoots, which treated with 25-100 mg/L of perlite and 25, 50 and 150 mg/L of MgO/perlite NCs. Our results provided the evidence that nanoperlite and MgO/perlite NCs at specific levels may act as a novel elicitor for in vitro biosynthesis of valuable secondary metabolites.

Melissa officinalis L: A Review Study With an Antioxidant Prospective.

Melissa officinalis is a plant cultivated in some parts of Iran. The leaves of lemon balm, Melissa officinalis L (Lamiaceae), are used in Iranian folk medicine for their digestive, carminative, antispasmodic, sedative, analgesic, tonic, and diuretic properties, as well as for functional gastrointestinal disorders. This review article was aimed not only to introduce Melissa officinalis (its growth condition, its chemical compounds, and its traditional usages) but also to overview its antioxidant properties in detail. This review was carried out by searching studies in PubMed, Medline, Web of Science, and IranMedex databases up to 2016. The search terms used were " Melissa officinalis L," "antioxidant properties," oxidative stress," "oxidative damage", "ROS." Articles whose full texts were not available were excluded from the study. In this study, firstly, traditional usage of this herb was reviewed, including antimicrobial activity (antiparasitic, antibacterial, antiviral, etc), antispasmodic, and insomnia properties. Then, its antioxidant properties were overviewed. Various studies have shown that Melissa officinalis L possesses high amount of antioxidant activity through its chemical compounds including high amount of flavonoids, rosmaric acid, gallic acid, phenolic contents. Many studies confirmed the antioxidative effects of Melissa officinalis; thus, its effect in preventing and treating oxidative stress-related diseases might be reliable.

A Comparative Study on Essential Oil Intraspecific and Seasonal Variations: Melissa romana Mill. and Melissa officinalis L. from Sardinia.

Two species of Melissa are currently present in Sardinia: Melissa officinalis L. and Melissa romana Mill. Our research can only count on a few supported evidences (as reported in Flora Italiana and Moris) and some notes on new stations in Sardinia that give us some information about morphology, distribution, bioecological, and ethnobotanical characteristics of both species. In this paper, we present the results of the research about morphological aspects of M. romana vs. M. officinalis, and their essential oils in different stations at different phenological periods. Moreover, we compared the essential oil of M. romana with the one obtained from M. officinalis growing in the few naturalized stations still present in Sardinia. The most evident morphological differences between the two entities are the long-stalked capitate glandular trichomes, shorts and inclined capitate trichomes, and peltate hairs. The chemical composition of essential oil presents several significant differences between the species. In fact, oils show that in none of the phenological stages, M. romana recalls in its composition M. officinalis. Major distinctions are also evident between dry and fresh plants, and among essential oils distilled in different seasons.

Effect of Arbuscular Mycorrhizal Fungi on the Growth and Polyphenol Profile of Marjoram, Lemon Balm, and Marigold.

The aim of this study is to examine the effect of arbuscular mycorrhizal fungi (AMF) colonization on biomass, polyphenol profile, and content of economically important herbs. A pot experiment was performed with marjoram, lemon balm, and marigold applying a commercially available AMF mixture for inoculation. Major polyphenols were identified using HPLC-UV-ESI-qTOFMS on the basis of their UV-vis and mass spectral characteristics, and selected ones were quantified. We showed that AMF can provide different services for each herb. Marjoram had the highest level of fungal colonization (82 M%) followed by lemon balm (62 M%) and marigold (17 M%). AMF inoculation significantly increased the biomass of marjoram (1.5-fold), the number of marigold flowers (1.2-fold), and the yield of rosmarinic acid and lithospermic acid isomers of marjoram (1.5-fold) and lemon balm (1.2-fold). Therefore, the quantity and quality of plant material could be improved by the application of optimized AMF inoculum.

Effect of water supply on growth and polyphenols of lemon balm (Melissa officinalis L.) and thyme (Thymus vulgaris L.).

A pot experiment was carried out with lemon balm (Melissa officinalis L.) and thyme (Thymus vulgaris L.). Different water supply was applied: 25%, 40% and 70% saturation of soil water capacity (SWC). Morphological traits, biomass and phenolic type active ingredients were investigated. Among the two species, main differences were registered in biomass and TPC. Lower SWC resulted in reduced biomass production of lemon balm, while the applied stress treatments did not effect the biomass of thyme. In lemon balm, highest TPC contents were measured in control plants both in shoots and roots but in thyme, the shoots showed a significantly increased TPC at the 25% SWC conditions. Neither the content of total flavonoids nor that of the rosmarinic acid was affected by the treatments. The antioxidant capacity proved to be in tight connection with the TPC in both species (r = 0.766-0.883). The rosmarinic acid content of lemon balm plants contributed to the antioxidant capacity, as well (r = 0.679-0.869).

Volatile constituents of Melissa officinalis leaves determined by plant age.

The present study investigated changes in the content and chemical composition of the essential oil extracted by hydrodistillation from air-dried Melissa officinalis L. (lemon balm) leaves in the first and second year of plant growth. The lemon balm oil was analysed by GC-MS and GC-FID. The presence of 106 compounds, representing 100% of the oil constituents, was determined in the oil. The predominant components were geranial (45.2% and 45.1%) and neral (32.8% and 33.8%); their proportions in the examined samples of the oil obtained from one- and two-year-old plants were comparable. However, the age of lemon balm plants affected the concentration of other constituents and the proportions of the following compounds were subject to especially high fluctuations: citronellal (8.7% and 0.4%), geraniol (trace amounts and 0.6%), and geranyl acetate (0.5% and 3.0%), as well as, among others, isogeranial, E-caryophyllene, caryophyllene oxide, germacrene D, and carvacrol. The essential oil of two-year-old plants was characterized by a richer chemical composition than the oil from younger plants.

Estabelecimento in vitro e aclimatização de Lippia alba (Mill. ) N. E. Brown / In vitro establishment and acclimatization of Lippia alba (Mill. ) N. E. Brown

O presente trabalho teve por objetivo estabelecer in vitro a espécie Lippia alba (Mill.) N. E. Brown e promover a aclimatização de mudas dessa espécie. Para isso, foi testada a influência de diferentes concentrações e tempos de imersão em hipoclorito de sódio na assepsia dos explantes. Segmentos nodais foram imersos em hipoclorito de sódio nas concentrações 0,4; 0,6; 0,8 e 1,0 % e nos tempos 8, 12 e 16 minutos. Após 30 dias avaliou-se a contaminação bacteriana (%), número de brotos, número de folhas, e a taxa de sobrevivência (%). A concentração de 1% de hipoclorito de sódio foi a mais eficiente no controle da contaminação. Ápices caulinares de L. alba foram estabelecidos in vitro em meio MS suplementado com diferentes doses de BAP (0; 0,5; 1,0; 1,5 mg L-1). Após 140 dias avaliou-se a contaminação (%), a taxa de sobrevivência (%), a oxidação (%) e o número de brotos. Os melhores resultados foram obtidos para a dose 1,5 mg L-1 deste regulador. Para a aclimatização foram testados quatro tipos de substratos: pó de coco + calcário (1 g L-1), Plantmax® + calcário (1 g L-1), vermiculita + calcário (1 g L-1) e pó de coco + Plantmax® + vermiculita (1:1:1) + calcário (1 g L-1). Avaliou-se a taxa de sobrevivência (%), comprimento da parte aérea (cm), comprimento da raiz (cm), massa fresca da parte aérea e da raiz (g), e a massa seca da parte aérea e da raiz (g). Os melhores resultados foram obtidos quando com o substrato comercial Plantmax®.

The present study aimed to establish the species Lippia alba (Mill.) N. E. Brown in vitro and promote the acclimatization of the seedlings of this species. Therefore, we tested the effect of different concentrations and immersiog times in sodium hypochloritetfor the disinfection of explants. Nodal segments were immersed in sodium hypochlorite at the different concentrations of ,.4; ,.6; ,.8 and ,.0% during the different times of 8, 12 and 16 minutes. After 30 days, bacterial contamination (%), number of shoots, number of leaves and survival rate (%) were evaluated. The 1% concentration of sodium hypochlorite was more effective in controlling contamination. Shoot apices of L. alba were established in vitro on MS medium supplemented with different concentrations of BAP (;, ,.5; ,.0; ,.5 mg L-1). After 140 days, we evaluated the contamination (%), survival rate (%), oxidation (%) and number of shoots. The best results were obtained when we used the dose of ,.5 mg L-1 of this regulator. For acclimatization, we tested four types of substrates: coconut powder + lime (1 g L-1), Plantmax® + lime (1 g L-1), vermiculite + lime (1 g L-1) and coconut powder + Plantmax® + vermiculite (1:1:1) + lime (1 g L-1). We evaluated the survival rate (%), shoot length (cm), root length (cm), fresh weight of shoot and root (g) and dry weight of shoot and root (g). The best results were obtained when using the substrate Plantmax®.

Effects of IAA, IBA, NAA, and GA3 on rooting and morphological features of Melissa officinalis L. stem cuttings.

This study analyzed the potential of producing Melissa officinalis L. using stem cuttings. Four different hormones (IAA, IBA, NAA, and GA3) were applied to the cuttings, with and without buds, in two doses (1000 mg/L and 5000 mg/L), and after 60 days, 10 morphological characteristics of newly generated plants were detected, and a statistical analysis was carried out. The results of the study show that the cuttings with at least one bud must be used in order to produce M. officinalis using stem cuttings. Even though the auxin group hormones (IAA, IBA, and NAA) do not have an apparent effect on rooting percentage, these hormones were detected to affect the morphological characteristics of the newly generated plants, especially root generation. GA3 application has a considerable effect on stem height.

Phenolic profiles of cultivated, in vitro cultured and commercial samples of Melissa officinalis L. infusions.

Melissa officinalis L. (lemon balm) is normally consumed as an infusion and presents therapeutic properties, such as sedative, carminative and antispasmodic, also being included in some pharmaceutical preparations. The phenolic profiles of different samples of lemon balm, prepared as infusions, were evaluated by HPLC-DAD-ESI/MS. The profiles were compared in order to understand the differences between cultivated, in vitro cultured and commercial (bags and granulated) samples. All the samples showed a similar phenolic profile, presenting differences only in the quantities found of each compound. Rosmarinic acid was the most abundant compound, being higher in commercial samples, especially in tea bag sample (55.68mg/g of infusion) and lower in in vitro cultured sample (15.46mg/g). Moreover, dimers, trimers and tetramers of caffeic acid were identified and quantified for the first time in lemon balm. Only one flavonoid, luteolin-3'-O-glucuronide was found in all the samples, ranging from 8.43mg/g in commercial granulate sample to 1.22mg/g in in vitro cultured sample. Overall, cultivated and in vitro cultured samples presented the lowest amounts of phenolic compounds (59.59 and 30.21mg/g, respectively); otherwise, commercial samples showed the highest contents (109.24mg/g for tea bag and 101.03mg/g for granulate sample). The present study shows that infusion of lemon balm can be a source of phenolic compounds, known for their bioactive effects.

Produção de biomassa de erva-cidreira [Lippia alba (Mill. ) N.E.Br. ] sob adubação com composto de capim elefante inoculado e sem inoculação de actinomicetos / Biomass production of Lippia alba (Mill. ) N.E.Br. under fertilization with compost inoculated elephant grass and without inoculation of actinomycetes

A erva-cidreira [Lippia alba (Mill.) N. E. Br.] é uma espécie da família Verbenaceae amplamente utilizada pela população brasileira, devido às propriedades calmante, espasmolítica suave, analgésica, sedativa, ansiolítica e levemente expectorante. Para implantação do experimento foi produzido composto orgânico de capim elefante + esterco, na proporção de 70% de material vegetal e 30% de esterco, sem inoculação e inoculados com os isolados de actinomicetos do gênero Streptomyces: AC16; AC26; AC92 e AC103. Os compostos foram acondicionados em sacos de polietileno contendo 10,0 kg de solo adicionados na proporção equivalente a 20 t ha-1 e foram reaplicados logo após a primeira colheita. As colheitas da parte aérea foram realizadas aos 75 e 165 dias após o transplante. As raízes foram colhidas logo após a segunda colheita. Os tratatamentos com adubação orgânica promoveram aumento médio na produção de biomassa da erva-cidreira de 184, 83, 125, 115 e 122% para biomassa seca da parte aérea (MSPA) na primeira colheita, de MSPA na segunda colheita, biomassa seca total da parte aérea (MSTPA), biomassa seca de raiz (MSR) e biomassa seca total da cultura (MST), respectivamente, quando comparados com o tratamento controle, porém não houve efeito da inoculação de actinomicetos no composto orgânico. Nas condições em que este estudo foi realizado foi possível concluir que a adubação com composto orgânico de capim elefante influenciou positivamente a produção de biomassa seca da L. alba, mas a inoculação dos compostos com actinomicetos não exerceu influência.

L. alba is a species of the family Verbenaceae widely used by the Brazilian population, because of the soothing, gentle antispasmodic, analgesic, expectorant, sedative, anxiolytic and smoothly. For the implementation of the experiment produced organic compound of elephant grass (Pennisetum purpureum Shum.) + manure at 70% plant material and 30% manure, uninoculated and inoculated with the actinomycete Streptomyces genus, AC16, AC26, AC92 and AC103. The compounds were placed in polyethylene bags containing 10.0 kg of soil added in a proportion equivalent to 20 t ha-1 and re-apply after the first harvest. The harvests of the shoots were taken at 75 and 165 days after transplantation. The roots were harvested after the second harvest. Dealing with organic fertilization promoted the average increase in biomass production of lemon grass 184, 83, 125, 115 and 122% for the dry stem biomass (MSPA) in the first harvest, second harvest of MSPA, total shoot biomass (MSTPA), dry biomass of the root (MSR) and the total crop biomass (MST), respectively, compared with the control, but no effect of inoculation of actinomycetes in compost. Under the conditions of this study was conducted, it was concluded that the organic compound fertilizer with elephant grass positively influenced the production of dry biomass of L. alba, but the injection of the compounds of the actinomycetes, without influence.

Cloning and characterisation of rosmarinic acid synthase from Melissa officinalis L.

Lemon balm (Melissa officinalis L.; Lamiaceae) is a well-known medicinal plant mainly due to two groups of compounds, the essential oil and the phenylpropanoid derivatives. The prominent phenolic compound is rosmarinic acid (RA), an ester of caffeic acid and 3,4-dihydroxyphenyllactic acid. RA shows a number of interesting biological activities. Rosmarinic acid synthase (RAS; 4-coumaroyl-CoA:hydroxyphenyllactic acid hydroxycinnamoyltransferase) catalyses the ester formation. Cell cultures of M. officinalis have been established in order to characterise the formation of RA in an important diploid medicinal plant. RAS activity as well as the expression of the RAS gene are closely correlated with the accumulation of RA in suspension cultures of M. officinalis. The RAS cDNA and gene (MoRAS) were isolated. The RAS gene was shown to be intron-free. MoRAS belongs to the BAHD superfamily of acyltransferases. Southern-blot analysis suggests the presence of only one RAS gene copy in the M. officinalis genome. The enzyme was characterised with respect to enzyme properties, substrate preferences and kinetic data in crude plant extracts and as heterologously synthesised protein from Escherichia coli.