Rosmarinic Acid Production Research Articles

Salvia officinalis L. resilience under chromium stress: An integrated study of growth, physiology, biochemical changes and rosmarinic acid production

Medicinal plants are increasingly challenged by rising chromium (Cr) levels in agricultural soil and water bodies due to industrialization and human activities. This research examines the impact of various chromium concentrations on Salvia officinalis L., a medicinal herb, over 3 specific time periods: 30, 60 and 90 days. As the duration of Cr exposure increases, various growth parameters showed an upward trend at the lowest concentrations, with the most robust growth observed in the 20 ppm Cr treatment group after 90 days. However, higher chromium concentrations resulted in reduced plant growth compared to untreated plants. Chromium primarily accumulates in the roots, stems and leaves, with the highest accumulation observed at 100 ppm. However, chlorophyll content declined with prolonged Cr exposure, particularly at higher concentrations. Carbohydrate levels initially increased at lower Cr concentrations but decreased with greater exposure, while protein content consistently decreased with elevated Cr levels. Proline levels exhibited mixed responses, rising at lower concentrations and declining at higher ones. Malondialdehyde (MDA) content increased with higher Cr levels and extended exposure. The enzymatic antioxidant system showed an initial increase followed by a decline with prolonged exposure. Rosmarinic acid content increased with chromium (Cr) exposure upto 60 ppm but subsequently decreased beyond that threshold. In the first 30 days, plants treated with Cr demonstrated a 17 % increase in rosmarinic acid production compared to the control (48.9 mg/g DW). However, with continued Cr exposure, there was a decline in rosmarinic acid production ranging from 10 % to 20 % compared to the control level (67.02 mg/g DW) at 90 days post-treatment. These findings underscore the complex and contrasting responses of Salvia officinalis to Cr toxicity, highlighting the necessity for extended study into the core mechanisms governing these responses and the development of strategies to alleviate heavy metal stress in plants.

RBOH-dependent signaling is involved in He-Ne laser-induced salt tolerance and production of rosmarinic acid and carnosol in Salvia officinalis

BackgroundIn the past two decades, the impacts of Helium-Neon (He–Ne) laser on stress resistance and secondary metabolism in plants have been studied, but the signaling pathway which by laser regulates this process remains unclear. Therefore, the current study sought to explore the role of RBOH-dependent signaling in He–Ne laser-induced salt tolerance and elicitation of secondary metabolism in Salvia officinalis. Seeds were primed with He–Ne laser (6 J cm− 2) and peroxide hydrogen (H2O2, 5 mM) and 15-old-day plants were exposed to two salinity levels (0, 75 mM NaCl).ResultsSalt stress reduced growth parameters, chlorophyll content and relative water content (RWC) and increased malodialdehyde (MDA) and H2O2 contents in leaves of 45-old-day plants. After 48 h of salt exposure, higher transcription levels of RBOH (encoding NADPH oxidase), PAL (phenylalanine ammonia-lyase), and RAS (rosmarinic acid synthase) were recorded in leaves of plants grown from seeds primed with He–Ne laser and/or H2O2. Despite laser up-regulated RBOH gene in the early hours of exposing to salinity, H2O2 and MDA contents were lower in leaves of these plants after 30 days. Seed pretreatment with He–Ne laser and/or H2O2 augmented the accumulation of anthocyanins, total phenol, carnasol, and rosmarinic acid and increased total antioxidant capacity under non-saline and more extensively at saline conditions. Indeed, these treatments improved RWC, and K+/Na+ ratio, enhanced the activities of superoxide dismutase and ascorbate peroxidase and proline accumulation, and significantly decreased membrane injury and H2O2 content in leaves of 45-old-day plants under salt stress. However, applying diphenylene iodonium (DPI as an inhibitor of NADPH oxidase) and N, N-dimethyl thiourea (DMTU as a H2O2 scavenger) after laser priming reversed the aforementioned effects which in turn resulted in the loss of laser-induced salt tolerance and secondary metabolism.ConclusionsThese findings for the first time deciphered that laser can induce a transient RBOH-dependent H2O2 burst, which might act as a downstream signal to promote secondary metabolism and salt stress alleviation in S. officinalis plants.

Exploring the impact of exogenous melatonin on agro-morphological characteristics, carvacrol, and rosmarinic acid production in Satureja rechingeri Jamzad under drought stress

Satureja rechingeri Jamzad (known as “Jatra” in Persian), which belongs to the Lamiaceae family, is a rich source of essential oil particularly carvacrol, and rosmarinic acid. Drought stress has a detrimental impact on the physiological and biochemical parameters of plants, leading to a decline in plant productivity. Melatonin (MT), a new plant growth regulator found abundantly in plants, has been found to enhance the plant's internal resistance to various environmental stresses. The present study aimed to examine the impact of exogenously applied MT on the agro-morphological, physio-biochemical, and phytochemical traits of S. rechingeri plants cultivated under different levels of drought stress. The results indicated that plants treated with 200 µM MT obtained the highest plant height, length and width of leaf, fresh, dry and drug weight under different drought stress levels. The highest values of relative water content (RWC) (93.5%) and chlorophyll content (15.4 mg/g FW) were recorded by MT 200 µM and 100 µM, respectively, in 100% FC. Drought stress treatments (40, 60, and 80% FC) without foliar spray of MT significantly enhanced the H2O2 content, electrolyte leakage, and malondialdehyde content in leaves, whereas MT treatment under drought stress significantly decreased the above parameters. The lowest H2O2 content (11.5 nmol/g), electrolyte leakage (3.08%), and malondialdehyde content (0.78 µM/g) were obtained by 200 µM MT at 100% FC. In contrast, drought stress treatment increased the total phenol content (TPC), rosmarinic acid (RA), essential oils (EOs) content and yield, and carvacrol. The maximum values of TPC (28.1 mg GAE/g DW), EOs content (3.63%) and yield (0.96%), and carvacrol (95.66%) were achieved by 200 µM MT at 40% FC. The highest RA content (7.43 mg GAE/g DW) was recorded in 100 µM MT at 40% FC. Thus, foliar spray MT has the potential to enhance plant growth through the mitigation of reactive oxygen species (ROS)-induced oxidative harm, as well as the augmentation of photosynthesis pigments, secondary metabolites such as phenolics, EOs levels, overall antioxidant scavenging capacity, and the preservation of RWC during periods of drought stress.Graphical

The total biosynthesis route of rosmarinic acid in Sarcandra glabra based on transcriptome sequencing

Sarcandra glabra is a widely distributed and valuable plant in food and daily chemical industries, and is also a common-used medicinal plant for treating inflammatory diseases and tumors. Rosmarinic acid (RA) with significant pharmacological activity is an abundant and important constituent in S. glabra, however, little information about key enzymes involving the biosynthesis of RA in S. glabra is available and the underlying biosynthesis mechanisms of RA in S. glabra remain undeciphered. Therefore, in this study, by full-length transcriptome sequencing analyses of S. glabra, we screened the RA biosynthesis candidate genes based on sequence similarity and conducted enzymatic function characterization in vitro and in vivo. As a result, a complete set of 7 kinds of enzymes (SgPALs, SgC4H, Sg4CL, SgTATs, SgHPPRs, SgRAS and SgC3H) involving the biosynthesis route of RA from phenylalanine and tyrosine, were identified and fully characterized. This research systematically revealed the complete biosynthesis route of RA in S. glabra, which helps us better understand the process of RA synthesis and accumulation, especially the substrate promiscuities of SgRAS and SgC3H provide the molecular biological basis for the efficient biosynthesis of specific and abundant RA in S. glabra. The 7 kinds of key enzymes revealed in this study can be utilized as tool enzymes for production of RA by synthetic biology methods.

Rosmarinic Acid Content and Antioxidant Activity in Ehretia asperula Zollinger et Moritzi Cell Suspension Cultures

Background: Ehretia asperula Zollinger et Moritzi is a medicinal tree abundant in rosmarinic acid, the primary phenolic component, with numerous beneficial biological properties, including antioxidant, antibacterial, antiviral, anti-allergy, anti-arthritis, asthma and anti-cancer. Methods: The cell suspension cultures of E. asperula Zollinger et Moritzi derived from the leaf in vitro callus were established in liquid B5 medium added with 0.4 mg/L NAA, 0.1 mg/L BA and 45 g/L glucose. After four weeks, 50 mg/L of chitosan was given to the cell suspension cultures to stimulate rosmarinic acid (RA) production after a 48-hour treatment period. RA content was analyzed using the HPLC and spectrophotometry method after four weeks and 48 hours of chitosan treatment. In addition, the antioxidant capacity of the extracts from E. asperula Zollinger et Moritzi was also tested using DPPH free radical scavenging assay. Result: The RA content and antioxidant capacity of E. asperula Zollinger et Moritzi’ extracts from the leaf in vitro greater than callus derived from the leaf in vitro greater than field-grown leaf greater than biomass of cell suspension cultures. These results suggested a strong correlation between RA concentration and antioxidant capacity. The use of E. asperula Zollinger et Moritzi cell suspension cultures with chitosan as an elicitor for RA production and evaluation of antioxidant activity is presented in this study for the first time. Our results suggest that cell suspension cultures and others may be a good source of RA, an antioxidant compound.

Improvement of Bioactive Polyphenol Accumulation in Callus of Salvia atropatana Bunge.

Callus cultures of the Iranian medicinal plant Salvia atropatana were initiated from three-week-old seedlings on Murashige and Skoog (MS) medium supplemented with α-naphthaleneacetic acid (NAA) and various cytokinins. Although all tested hormonal variants of the medium and explant enabled callus induction, the most promising growth was noted for N-(2-chloro-4-pyridyl)-N'-phenylurea (CPPU)-induced calli. Three lines obtained on this medium (cotyledon line-CL, hypocotyl line-HL, and root line-RL) were preselected for further studies. Phenolic compounds in the callus tissues were identified using UPLC-MS (ultra-performance liquid chromatography-mass spectrometry) and quantified with HPLC (high-performance liquid chromatography). All lines exhibited intensive growth and contained twelve phenolic acid derivatives, with rosmarinic acid predominating. The cotyledon-derived callus line displayed the highest growth index values and polyphenol content; this was exposed to different light-emitting diodes (LED) for improving biomass accumulation and secondary metabolite yield. Under LED treatments, all callus lines exhibited enhanced RA and total phenolic content compared to fluorescent light, with the highest levels observed for white (48.5-50.2 mg/g dry weight) and blue (51.4-53.9 mg/g dry weight) LEDs. The selected callus demonstrated strong antioxidant potential in vitro based on the 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS), 2,2-diphenyl-1-picrylhydrazyl (DPPH), and ferric reducing antioxidant power (FRAP) tests. Our findings confirm that the S. atropatana callus system is suitable for enhanced rosmarinic acid production; the selected optimized culture provide high-quality plant-derived products.

Distinction of chia varieties in vivo and in vitro based on the flow cytometry and rosmarinic acid production

Flow cytometry has made a significant contribution to the study of several complex fundamental mechanisms in plant cytogenetics, becoming a useful analytical tool to understand several mechanisms and processes underlying plant growth, development, and function. In this study, the genome size, DNA ploidy level, and A-T/G-C ratio were measured for the first time for two genotypes of chia, Salvia hispanica, an herbaceous plant commonly used in phytotherapy and nutrition. This study also evaluated, for the first time by flow cytometry, the capacity to produce organic acids of tissues stained with LysoTracker Deep Red after elicitation with either yeast extract or cadmium chloride. Rosmarinic acid content differed between the two chia varieties treated with different elicitor concentrations, compared with non-elicited plant material. Elicited tissues of both varieties contained a higher content of rosmarinic acid compared with non-elicited cultures, and cadmium chloride at 500 μM was much better than that at 1000 μM, which led to plant death. For both genotypes, a dose-response was observed with yeast extract, as the higher the concentration of elicitor used, the higher rosmarinic acid content, resulting also in better results and a higher content of rosmarinic acid compared with cadmium chloride. This study demonstrates that flow cytometry may be used as a taxonomy tool, to distinguish among very close genotypses of a given species and, for the first time in plants, that this approach can also be put to profit for a characterization of the cytoplasmic acid phase and the concomitant production of secondary metabolites of interest in vitro, with or without elicitation.Key points• Genome size, ploidy level, A-T/G-C ratio, and cytoplasm acid phase of S. hispanica• Cytometry study of cytoplasm acid phase of LysoTracker Deep Red-stained plant cells• Yeast extract or cadmium chloride elicited rosmarinic acid production of chia tissuesGraphical

Foliar-applied iron and zinc nanoparticles improved plant growth, phenolic compounds, essential oil yield, and rosmarinic acid production of lemon balm (Melissa officinalis L.).

Metallic nanoparticles (NPs) have been highlighted to improve plant growth and development in the recent years. Although positive effects of some NPs have been reported on medicinal plants, the knowledge for stimulations application of iron (Fe) and zinc (Zn) NPs is not available. Hence, the present work aimed to discover the effects of Fe NPs at 10, 20, and 30 mg L-1 and Zn NPs at 60 and 120 mg L-1 on growth, water content, photosynthesis pigments, phenolic content, essential oil (EO) quality, and rosmarinic acid (RA) production of lemon balm (Melissa officinalis L.). The results showed that Fe NPs at 20 and 30 mg L-1 and Zn NPs at 120 mg L-1 significantly improved biochemical attributes. Compared with control plants, the interaction of Fe NPs at 30 mg-1 and Zn NPs at 120 mg L-1 led to noticeable increases in shoot weight (72%), root weight (92%), chlorophyll (Chl) a (74%), Chl b (47%), RA (66%), proline (81%), glycine betaine (GB, 231%), protein (286%), relative water content (8%), EO yield (217%), total phenolic content (63%), and total flavonoid content (57%). Heat map analysis revealed that protein, GB, EO yield, shoot weight, root weight, and proline had the maximum changes upon Fe NPs. Totally, the present study recommended the stimulations application of Fe NPs at 20-30 mg L-1 and Zn NPs at 120 mg L-1 to reach the optimum growth and secondary metabolites of lemon balm.

Cyanobacterial elicitor enhances the biomass of Mentha piperita L. and improves the production of high-value rosmarinic acid under in vitro culture of apical meristem

BackgroundRosmarinic acid (RA), like other phenolic compounds, is sources of antioxidants and anti-inflammatory agents in medicinal plants. In vitro culture of plants can improve the medicinal plants’ metabolite profile and phenolic compound quantity. To date, various methods have been proposed to increase this medicinal metabolite in plants, among which the use of bioelicitors can be mentioned. In the present study, a native isolate of heterocystous cyanobacteria, Nostoc spongiaeforme var. tenue ISB65, was used to stimulate the production of biomass and content of RA in Mentha piperita L. (peppermint) grown in vitro from apical meristem. Mentha piperita L. explants were inoculated in half strength Murashige and Skoog (1/2 MS) medium containing cyanobacterial lysate (CL). After 50 days of culturing, the growth indices, the content of photosynthetic pigments, and RA in control and treated plants were measured.ResultsCL inoculation resulted in a significant enhancement in the vegetative growth indices of peppermint, including root and shoot length, plant biomass and leaf number. The content of photosynthetic pigments also increased in cyanobacteria-treated plants. Inoculation with CL increased the RA content by 2.3-fold, meaning that the plants treated with CL had the highest RA content (7.68 mg. g− 1 dry weight) compared to the control (3.42 mg. g− 1 dry weight). Additionally, HPLC analysis revealed the presence of several auxins in CL.ConclusionsThe presence of auxins and the chemical content of CL such as K+ and Ca2+, as regulators of metabolic pathways and molecular activities of cells, may be responsible for the enhanced growth and phenolic compounds of plants under tissue culture conditions. An improvement in RA content in the tissue culture of medicinal plants treated with CL was reported for the first time in this investigation.

Hairy roots as a potential source for the production of rosmarinic acid from genus Salvia

The Salvia genus, a member of the Lamiaceae family, exhibits a rich array of secondary metabolites, including di- and triterpenoids, phenols, polyphenols, and essential oil compounds. These constituents contribute to valuable pharmacological activities such as antibacterial, antiviral, anti-inflammatory, and antioxidant properties. Among these metabolites, rosmarinic acid stands out as a particularly promising compound, deriving from the precursors phenylalanine and tyrosine. It belongs to the phenolic compound class and acts as an ester of caffeic acid, showcasing diverse therapeutic potentials like antifungal, antibacterial, antiviral, antioxidant, anticancer, anti-ageing, anti-inflammatory, and anti-diabetic effects. To facilitate the production of such secondary metabolites, plant tissue culture techniques have played a pivotal role, with hairy root cultures being one of the preferred methods. This review provides an extensive examination of the biosynthetic pathway of rosmarinic acid and its successful generation using hairy root cultures. Additionally, the review highlights the utilization of genetic modification tools and various biotic and abiotic elicitors, including yeast extract, methyl jasmonate, and silver ion (Ag+), in hairy root cultures of diverse Salvia species to enhance the production of rosmarinic acid.

Enhancing rosmarinic acid production and regulating enzyme activity in Melissa officinalis L. using spectrally tunable light-emitting diodes

Melissa officinalis L. (lemon balm) is a renowned medical herb enriched with phenolic compounds, particularly rosmarinic acid (RA) which is known for its antimicrobial and antioxidant activities. This study aimed at improving the medicinal characteristics of M. officinalis by enhancing the production of the total phenolic content (TPC) and RA as well as evaluating the activities of RA-regulatory enzymes, following a series of light treatments, using i) three different spectrally tuned light treatments, ii) white light-emitting diode (LED), and iii) sunlight. Phytochemical analyses revealed that the blue-enriched light treatment (40% red, 35% blue, 15% green and 10% far-red) significantly elevated the TPC, RA, and antioxidant capacity of M. officinalis. Additionally, the application of spectrally tunable LEDs also induced the malondialdehyde level and sugar concentration with minimal effect on the chlorophyll content and antimicrobial activity. Increased activities of RA-regulatory enzymes, namely phenylalanine ammonia-lyase, tyrosine aminotransferase, and hydroxyphenylpyruvate reductase, were associated with RA production. These findings suggest that the elicitation based on spectrally tunable LEDs has promising potential for promoting phenolic compounds production in M. officinalis.

The Effect of Light and Dark Treatment on the Production of Rosmarinic Acid and Biological Activities in Perilla frutescens Microgreens.

This study aimed to investigate the effect of light [a long-day photoperiod (16 h light/8 h dark cycle)] and dark treatment on the production of rosmarinic acid in P. frutescens microgreens and to determine its antioxidant and antibacterial activities. Microgreens of P. frutescens were grown under light and dark conditions and harvested after 10, 15, 20, and 25 days of each treatment. Although dry weight values of microgreens gradually increased from 10 to 25 days of both treatments, the microgreens grown under light treatment possessed slightly higher levels of dry weight than those grown in the dark. Rosmarinic acid and total phenolic content (TPC) were also analyzed using high-performance liquid chromatography (HPLC) and Folin-Ciocalteu assay. The accumulation patterns of rosmarinic acid and TPC gradually increased and decreased, respectively, in P. frutescens microgreens grown in continuous darkness. The highest accumulation was observed in microgreens grown for 20 days. However, rosmarinic acid and TPC values were not significantly different in microgreens grown under light conditions. According to the 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical inhibition assay, the extracts of P. frutescens microgreens were confirmed to be strong antioxidants, and their ability to scavenge DPPH radicals was positively correlated with the total phenolic content in the microgreens after 10, 15, 20, and 25 days of both treatments. Considering the relatively higher values of dry weight, rosmarinic acid, TPC, and DPPH assay, P. frutescens microgreens after 20 days of darkness and 20 days of light treatment, respectively, were selected for screening antibacterial activity using nine pathogens. Both microgreen extracts showed strong antibacterial activity against pathogens. In particular, the extracts of microgreens grown for 20 days under light treatment showed higher antimicrobial effects. Therefore, the light treatments for 20 days, as well as the darkness treatment for 20 days, were the best conditions for P. frutescens microgreen production because of their high levels of dry weight, phenolics, and biological activities.

Effects of Carbohydrates on Rosmarinic Acid Production and In Vitro Antimicrobial Activities in Hairy Root Cultures of Agastache rugosa.

Agastache rugosa (popularly known as Korean mint) belongs to the Lamiaceae family and comprises 22 species of perennial aromatic medicinal species native to East Asian countries, such as Korea, Taiwan, Japan, and China. A. rugosa contains many phenolic compounds that exhibit pharmacological and physiological activities, including antioxidant, anticancer, antiviral, antifungal, and antibacterial activities. The highest concentrations of rosmarinic acid and its isomers have been reported in the roots of A. rugosa. In this in vitro study, hairy roots of A. rugosa were obtained and the carbohydrates (sorbitol, mannitol, glucose, maltose, galactose, mannose, and sucrose) were evaluated to determine those that were optimal for rosmarinic acid production and hairy root growth. Antioxidant and antibacterial activities of extracts of A. rugosa were also assessed. The best carbon source for A. rugosa hairy root cultures was sucrose, considering biomass productivity (0.460 ± 0.034 mg/30 mL), rosmarinic acid production (7.656 ± 0.407 mg/g dry weight), and total phenolic content (12.714 ± 0.202 mg/g gallic acid equivalent). Antioxidant and antimicrobial activities were displayed by A. rugosa hairy roots cultured in liquid medium supplemented with 100 mM sucrose. Twenty-five bacterial strains, including multidrug-resistant bacteria and one pathogenic yeast strain, were used for antimicrobial screening of A. rugosa hairy roots. The hairy root extracts displayed antibacterial activity against Micrococcus luteus (KCTC 3063) and Bacillus cereus (KCTC 3624). The inhibition of these bacteria was greater using A. rugosa hairy roots with the highest levels of phenolic compounds cultured in the presence of sucrose, compared to hairy roots with the lowest levels of phenolic compounds cultured in the presence of fructose. Considering hairy root biomass, phenolic compound production, and antibacterial activity, sucrose is the best carbon source for A. rugosa hairy root cultures.

Rosmarinic Acid Production from Origanum dictamnus L. Root Liquid Cultures In Vitro.

In the present work Origanum dictamnus L. was studied as a suitable in vitro adventitious root culture system for the production of important bioactive molecules, such as rosmarinic acid (RA). Callus culture was initiated from leaf, petiole and root explants on solid MS medium supplemented with either 5 μM NAA + 5 μM kinetin (ODK3) or 5 μM NAA + 0.5 μM kinetin (ODK4). New roots formed from leaf, petiole and root calluses were aseptically transferred into Erlenmeyer flasks containing 100 mL liquid medium and shaken at 120 rpm in the dark. The liquid medium used was the MS supplemented either with 35 μM IBA + 2.5 μM kinetin (ODY1) or 5 μM NAA + 0.5 μM kinetin (ODY2). Biomass production parameters, RA content (%) and yield index (YI) were recorded for each treatment explant type, medium composition and incubation period. Results showed, in every case, the production of RA in vitro. Between the two liquid media (ODY1, ODY2) and the different culture periods, the ODY1 medium and the longest 200-day-culture period were more effective for RA and biomass production, regardless of the initial explant type used. The combination of ODK4-ODY1 resulted in higher RA (5.1% and 4.7%), fresh biomass production (19.0 g and 11.6 g), mean YI (93.7 mg and 51.4 mg) and YI per explant (3.75 mg and 2.06 mg) for roots derived from leaf calluses and root calluses, respectively. However, the solid ODK3 (200 days)-liquid ODY1 (40 days) transition treatment was more beneficial for roots derived from petiole calluses leading to an 18.8-fold increase in fresh biomass growth rate. RA accumulation and YIs were also significantly influenced by explant type, with the highest value produced from root petiole calluses (6.6% RA dry weight, 115.3 mg mean YI and 4.61 mg YI per explant) after 240 days.

Coordinating caffeic acid and salvianic acid A pathways for efficient production of rosmarinic acid in Escherichia coli

Rosmarinic acid is a natural hydroxycinnamic acid ester used widely in the food and pharmaceutical industries. Although many attempts have been made to screen rate-limiting enzymes and optimize modules through co-culture fermentation, the titer of rosmarinic acid remains at the microgram level by microorganisms. A de novo biosynthetic pathway for rosmarinic acid was constructed based on caffeic acid synthesis modules in Escherichia coli. Knockout of competing pathways increased the titer of rosmarinic acid and reduced the synthesis of rosmarinic acid analogues. An L-amino acid deaminase was introduced to balance metabolic flux between the synthesis of caffeic acid and salvianic acid A. The ratio of FADH2/FAD was maintained via the coordination of deaminase and HpaBC, which is responsible for caffeic acid synthesis. Knockout of menI, encoding an endogenous thioesterase, increased the stability of caffeoyl-CoA. The final strain produced 5780.6 mg/L rosmarinic acid in fed-batch fermentation, the highest yet reported for microbial production. The strategies applied in this study lay a foundation for the synthesis of other caffeic acid and rosmarinic acid derivatives.

Auxin sensitivity improves production of rosmarinic acid in transformed hairy roots of <em>Lavandula angustifolia</em>

Hairy root culture is a promising approach to improve production of plant secondary metabolites. The genes, which are located in T-DNA of a root-inducing plasmid, regulate auxin sensitivity of hairy roots. Therefore, this study was aimed to improve the growth and rosmarinic acid production of Lavandula angustifolia hairy roots. Lateral branches of hairy roots were transferred to ½ MS and ½ B5 liquid media. To assess auxin sensitivity, indole-3-acetic acid (IAA) and indole-3-butyric acid (IBA) with four different concentrations (0, 0.1, 0.5 and 1 mg/l) were also applied. The growth of hairy roots in ½ MS medium was two-fold higher than in ½ B5 medium. In addition, both auxins were found to significantly improve the growth of hairy roots whereas non-transformed roots stopped growing in the presence of the auxins. The highest dry weight and rosmarinic acid production of hairy roots were obtained from ½ MS medium supplemented with IBA irrespective of its concentration. As a result, the hairy roots grown in ½ MS medium supplemented with IBA produced the maximum amount of rosmarinic acid (7.98 mg/g dry weight of hairy roots). This first report of rosmarinic acid production in L. angustifolia hairy roots provides new insights into the auxin sensitivity of L. angustifolia hairy roots.

Rosmarinic acid production in hairy root cultures of Salvia nemorosa L. (Lamiaceae)

Rosmarinic acid (RA) is a widely known and interesting medicinal phenolic acid produced in plant, cell, tissue, and organ in vitro cultures. In this study, hairy roots were induced using various strains of Agrobacterium rhizogenes (ATCC15834, A4, R1000, and GM1534). Furthermore, the effect of sucrose, IBA anddifferent in vitro basal medium (MS, ½ MS) on the hairy root generation and RA production was studied. ATCC15834 strain showed the maximum transformation efficiency (76%). Visible growth of the roots was observed in liquid MS medium containing 0.1 mg/L IBA. Based on a time-course experiment, the highest biomass production and RA content were observed after 2 weeks. The highest biomass (9.28 g/L DW) and RA content (15.9 mg/g DW) from the hairy root culturs were obtained in ½ MS medium supplemented with 1% and 3% sucrose, respectively. The results demonstrated high possible potential of S. nemorosa hairy root cultures on RA production.

Quantification of rosmarinic acid from different plant species of lower Himalayan region and expression analysis of underlying L-Phenylalanine pathway.

This study adopts a very effective high-performance liquid chromatography (HPLC) technique for the quantitative determination of rosmarinic acid (RA) and PCR-based amplification of biosynthetic key regulators in Isodon rugosus, Daphne mucronata, and Viburnum grandiflorum from the lower Himalayan regions. Rosmarinic acid is engaged in a variety of biological processes and has significant industrial significance. In this study, it was identified from crude methanolic extract using thin-layer chromatography with a standard, and its content was quantified using HPLC without interrupting spikes using a mixture of methanol and deionized water containing acetonitrile (70:30 v/v) and acetic acid (0.1% v/v) at UV 310 nm absorption. We used RT-PCR to identify cDNAs encoding PAL, C4H, and RAS, and Image J's semi-quantitative analysis to quantify the expression levels of genes involved in RA production from chosen plant material. The highest levels of PAL, C4H, and RAS were detected, by band intensity, in the leaves and flowers of I. rugosus, which also exhibited a substantial quantity of RA. However, in V. grandiflorum and D. mucronata the transcript of the given genes was low. The concentration of RA ranged from 187.7 to 21.2mg g-1 for I. rugosus, 17.42 to 5.42 mg g-1 for V. grandiflorum, and 15.19 mg g-1 for D. mucronata. This study demonstrated that the method for quantifying RA from a crude methanolic extract was effective, indicating that I. rugosus might be used as an indigenous alternative source of RA.

Production of rosmarinic acid and biomass from adventitious root cultures of Ocimum basilicum by optimization of medium components in airlift bioreactors

Production of rosmarinic acid and biomass from adventitious root cultures of Ocimum basilicum by optimization of medium components in airlift bioreactors

In Vitro Strategy for the Enhancement of the Production of Bioactive Polyphenols in Transformed Roots of Salvia bulleyana.

The underground parts of Salvia bulleyana, a rare Chinese plant species, have long been used in traditional Chinese medicine. The Rhizobium rhizogenes-transformed root culture obtained from this plant might be a promising novel source of valuable phenolics, including rosmarinic acid. The present study identifies for the first time, the optimal growth conditions of S. bulleyana hairy roots regarding production efficiency. The comprehensive optimization comprised cultivation in different basal media (B5, SH, MS, and WP) with full- and half-strength macro- and microelements, different vitamin contents (full, half, one-quarter part, and without) and sucrose concentrations (2, 3, 4, 5%), and under different light conditions: in dark, under blue LED (λ = 430 nm), red LED (λ = 670 nm), mixed blue and red LED (30%:70%), and white LED (390–670 nm). Hairy root growth and bioactive compound accumulation were also detailed every five days over the 50-day culture cycle. The optimal conditions were determined using a technique for order preference by similarity to the ideal solution (TOPSIS). The most efficient combination for root growth and polyphenol content was found to be ½SH liquid medium with half vitamin concentration and 3% sucrose when grown in the dark. The biomass yield during the growth cycle was 6.1 g (fresh weight—FW) and 0.92 g (dry weight—DW) on one Erlenmeyer flask: a 14.3-fold increase in FW and 16.1-fold increase in DW in relation to the inoculum. The highest mean total phenolic content was 93.6 mg/g DW including about 70 mg/g DW rosmarinic acid, reached on day 40 of culture; compared to roots of two-year-old plants grown under field conditions, the total phenolic acid content was four times higher and rosmarinic acid eight times higher. The obtained results place the investigated culture among the best hair root cultures for rosmarinic acid production.