Seseli tortuosum L. subsp. tortuosum Essential Oils and Their Principal Constituents as Anticancer Agents.

Seseli tortuosum L. subsp. tortuosum, belonging to the Apiaceae family, is a species that grows in Europe, mainly in the Mediterranean regions. The history of its application in traditional medicine highlights its various biological properties. Trying to explore the phytochemistry and pharmacological aspects of this species, the essential oils (EOs) extracted from flowers, stems, and roots of a locally wild accession, never previously investigated, growing in Sicily, Italy, were investigated. The chemical composition of all EOs, obtained by the hydrodistillation method, was evaluated by GC-MS. The most abundant class of all investigated samples was that of monoterpene hydrocarbons (79.98-91.21%) with p-cymene, α-pinene, ß-pinene, and ß-ocimene as major compounds. These EOs, and their main components, were tested for their possible anticancer activity. Obtained data provided evidence that among the different EOs tested, at the dose of 100 µg/mL, those extracted from stems and roots were particularly effective, already at 24 h of treatment, in reducing the cell viability of 42% and 95%, respectively, in HCT116 colon cancer cell line. These EOs also exerted a remarkable cytotoxic effect that was accompanied by morphological changes represented by cell shrinkage as well as a reduction in residual cell population. Differently, modest effects were found when EOs extracted from flowers were tested in the same experimental conditions. The evaluation of the phytocompounds mainly represented in the EOs extracted from different parts of the plant and tested in a range of concentrations between 20 and 200 µg/mL, revealed that α-pinene, ß-pinene, and p-cymene exerted only modest effects on cell viability. Differently, a remarkable effect was found when ß-ocimene, the most abundant phytocomponent in EOs from roots, was tested on colon cancer cells. This phytocompound, among those identified in EOs from Seseli tortuosum L. subsp. tortuosum, was found to be the most effective in reducing colon cancer cell viability with IC50 = 64.52 µg/mL at 24 h of treatment. All together, these data suggest that ß-ocimene could be responsible for the effects observed in colon cancer cells.

Testing the effect of individual scent compounds on pollinator attraction in nature using quasi-isogenic Capsella lines.

PREMISE: Floral scent, usually consisting of multiple compounds, is a complex trait, and its role in pollinator attraction has received increasing attention. However, disentangling the effect of individual floral scent compounds is difficult due to the complexity of isolating the effect of single compounds by traditional methods. METHODS: Using available quasi-isogenic lines (qILs) that were generated as part of the original mapping of the floral scent volatile-related loci CNL1 (benzaldehyde) and TPS2 (ß-ocimene) in Capsella, we generated four genotypes that should only differ in these two compounds. Plants of the four genotypes were introduced into a common garden outside the natural range of C. rubella or C. grandiflora, with individuals of a self-compatible C. grandiflora line as pollen donors, whose different genetic background facilitates the detection of outcrossing events. Visitors to flowers of all five genotypes were compared, and the seeds set during the common-garden period were collected for high-throughput amplicon-based sequencing to estimate their outcrossing rates. RESULTS: Benzaldehyde and ß-ocimene emissions were detected in the floral scent of corresponding genotypes. While some pollinator groups showed specific visitation preferences depending on scent compounds, the outcrossing rates in seeds did not vary among the four scent-manipulated genotypes. CONCLUSIONS: The scent-manipulated Capsella materials constructed using qILs provide a powerful system to study the ecological effects of individual floral scent compounds under largely natural environments. In Capsella, individual benzaldehyde and ß-ocimene emission may act as attractants for different types of pollinators.

CsTPS21 encodes a jasmonate-responsive monoterpene synthase producing ß-ocimene in citrus against Asian citrus psyllid.

Huanglongbing (HLB), spread by the Asian citrus psyllid (ACP), is a widespread, devastating disease that causes significant losses in citrus production. Therefore, controlling the ACP infestation and HLB infection is very important for citrus production. The aim of our study was to identify any citrus volatile which could be used as a repellent or less attractant towards ACP, and to envisage the potential of this strategy to control HLB spread. The present study identified a terpene synthase (TPS)-encoding gene CsTPS21 in citrus plants, and this gene was predicted to encode a monoterpene synthase and had an amino acid sequence similar to ß-ocimene synthase. CsTPS21 was significantly upregulated by ACP infestation and methyl jasmonic acid (MeJA) treatment but downregulated by salicylic acid (SA). Further heterologous gene expression studies in yeast cells and tobacco plants indicated that the protein catalyzed the formation of ß-ocimene, which acted as an ACP repellent. Detailed analysis of tobacco overexpressing CsTPS21 showed that CsTPS21 synthesizing ß-ocimene regulated jasmonic acid (JA)-associated pathways by increasing the JA accumulation and inducing the JA biosynthetic gene expression to defend against insect infestation. These findings provide a basis to plan strategies to manage HLB in the field using ß-ocimene and CsTPS21 as candidates.

Cytotoxic and Antileishmanial Effects of the Monoterpene ß-Ocimene.

Leishmaniasis is a group of infectious-parasitic diseases with high mortality rates, and endemic in many regions of the globe. The currently available drugs present serious problems such as high toxicity, costs, and the emergence of drug resistance. This has stimulated research into new antileishmania drugs based on natural products and their derivatives. ß-Ocimene is a monoterpene found naturally in the essential oils of many plant species which presents antileishmanial activity, and which has not yet been evaluated for its potential to inhibit the etiological agent of leishmaniasis. The aim of this work was to evaluate the activity of ß-ocimene against Leishmania amazonensis, its cytotoxicity, and potential mechanisms of action. ß-Ocimene presented direct activity against the parasite, with excellent growth inhibition of promastigotes (IC50 = 2.78 µM) and axenic amastigotes (EC50 = 1.12 µM) at concentrations non-toxic to RAW 264.7 macrophages (CC50 = 114.5 µM). The effect is related to changes in membrane permeability and resulting abnormalities in the parasitic cell shape. These were, respectively, observed in membrane integrity and atomic force microscopy assays. ß-Ocimene was also shown to act indirectly, with greater activity against intra-macrophagic amastigotes (EC50 = 0.89 µM), increasing TNF-α, nitric oxide (NO), and reactive oxygen species (ROS), with lysosomal effects, as well as promoting decreases in IL-10 and IL-6. Against intra-macrophagic amastigote forms the selectivity index was higher than the reference drugs, being 469.52 times more selective than meglumine antimoniate, and 42.88 times more selective than amphotericin B. Our results suggest that ß-ocimene possesses promising in vitro antileishmania activity and is a potential candidate for investigation in in vivo assays.

The Identification of ß-Ocimene Biosynthetic Pathway Through Mevalonate Acid (MVA) and 1-Deoxy-D-Xylulose 5-Phosphate (DXP) Pathways Using Crude Enzyme Extracts in Indonesian Bay Leaf/Salam Leaf (Syzygium polyanthum).

Salam leaf has a ß-ocimene as a key volatile compound that gives a fresh aroma to the food when the salam leaves are involved in the cooking process. As a secondary metabolic product, enzymatic biosynthesis as the early stage of ß-ocimene is a factor that needs to be known. Thus, this study was done to identify the mechanism of the two well-known terpenoid biosynthetic pathways, namely Mevalonate Acid (MVA) and 1-Deoxy-D-Xylulose 5-Phosphate (DXP) pathways, in the biosynthesis of ß-ocimene in salam leaves. The activity of the 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR)-MVA pathway-determining enzyme and 1-deoxy-D-xylulose-5-phosphate synthase (DXS)-DXP pathway-determining enzyme in the crude enzyme and their derivative products of salam leaves were analysed for their changes by differences of substrate ratios and enzyme inhibitors. The results showed that the activity of the HMGR enzyme was lower significantly than the DXS enzyme based on the addition of variations to the substrate ratio. These results were also supported by the enzyme and substrate reaction products, MVA and Isopentenyl diphosphate (IPP) intermediates from the MVA pathway, which were significantly lower when compared to DXP and IPP intermediates from the DXP pathway. As the end product of the reaction, ß-ocimene gave a significantly higher value of the DXP pathway than the MVA pathway. Therefore, it can conclude that the mechanism of the biosynthetic pathway of ß-ocimene in salam leaves was synthesised via the DXP pathway. The production of ß-ocimene could have crosstalk-pathway through the MVA pathway, especially when the DXP pathway was blocked.

Elucidation of the Regular Emission Mechanism of Volatile ß-Ocimene with Anti-insect Function from Tea Plants (Camellia sinensis) Exposed to Herbivore Attack.

Herbivore-induced plant volatiles (HIPVs) play an important role in insect resistance. As a common HIPV in tea plants (Camellia sinensis), ß-ocimene has shown anti-insect function in other plants. However, whether ß-ocimene in tea plants also provides insect resistance, and its mechanism of synthesis and emission are unknown. In this study, ß-ocimene was confirmed to interfere with tea geometrid growth via signaling. Light was identified as the key factor controlling regular emission of ß-ocimene induced by the wounding from tea geometrids. ß-Ocimene synthase (CsBOS1) was located in plastids and catalyzed ß-ocimene formation in overexpressed tobacco. CsBOS1 expression in tea leaves attacked by tea geometrids showed a day-low and night-high variation pattern, while CsABCG expression involved in volatile emission showed the opposite pattern. These two genes might regulate the regular ß-ocimene emission from tea plants induced by tea geometrid attack. This study advances the understanding on HIPV emission and signaling in tea plants.

Genome-wide analysis of ARF transcription factors reveals HcARF5 expression profile associated with the biosynthesis of ß-ocimene synthase in Hedychium coronarium.

KEY MESSAGE: Herein, 37 ARF genes were identified and analyzed in Hedychium coronarium and HcARF5 showed a potential role in the regulation of HcTPS3. Auxin is an important plant hormone, implicated in various aspects of plant growth and development processes especially in the biosynthesis of various secondary metabolites. Auxin response factors (ARF) belong to the transcription factors (TFs) gene family and play a crucial role in transcriptional activation/repression of auxin-responsive genes by directly binding to their promoter region. Nevertheless, whether ARF genes are involved in the regulatory mechanism of volatile compounds in flowering plants is largely unknown. ß-ocimene is a key floral volatile compound synthesized by terpene synthase 3 (HcTPS3) in Hedychium coronarium. A comprehensive analysis of H. coronarium genome reveals 37 candidate ARF genes in the whole genome. Tissue-specific expression patterns of HcARFs family members were assessed using available transcriptome data. Among them, HcARF5 showed a higher expression level in flowers, and significantly correlated with the key structural ß-ocimene synthesis gene (HcTPS3). Furthermore, transcript levels of both genes were associated with the flower development. Under hormone treatments, the response of HcARF5 and HcTPS3, and the emission level of ß-ocimene contents were evaluated. Subcellular and transcriptional activity assay showed that HcARF5 localizes to the nucleus and possesses transcriptional activity. Yeast one-hybrid (Y1H) and dual-luciferase assays revealed that HcARF5 directly regulates the transcriptional activity of HcTPS3. Yeast two-hybrid (Y2H) and bimolecular fluorescence complementation (BiFC) assays showed that HcARF5 interacts with scent-related HcIAA4, HcIAA6, and HcMYB1 in vivo. Overall, these results indicate that HcARF5 is potentially involved in the regulation of ß-ocimene synthesis in H. coronarium.

Using ß-ocimene to increase the artemisinin content in juvenile plants of Artemisia annua L.

OBJECTIVE: The anti-malarial drug, artemisinin, is harvested from the leaves of adult Artemisia annua L. plants. As its concentration in juvenile plants is very low, the present study aimed to assess if the airborne signaling molecule, ß-ocimene, could be used to enhance artemisinin accumulation in juvenile A. annua plants. RESULTS: Application of exogenous ß-ocimene increased artemisinin accumulation in A. annua. Treatment with 10 µM ß-ocimene for 4 days resulted in juvenile plants accumulating artemisinin contents of up to 25 mg/g (2.5%) of dry weight. The expression levels of key genes encoding enzymes involved in both precursor biosynthetic pathways and artemisinin biosynthetic pathways induced by ß-ocimene were upregulated. Glandular secretory trichome (GST) size and density increased by 49.2% and 38.2%, respectively, along with the upregulation of genes associated with GST development. CONCLUSION: ß-ocimene enhances artemisinin accumulation in juvenile A. annua plants by modulating artemisinin biosynthetic pathways and GST development.

Scenarios of Genes-to-Terpenoids Network Led to the Identification of a Novel α/ß-Farnesene/ß-Ocimene Synthase in Camellia sinensis.

Terpenoids play vital roles in tea aroma quality and plants defense performance determination, whereas the scenarios of genes to metabolites of terpenes pathway remain uninvestigated in tea plants. Here, we report the use of an integrated approach combining metabolites, target gene transcripts and function analyses to reveal a gene-to-terpene network in tea plants. Forty-one terpenes including 26 monoterpenes, 14 sesquiterpenes and one triterpene were detected and 82 terpenes related genes were identified from five tissues of tea plants. Pearson correlation analysis resulted in genes to metabolites network. One terpene synthases whose expression positively correlated with farnesene were selected and its function was confirmed involved in the biosynthesis of α-farnesene, ß-ocimene and ß-farnesene, a very important and conserved alarm pheromone in response to aphids by both in vitro enzymatic assay in planta function analysis. In summary, we provided the first reliable gene-to-terpene network for novel genes discovery.

Functional characterization and expression analysis of two terpene synthases involved in floral scent formation in Lilium 'Siberia'.

MAIN CONCLUSION: Floral scent formation in Lilium 'Siberia' is mainly due to monoterpene presence in the floral profile. LoTPS1 and LoTPS3 are responsible for the formation of (±)-linalool and ß-ocimene in Lilium 'Siberia'. Lilium 'Siberia' is a perennial herbaceous plant belonging to Liliaceae family, cultivated both as a cut flower and garden plant. The snowy white flower emits a pleasant aroma which is mainly caused by monoterpenes present in the floral volatile profile. Previously terpene synthase (TPS) genes have been isolated and characterized from various plant species but less have been identified from Liliaceae family. Here, two terpene synthase genes (LoTPS1 and LoTPS3), which are highly expressed in sepals and petals of Lilium 'Siberia' flower were functionally characterized recombinant LoTPS1 specifically catalyzes the formation of (Z)-ß-ocimene and (±)-linalool as its main volatile compounds from geranyl pyrophosphate (GPP), whereas LoTPS3 is a promiscuous monoterpene synthase which utilizes both GPP and farnesyl pyrophosphate (FPP) as a substrate to generate (±)-linalool and cis-nerolidol, respectively. Transcript levels of both genes were prominent in flowering parts, especially in sepals and petals which are the main source of floral scent production. The gas chromatography-mass spectrometry (GC-MS) and quantitative real-time PCR analysis revealed that the compounds were emitted throughout the day, prominently during the daytime and lower levels at night following a strong circadian rhythm in their emission pattern. Regarding mechanical wounding, both genes showed considerable involvement in floral defense by inducing the emission of (Z)-ß-ocimene and (±)-linalool, elevating the transcript accumulation of LoTPS1 and LoTPS3. Furthermore, the subcellular localization experiment revealed that LoTPS1 was localized in plastids, whilst LoTPS3 in mitochondria. Our findings on these two TPSs characterized from Lilium 'Siberia' provide new insights into molecular mechanisms of terpene biosynthesis in this species and also provide an opportunity for biotechnological modification of floral scent profile of Lilium.

Volatile ß-Ocimene Can Regulate Developmental Performance of Peach Aphid Myzus persicae Through Activation of Defense Responses in Chinese Cabbage Brassica pekinensis.

In nature, plants have evolved sophisticated defense mechanisms against the attack of pathogens and insect herbivores. Plant volatile-mediated plant-to-plant communication has been assessed in multitrophic systems in different plant species and different pest species. ß-ocimene is recognized as an herbivore-induced plant volatile that play an important role in the chemical communication between plants and pests. However, it is still unclear whether ß-ocimene can active the defense mechanism of Chinese cabbage Brassica pekinensis against the peach aphid Myzus persicae. In this study, we found that treatment of Chinese cabbage with ß-ocimene inhibited the growth of M. persicae in terms of weight gain and reproduction. Moreover, ß-ocimene treatment negatively influenced the feeding behavior of M. persicae by shortening the total feeding period and phloem ingestion and increasing the frequency of stylet puncture. When given a choice, winged aphids preferred to settle on healthy Chinese cabbage compared with ß-ocimene-treated plants. In addition, performance of the parasitoid Aphidius gifuensis in terms of Y-tube olfaction and landings was better on ß-ocimene-treated Chinese cabbage than on healthy plants. Furthermore, ß-ocimene significantly increased the expression levels of salicylic acid and jasmonic acid marker genes and the accumulation of glucosinolates. Surprisingly, the transcriptional levels of detoxifying enzymes (CYP6CY3, CYP4, and GST) in aphids reared on ß-ocimene-treated Chinese cabbage were significantly higher than those of aphids reared on healthy plants. In summary, our results indicated that ß-ocimene can activate the defense response of Chinese cabbage against M. persicae, and that M. persicae can also adjust its detoxifying enzymes machinery to counter the host plant defense reaction.

Chemical composition, antimicrobial and antioxidant activities of Echinophora platyloba essential oil / Composición química y actividades antimicrobianas y antioxidantes del aceite esencial de Echinophora platyloba

Objetivo: Echinophora platyloba se utiliza tradicionalmente como agente antimicrobiano con el fin de preservar los productos caseros de deterioro. El objetivo de este estudio fue evaluar la composición química del aceite esencial, actividad antimicrobiana y antioxidante del aceite esencial de E. platyloba de la provincia de Shahr-e-Kord. Materiales y métodos: la composición química del aceite esencial de E. platyloba mediante técnicas de GC-MS y GC y evaluar su efecto antibacteriano frente a Staphylococcus aureus, Salmonella enterica y Helicobacter pylori por ensayos de difusión en disco y de micro dilución en caldo. La actividad antioxidante del aceite esencial de E. platyloba se evaluó por los radicales ABST. β-ocimeno y α-cariofileno eran los componentes principales del aceite esencial de E. platyloba. El aceite esencial mostró la actividad antibacteriana prometido contra S. aureus, seguido de S. enterica y H. pylori. El aceite esencial presenta actividad antioxidante del aceite esencial mostró la IC50 de 0. 32 mg/ml que fue mayor que la IC50 de ácido ascórbico (0.20 mg/ml). En conclusión : Aceite esencial de E. platyloba ha se recomienda ß-ocimeno y quimiotipo α-cariofileno y su uso como agentes antioxidantes y antibacterianas como sus usos tradicionales.

Objective: Echinophora platyloba is traditionally used as antimicrobial agent in order to preserve the home-made products from deterioration. The aim of this study was to evaluate chemical composition of essential oil, antimicrobial and antioxidant activity of E. platyloba essential oil from Shahr-E-Kord city, Chaharmahal and Bakhtiari Province. Materials and methods: chemical composition of E. platyloba essential oil by GC and GC-MS techniques and evaluate its antibacterial effect against Staphylococcus aureus, Salmonella enterica and Helicobacter pylori by disc diffusion and micro broth dilution assays. The antioxidant activity of E. platyloba essential oil was evaluated by ABST radicals. β-ocimene and α-caryophyllene were the main components of E. platyloba essential oil. The essential oil showed the promised antibacterial activity against S. aureus, followed by S. enterica and H. pylori. The antioxidant activity evaluation of essential oil showed the IC50 of 0.32 mg/ml that was higher than the IC50 of ascorbic acid (0.20 mg/ml). In conclusion , E. platyloba essential oil has β-ocimene and α-caryophyllene chemotype and its use as antioxidant and antibacterial agents is recommended as its traditional uses.

Genetic Analysis of Natural Variation in Antirrhinum Scent Profiles Identifies BENZOIC ACID CARBOXYMETHYL TRANSFERASE As the Major Locus Controlling Methyl Benzoate Synthesis.

The Antirrhinum genus has a considerable complexity in the scent profiles produced by different species. We have analyzed the genetic differences between A. majus and A. linkianum, two species divergent in the emission of methyl benzoate, methyl cinnamate, acetophenone, and ocimene. The genetic analysis showed that all compounds segregated in a Mendelian fashion attributable to one or two loci with simple or epistatic interactions. Several lines lacked methyl benzoate, a major Volatile Organic Compound emitted by A. majus but missing in A. linkianum. Using a candidate gene approach, we found that the BENZOIC ACID CARBOXYMETHYL TRANSFERASE from A. linkianum appeared to be a null allele as we could not detect mRNA expression. The coding region did not show significant differences that could explain the loss of expression. The intron-exon boundaries was also conserved indicating that there is no alternative splicing in A. linkianum as compared to A. majus. However, it showed multiple polymorphisms in the 5' promoter region including two insertions, one harboring an IDLE MITE transposon with additional sequences with high homology to the PLENA locus and a second one with somewhat lower homology to the regulatory region of the VENOSA locus. It also had a 778 bp deletion as compared to the A. majus BAMT promoter region. Our results show that the differences in scent emission between A. majus and A. linkianum may be traced back to single genes involved in discrete biosynthetic reactions such as benzoic acid methylation. Thus, natural variation of this complex trait maybe the result of combinations of wild type, and loss of function alleles in different genes involved in discrete VOCs biosynthesis. Furthermore, the presence of active transposable elements in the genus may account for rapid evolution and instability, raising the possibility of adaptation to local pollinators.