Boraginaceae Species Research Articles

Restricted‐Range Boraginaceae Species Constitute Potential Sources of Valuable Fatty Acids

AbstractThe aim of this work was to establish the richness in γ‐linolenic acid (GLA, 18:3n‐6) and stearidonic acid (SDA, 18:4n‐3) of the seed oil of several restricted‐range Boraginaceae species, in a search for new valuable oils as advantageous alternatives to the commercially available sources of both polyunsaturated fatty acids. To this end, seeds of selected Boraginaceae species were collected and analyzed. The highest GLA contents (% total fatty acids) were found in the seed oils of Symphytum caucasicum M.Bieb. (22.9 %), Anchusa undulata subsp. undulata (Ten.) Cout. (22.0 %), Anchusa puechii Valdés (20.0 %), Glandora nitida Thomas (19.2 %), Echium pininana Webb & Berth. (17.1 %) and Pentaglottis sempervirens (L.) L. H. Bailey (17.0 %). With regard to SDA, the highest percentage was found in the seed oil of Echium cantabricum (M. Laínz) Fdez. Casas & M. Laínz (14.7 %), followed by Lappula patula (Lehm.) Asch ex Gürke (13.6 %). It is noticeable that several GLA‐enriched species stand under a great threat of extinction, thus revealing the importance of the preservation of the natural ecosystems for endangered species.

A phylogenetic analysis of morphological and molecular characters of Boraginaceae: evolutionary relationships, taxonomy, and patterns of character evolution.

The angiosperm family Boraginaceae includes ca. 1600 species distributed among ca. 110 genera. Some floral features are constant within the family, but many vegetative, floral, pollen, and nutlet traits vary. Utilizing 224 species of Boraginaceae and related taxa, five matrices were constructed with various combinations of morphological characters, three chloroplast DNA regions, and one nuclear ribosomal DNA region. Phylogenetic analyses were conducted for these matrices, and patterns of character evolution were examined. Boraginaceae is resolved as monophyletic, with Wellstedia as its sister. Codon is sister to Boraginaceae+Wellstedia. Although most of the investigated morphological characters have a low consistency index, particular character states are synapomorphies for large clades in each of the tribes of the family. In Boraginaceae, the breeding system heterostyly evolved at least 12 times, which is the largest number of origins resolved in any family; therefore Boraginaceae can serve as a model for the evolution and development of heterostyly. Nutlet ornamentation is most diverse in Cynoglosseae and Trichodesmeae, while pollen and floral features are most variable in Boragineae and Lithospermeae. Phylogenetic relationships and patterns of character evolution identified in the present study set the stage for future work creating an updated taxonomic system of Boraginaceae.

New seed oils of Boraginaceae rich in stearidonic and gamma-linolenic acids from the Maghreb region

The main goal of this work was to find new seed oils rich in γ-linolenic acid (GLA, 18:3n-6) and stearidonic acid (SDA, 18:4n-3) as alternatives to the commercially available oils, such as those from fish and borage. To this end, seeds of selected Boraginaceae species growing wild in the North Africa region (Maghreb) were collected and analyzed. The key finding is that two Echium species have been identified as new sources of valuables fatty acids (FAs): Echium sericeum and Echium parviflorum, showing 18.3% GLA and 17.3% SDA on total FAs respectively. Both of them could be cultivated to provide functional oils, polyunsaturated FA (PUFA) concentrates or pure FAs, among other uses. It is also noteworthy that the seed oils of Borago officinalis and Echium plantagineum gathered in different locations showed different GLA and SDA percentages, respectively.

Essential oil composition of Anchusa italica from Iran

The family Boraginaceae consists of about 100 genera and 2000 species distributed in the temperate zone, especially in the Mediterranean and tropical regions [1]. Five species of the genus Anchusa (Boraginaceae) are found in Iran [2]. The Borage plants are one of the most important folk medicine plants in Iran. They are of value in treating common cold symptoms, depression and stress, and are anti-inflammatory and antibacterial [3, 4]. Anchusa italica Retz. plants, named “Gav zaban” in Persian, grows in the mountains of some provinces of Iran. The people of that area use the leaves and flowers of A. italica specifically to treat flu symptoms. The anti-influenza activity of alcoholic and aqueous extracts of A. italica has been investigated in a previous report [3]. The findings of this study indicated that A. italica exhibited activity against the influenza virus. In other research, a qualitative phytochemical analysis of 55 Iranian plants, especially A. italica, has been performed for the presence of alkaloids, tannins, saponins, and flavonoids [4]. The results showed the existence of alkaloids, saponins, and flavonoids. The extract of A. italica plant has also been used as an emollient. Likewise, poly[3-(3,4-dihydroxyphenyl)glyceric acid] from A. italica roots has been reported [5]. The lipids and lipophilic components of seeds of three Boraginaceae species, including A. italica, have also been studied [6]. The seed oil of A. italica contains significant quantities of -6 and -3-octadecatrionic acids. In this study, the essential oil composition of A. italica from Iran was investigated for the first time. The essential oil constituents from the aerial part of A. italica are listed in Table 1, in which the percentages and Kovats indices of the components are given. As shown in Table 1, the oil of the aerial parts of A. italica consist of 34 compounds, representing 91.4% of the oil. The main constituents of the oil were diisobutyl phthalate (14.6%), dibutyl phthalate (9.0%), and hexahydrofarnesylacetone (8.1%). The aerial parts of A. italica were collected during the flowering stage in the Alamut region, Province of Qazvin, Iran, in May 2010. Voucher specimens (No. 88274) have been deposited at the Herbarium of the Research Institute of Forests and Rangelands (TARI), Tehran, Iran. The aerial parts (150.0 g) of A. italica were subjected to water distillation using a Clevenger-type apparatus and extracted by diethyl ether for 3 h. After decanting, drying over anhydrous sodium sulfate, and evaporating the solvent, we recovered the corresponding yellowish colored oil in a yield of 0.02% w/w. GC-FID analysis was performed on a Shimadzu 15A gas chromatograph with a split/splitless (ratio 1:30), injector (250 C) and a flame ionization detector (250 C); nitrogen was used as the carrier gas (1 mL/min). A DB-5 capillary column (50 m 0.2 mm, film thickness 0.32 m) was used. The column was kept at 60 C for 3 min, then heated to 220 C with a 5 C/min rate and kept constant at 220 C for 5 min. Relative percentage amounts were calculated from the peak area using a Shimadzu C-R4A chromatopac data processor without the use of correction factors. GC-MS analysis was performed using a Hewlett-Packard 5973 with an HP-5MS column (30 m 0.25 mm, film thickness 0.25 m). The column temperature was kept at 60 C for 3 min and programmed to 220 C at a rate of 5 C/min and kept constant at 220 C for 5 min. The flow rate of helium as carrier gas was 1 mL/min. MS were taken at 70 eV, mass range 30 to 350 amu, and scan time 2 scans/sec.

Pectocarya anisocarpa, a new species of Boraginaceae, and a revised key for the genus in western North America

Pectocarya is a genus of 13 currently accepted taxa in Boraginaceae, subtribe Cryptanthinae. The genus has an amphitropical distribution in the New World, with eight taxa in western North America, seven in South America, and two occurring in both continents. Members of Pectocarya are distinctive in having a bilaterally or radially symmetrical fruit of four strongly divergent nutlets. Taxa within the genus are distinguished largely by the morphology of the nutlets, including shape, size, margin, posture, and variability both within a fruit and/or within a plant. Pectocarya "anisocarpa" (Boraginaceae) was previously named and described, but not validly published. Along with taxon-specific nutlet ornamentation, P. “anisocarpa” has fruits within a plant that are monomorphic and nutlets within a fruit that are heteromorphic. It is common throughout its range, which in the United States includes the southern half of California, Arizona, and southern Utah to the east. The species’ southernmost extent is in northwestern Baja California, Mexico. It is often encountered in mixed populations with congeners, and has been confused historically with P. penicillata and P. linearis var. ferocula in California and Baja California and P. heterocarpa in Arizona and Utah. Here we validly publish the name P. anisocarpa, as well as provide illustrations, an image of the holotype specimen, and a range map of the new taxon. A revised taxonomic key and photographs of the fruits of the North American Pectocarya taxa are also included.

Effect of l-phenylalanine on PAL activity and production of naphthoquinone pigments in suspension cultures of Arnebia euchroma (Royle) Johnst

The effects of l-phenylalanine (PHE) on cell growth and production of shikonin and its derivatives, acetylshikonin (ACS) and isobutyrylshikonin (IBS), in suspension cultures of Arnebia euchroma were examined. Supplementing media using PHE have been successfully utilized to enhance shikonin production in cell cultures of other species of Boraginaceae. l-Phenylalanine, the key compound in the phenylpropanoid pathway, is converted by phenylalanine ammonia lyase (PAL) to trans-cinnamic acid, which is the precursor of p-hydroxybenzoic acid (PHB). Coupling of PHB and geranyl pyrophosphate (derived from mevalonate pathway) by p-hydroxybenzoate-m-geranyltransferase leads later to biosynthesis of shikonins. The addition of 0.01 or 0.1 mM PHE to the culture medium stimulated cell proliferation, where the highest observed increase in fresh cell biomass (measured as a ratio of final weight to initial weight) was 12-fold, in contrast to an eightfold increase in control cultures. Whereas, growth media supplemented with 1 mM PHE markedly reduced the rate of cell growth (to only twofold). Precursor feeding had detrimental effects on both ACS and IBS production in all PHE-supplemented media. The highest total content (intracellular + extracellular) of the investigated red pigments (9.5 mg per flask) was detected in the control culture without PHE. ACS was the major component of the naphthoquinone fraction determined in cells and post-culture media. Shikonin itself was found only in the post-culture media from cultures supplemented with 0.01 or 0.1 mM PHE. Increases in PAL activity corresponded well with the accumulation of investigated naphthoquinones in control culture. However, peak PAL activity did not directly correlate with maximum production of shikonin derivatives. Cytotoxicity of extracts, prepared from the cells cultivated in the presence of PHE or in control cultures, was tested on three cancer cell lines: HL-60, HeLa, and MCF-7. The extracts prepared from the untreated control cultures proved to be the most potent against the examined cancer cell lines. The mean inhibitory concentration values were 0.3, 13, and 8 μg ml−1 for the HL-60, HeLa, and MCF-7 cells, respectively.

Searching for health beneficial n‐3 and n‐6 fatty acids in plant seeds

Various plant seeds have received little attention in fatty acid research. Seeds from 30 species mainly of Boraginaceae and Primulaceae were analysed in order to identify potential new sources of the n-3 PUFA α-linolenic acid (ALA) and stearidonic acid (SDA) and of the n-6 PUFA γ-linolenic acid (GLA). The fatty acid distribution differed enormously between genera of the same family. Echium species (Boraginaceae) contained the highest amount of total n-3 PUFA (47.1%), predominantly ALA (36.6%) and SDA (10.5%) combined with high GLA (10.2%). Further species of Boraginaceae rich in both SDA and GLA were Omphalodes linifolia (8.4, 17.2%, resp.), Cerinthe minor (7.5, 9.9%, resp.) and Buglossoides purpureocaerulea (6.1, 16.6%, resp.). Alkanna species belonging to Boraginaceae had comparable amounts of ALA (37.3%) and GLA (11.4%) like Echium but lower SDA contents (3.7%). Different genera of Primulaceae (Dodecatheon and Primula) had varying ALA (14.8, 28.8%, resp.) and GLA portions (4.1, 1.5%, resp.), but similar amounts of SDA (4.9, 4.5%, resp.). Cannabis sativa cultivars (Cannabaceae) were rich in linoleic acid (57.1%), but poor in SDA and GLA (0.8, 2.7%, resp.). In conclusion, several of the presented plant seeds contain considerable amounts of n-3 PUFA and GLA, which could be relevant for nutritional purposes due to their biological function as precursors for eicosanoid synthesis.Practical applicationsN-3 PUFA are important for human health and nutrition. Unfortunately, due to the increasing world population, overfishing of the seas and generally low amounts of n-3 PUFA in major oil crops, there is a demand for new sources of n-3 PUFA. One approach involves searching for potential vegetable sources of n-3 PUFA; especially those rich in ALA and SDA. The conversion of ALA to SDA in humans is dependent on the rate-limiting Δ6-desaturation. Plant-derived SDA is therefore a promising precursor regarding the endogenous synthesis of n-3 long-chain PUFA in humans. The present study shows that, in addition to seed oil of Echium, other species of Boraginaceae (Cerinthe, Omphalodes, Lithospermum, Buglossoides) and Primulaceae (Dodecatheon, Primula), generally high in n-3 PUFA (30–50%), contain considerable amounts of SDA (5–10%). Therefore, these seed oils could be important for nutrition.

Echium acanthocarpum hairy root cultures, a suitable system for polyunsaturated fatty acid studies and production

BackgroundThe therapeutic and health promoting role of highly unsaturated fatty acids (HUFAs) from fish, i.e. eicosapentaenoic acid (EPA, 20:5n-3) and docosahexaenoic acid (DHA, 22:6n-3) are well known. These same benefits may however be shared by some of their precursors, the polyunsaturated fatty acids (PUFAs), such as stearidonic acid (SDA, 18:4 n-3). In order to obtain alternative sources for the large-scale production of PUFAs, new searches are being conducted focusing on higher plants oils which can contain these n-3 and n-6 C18 precursors, i.e. SDA and GLA (18:3n-6, γ-linolenic acid).ResultsThe establishment of the novel Echium acanthocarpum hairy root cultures represents a powerful tool in order to research the accumulation and metabolism of fatty acids (FAs) in a plant particularly rich in GLA and SDA. Furthermore, this study constitutes the first example of a Boraginaceae species hairy root induction and establishment for FA studies and production. The dominant PUFAs, 18:2n-6 (LA, linoleic acid) and 18:3n-6 (GLA), accounted for about 50% of total FAs obtained, while the n-3 PUFAs, 18:3n-3 (ALA, α-linolenic acid) and 18:4n-3 (SDA), represented approximately 5% of the total. Production of FAs did not parallel hairy root growth, and the optimal productivity was always associated with the highest biomass density during the culture period. Assuming a compromise between FA production and hairy root biomass, it was determined that sampling times 4 and 5 gave the most useful FA yields. Total lipid amounts were in general comparable between the different hairy root lines (29.75 and 60.95 mg/g DW), with the major lipid classes being triacylglycerols. The FAs were chiefly stored in the hairy roots with very minute amounts being released into the liquid nutrient medium.ConclusionsThe novel results presented here show the utility and high potential of E. acanthocarpum hairy roots. They are capable of biosynthesizing and accumulating a large range of polyunsaturated FAs, including the target GLA and SDA fatty acids in appreciable quantities.

Lipids and lipophilic components from seeds of three Boraginaceae species

In continuation of the search for lipid-bearers containing -3 and -6 acids, we studied lipids from seeds of Lindelofia macrostyla, Trichodesma incanum, and Anchusa italica (Boraginaceae) collected in Tashkent Oblast. These plants have attracted interest because many of their seed oils contain very active essential -3 and -6 fatty acids. Total neutral lipids were isolated by extraction with benzine (bp 72–85°C) in a Soxhlet apparatus. Bound lipids were extracted from the remaining pulp according to Folch [1]. Because a part of the NL was found in the bound fraction, they were separated from the latter by column chromatography with elution by CHCl3; GL, by acetone; PL, by MeOH. The NL obtained from the bound fraction were combined with those previously extracted. The yield of NL from oil of L. macrostyla was 13.9%; from T. incanum, 10.8; from A. italica, 22.6. The maximum content of NL was found in seed oil of A. italica. GL and PL dominated oil from T. incanum seeds. NL from the three studied species contained hydrocarbons, triacylglycerides, esters of cyclic alcohols with fatty acids, free fatty acids, sterols, and diacylglycerides according to TLC on silica gel using hexane:Et2O (4:1 and 1:1). GL were analyzed by TLC on silica gel using CHCl3:(CH3)2CO:MeOH:HOAc:H2O (65:20:10:10:3). It was found that GL from L. macrostyla seed oil consisted of sterolglycosides, esters of sterolglycosides, cerebrosides, esters of cerebrosides, digalactosyldiglycerides, and monogalactosyldiglycerides. GL from seed oil of T. incanum and A. italica had the same composition as that from L. macrostyla but did not contain cerebrosides and their esters. Two-dimensional chromatography was used to separate PL. The first direction used CHCl3:MeOH:NH4OH (25%) (65:25:5); the second, CHCl3:MeOH:HOAc:H2O (14:5:1:1). Phhosphatidylcholines (PC), phosphatidylinositols (PI), phosphatidylethanolamines (PE), phosphatidic acids (PA), and N-acyl-lyso-phosphatidylethanolamines were observed in PL of L. macrostyla seed oil. PL from T. incanum and A. italica had the same composition with the exception of N-acyl-lyso-phosphatidylethanolamines. The emulsions obtained after alkaline hydrolysis of NL, GL, and PL were decomposed by H2SO4 (10%). The released fatty acids were extracted with Et2O, methylated by diazomethane, and analyzed by GC on a Chrom-2 instrument [2]. Table 1 lists the composition of the fatty acids. It can be seen that the qualitative fatty-acid compositions of L. macrostyla and T. incanum seed oils were identical and consisted of 13 components whereas fatty acids from A. italica seed oil numbered 9 components. The principal saturated acid was 16:0; unsaturated, 18:1 and 18:2. Furthermore, seed oil of T. incanum and A. italica also contained significant quantities of -6 and -3-octadecatrienoic acids.

The phylogenetic significance of vestured pits in Boraginaceae

AbstractThe bordered pit structure in tracheary elements of 105 Boraginaceae species is studied using scanning electron microscopy to examine the systematic distribution of vestured pits. Forty–three species out of 16 genera show a uniform presence of this feature throughout their secondary xylem. Most vestures are small, unbranched and associated with the outer pit aperture of bordered intervessel pits. The feature is likely to have originated independently in the distantly related subfamilies Boraginoideae (tribe Lithospermeae) and Ehretioideae. The distribution of vestures in Ehretia agrees with recent molecular phylogenies: (1) species with vestured pits characterise the Ehretia I group (incl. Rotula), and (2) species with non–vestured pits belong to the Ehretia II group (incl. Carmona). The occurrence of vestured pits in Hydrolea provides additional support for excluding this genus from Hydrophylloideae, since Hydrolea is the only species of this subfamily with vestured pits. Functional advantages of vestured pits promoting parallel evolution of this conservative feature are suggested. The hydraulic benefits of poorly developed vestures remain underinvestigated.

Determination of the phenolic content and antioxidant potential of crude extracts and isolated compounds from leaves of Cordia multispicata and Tournefortia bicolor

In this work, the total phenolic content and antioxidant activity of extracts and four flavonoids isolated from leaves of two Boraginaceae species (Cordia multispicata Cham. and Tournefortia bicolor Sw.) were evaluated using Folin-Ciocalteu reagent, DPPH free radical scavenging and inhibition of peroxidation of linoleic acid by FTC method. For comparison, ascorbic acid, α-tocopherol and BHT were used. In general, extracts from T. bicolor (68.8 ± 0.001 to > 1000 mg/g) showed higher phenolic content than C. multispicata (66.1 ± 0.009 to 231 ± 0.07 mg/g), and also scavenged radicals (IC50 12.8 ± 2.5 to 437 ± 3.5 mg/L) and inhibited lipid peroxide formation (IC50 51.2 ± 2.29 to 89 ± 0.59 mg/L). For these extracts a good correlation between the phenolic content and antioxidant activity was observed, suggesting that T. bicolor is richer in phenolic compounds and that it could serve as a new source of natural antioxidants or nutraceuticals with potential applications. Chromatographic procedures monitored by antioxidant assays afforded seven compounds, which were identified by spectral analyses (IR, MS and 1D and 2D NMR) and comparison with reported data as being trans-phytol (1), taraxerol (2), 3,7,4’-trimethoxyflavone (3), 5,3’-dihydroxy-3,7,4’-trimethoxyflavone (4), quercetin (5), tiliroside (6), and rutin (7). Compounds (4-7) were also evaluated and were effective as DPPH quenching (IC50 7.7 ± 3.6 to 79.3 ± 3.4 mg/L) and as inhibition of lipid peroxidation (IC50 80.1 ± 0.98 to 88.7 ± 3.62 mg/L). This is the first report on the total phenolic content, radical-scavenging and antioxidant activities of these species.

Lithospermic acid B is more responsive to silver ions (Ag+) than rosmarinic acid in Salvia miltiorrhiza hairy root cultures

LAB (lithospermic acid B) is a dimer of RA (rosmarinic acid) and has been suggested to be derived from RA, but the detailed biosynthesis process has not yet been identified. The accumulation of RA has been intensively investigated in the plant species of Boraginaceae and Lamiaceae. In the present study, we report that silver ions (Ag+; 15 microM), an abiotic elicitor, did not stimulate RA accumulation but dramatically enhanced LAB from approx. 5.4% to 18.8% of dry weight in Salvia miltiorrhiza hairy root cultures, and the rise in LAB was found to be coincident with the decline of RA content at each time point after treatment. Meanwhile, a profiling analysis of genes and metabolites (intermediates) involved in the RA synthesis pathway was performed; the result indicated that several gene transcripts and metabolite accumulations show temporal changes in abundance consistent with LAB production. Thus a potential (putative) biosynthetic route from RA to LAB was presumed, which was suggested to be significantly activated by Ag+ in S. miltiorrhiza hairy root cultures. Further intermediate monitoring and compound feeding experiments were performed to rank the strength of this hypothesis. Our study, for the first time, provides evidence that RA is a precursor leading to LAB synthesis.

Evolutionary transitions of style polymorphisms in Lithodora (Boraginaceae)

Floral polymorphisms provide suitable model systems to test hypotheses concerning the evolution of outbreeding in plants. Although heterostyly has evolved in more than 28 angiosperm families, the evolutionary pathways involving related floral conditions have not yet been fully resolved. In this study, the reconstruction of ancestral states of style polymorphism, with both parsimony and maximum likelihood methods, was carried out for Boraginaceae species in the tribe Lithospermeae, particularly in the genus Lithodora sensu lato, where species present a wide variety of stylar conditions. Detailed floral morphometric analysis confirm different types of style polymorphism within Lithodora. They also reveal a novel style polymorphism (relaxed style dimorphism) in which anther height is variable within a flower (each anther being at a different height), which contrasts to regular distyly (constant anther height within flowers). Style monomorphism is likely to be the ancestral condition in Lithospermeae where the evolution of distyly has occurred several times. Style dimorphism is probably ancestral to distyly, as predicted by certain evolutionary models proposed for heterostyly. However, a reversion from distyly to style dimorphism also appears to occur in this tribe. This is the first documented occurrence of such a transition. This secondary style dimorphism is of the relaxed type and demonstrates the labile nature of floral polymorphisms, which are not necessarily a transition towards heterostyly. We discuss the selective forces involved in the evolution, maintainance and loss of style polymorphisms.

Pollen deposition rates and the functioning of distyly in the perennial Pulmonaria officinalis (Boraginaceae)

Distyly typically involves reciprocal polymorphism in stamen and style lengths and a set of associated polymorphisms of pollen and stigma characters. This flower polymorphism has been regarded as a strategy to reduce the likelihood of self- and intramorph pollination and to promote legitimate intermorph pollination. Although most distylous plants are characterized by a physiological self- and intramorph-incompatibility system, previous work on Pulmonaria affinis and other distylous Boraginaceae species have shown that self-incompatibility was not strict. In this study, we examined pollen deposition rates and the functioning of the distylous breeding system in Pulmonaria officinalis. Flowers exhibited reciprocal herkogamy and several other ancillary features of heterostyly. Controlled pollinations clearly showed weak self-incompatibility, with the LS-morph showing higher rates of seed set following self-, intramorph- and intermorph-pollinations than the SS-individuals. On the other hand, SS-pollen showed higher germination rates and pollen tube growth than that of LS-pollen. Under natural conditions, both the proficiency of legitimate pollen transfer and the proportion of deposited pollen were asymmetrical. Although SS-pollen grains showed a higher proficiency for legitimate transfer than that of LS-pollen on a per pollen basis, the ratio of legitimate to illegitimate deposited pollen on stigmas of SS-flowers markedly exceeded that of LS-flowers. The latter was explained by the higher pollen production rates of LS-individuals compared to SS-individuals. High levels of illegitimate pollen deposition on LS-stigmas, on the other hand, were attributed to the limited proboscis length of the principal pollinators and the occurrence of floral hairs at the corolla entrance of LS-flowers. This study thus indicates asymmetrical pollen transport patterns and morph-specific differences in the strength of the incompatibility system; two aspects that may affect female reproductive success and morph ratio variation under pollen limited conditions.

Cloning and molecular characterisation of a Δ8-sphingolipid-desaturase from Nicotiana tabacum closely related to Δ6-acyl-desaturases

Investigation on the absence of Delta(6)-desaturase activity in Nicotiana tabacum has led to the cloning of a new desaturase gene from this organism (NTDXDES) that exhibited unexpected biochemical activity. Cladistic analysis shows clustering of NTDXDES together with functional Delta(6)-acyl-desaturases of near Solanales plants, such as Borago and Echium. This group lies apart from that of previously characterised Delta(8)-sphingolipid-desaturases, which also includes two putative tobacco members identified in this study. Moreover, strong expression of NTDXDES is found in leaves, flowers, fruits and developing seeds of tobacco plants that is highly dependent on the development phase, with transcriptional activity being higher at stages of active tissue growth. This pattern is similar to that showed by Delta(6)-acyl-desaturases characterised in Boraginaceae species. However, functional assays using a yeast expression system revealed that the protein encoded by NTDXDES lacks Delta(6)-desaturase activity, but instead it is able to desaturate sphingolipid substrates by introducing a double bond on the Delta(8)-position. These data indicate that NTDXDES represent a novel desaturase gene placed in a different evolutionary lineage to that of previously characterised Delta(8)-desaturases.

Tissue distribution, core biosynthesis and diversification of pyrrolizidine alkaloids of the lycopsamine type in three Boraginaceae species

Three species of the Boraginaceae were studied: greenhouse-grown plants of Heliotropium indicum and Agrobacterium rhizogenes transformed roots cultures (hairy roots) of Cynoglossum officinale and Symphytum officinale. The species-specific pyrrolizidine alkaloid (PA) profiles of the three systems were established by GC–MS. All PAs are genuinely present as N-oxides. In H. indicum the tissue-specific PA distribution revealed the presence of PAs in all tissues with the highest levels in the inflorescences which in a flowering plant may account for more than 70% of total plant alkaloid. The sites of PA biosynthesis vary among species. In H. indicum PAs are synthesized in the shoot but not roots whereas they are only made in shoots for C. officinale and in roots of S. officinale. Classical tracer studies with radioactively labelled precursor amines (e.g., putrescine, spermidine and homospermidine) and various necine bases (trachelanthamidine, supinidine, retronecine, heliotridine) and potential ester alkaloid intermediates (e.g., trachelanthamine, supinine) were performed to evaluate the biosynthetic sequences. It was relevant to perform these comparative studies since the key enzyme of the core pathway, homospermidine synthase, evolved independently in the Boraginaceae and, for instance, in the Asteraceae [Reimann, A., Nurhayati, N., Backenkohler, A., Ober, D., 2004. Repeated evolution of the pyrrolizidine alkaloid-mediated defense system in separate angiosperm lineages. Plant Cell 16, 2772–2784.]. These studies showed that the core pathway for the formation of trachelanthamidine from putrescine and spermidine via homospermidine is common to the pathway in Senecio ssp. (Asteraceae). In both pathways homospermidine is further processed by a β-hydroxyethylhydrazine sensitive diamine oxidase. Further steps of PA biosynthesis starting with trachelanthamidine as common precursor occur in two successive stages. Firstly, the necine bases are structurally modified and either before or after this modification are converted into their O 9-esters by esterification with one of the stereoisomers of 2,3-dihydroxy-2-isopropylbutyric acid, the unique necic acid of PAs of the lycopsamine type. Secondly, the necine O 9-esters may be further diversified by O 7- and/or O 3′-acylation.

TWO NEW SPECIES OF BORAGINACEAE (TRIBE CYNOGLOSSEAE) FROM TURKEY

Two new species of Boraginaceae are described from Turkey: Paracaryum hedgei and Rindera dumanii. Their affinities and phytogeography are discussed. Both species are illustrated and their distributions mapped. Rindera sect. Bilegnum is raised to subgeneric rank as Rindera subgen. Bilegnum.

Acquired and partially de novo synthesized pyrrolizidine alkaloids in two polyphagous arctiids and the alkaloid profiles of their larval food-plants.

The profiles of pyrrolizidine alkaloids (PAs) in the two highly polyphagous arctiids Estigmene acrea and Grammia geneura and their potential PA sources in southeastern Arizona were compiled. One of four species of Boraginaceae, Plagiobothrys arizonicus, contained PAs; this is the first PA record for this plant species. The principle PA sources are Senecio longilobus (Asteraceae) and Crotalaria pumila (Fabaceae). The known PA pattern of S. longilobus was extended; the species was found to contain six closely related PAs of the senecionine type. Three novel PAs of the monocrotaline type, named pumilines A-C, were isolated and characterized from C. pumila, a species not studied before. The pumilines are the major PAs in the seeds, while in the vegetative organs they are accompanied by the simple necine derivatives supinidine and as the dominant compound subulacine (1beta,2beta-epoxytrachelanthamidine). In both plant species, the PAs are stored as N-oxides, except C. pumila seeds, which accumulate the free bases. Great variation in PA composition was observed between local populations of C. pumila. The PA profiles were established for larvae and adults of E. acrea that as larvae had fed on an artificial diet supplemented with crotalaria-powder and of G. geneura fed with S. longilobus. In both experiments, the larvae had a free choice between the respective PA source and diet or food plants free of PAs. The profiles compiled for the two species reflect the alkaloid profiles of their PA sources with one exception, subulacine could never be detected in E. acrea. Besides acquired PAs, insect PAs synthesized from acquired necine bases and necic acids of insect origin were detected in the two arctiid species. These insect PAs that do not occur in the larval food sources accounted for some 40-70% (E. acrea) and 17-37% (G. geneura) of total PAs extracted from the insects. A number of novel insect PAs were identified. Plant-acquired and insect PAs were found to accumulate as N-oxides. The results are discussed in relation to specific biochemical, electrophysiological, and behavioral mechanisms involved in PA sequestration by arctiids.

CHARACTERISATION AND EVALUATION OF SPECIES OF THE BORAGINACEAE FAMILY AS SOURCE OF GAMMA-LINOLENIC ACID FOR MEDITERRANEAN CONDITIONS

Trabajo presentado en XXVI International Horticultural Congress: The Future for Medicinal and Aromatic Plants, celebrado en Toronto (Canada) el 03 de enero de 2004

γ-Linolenic Acid Enrichment from Borago officinalis and Echium fastuosum Seed Oils and Fatty Acids by Low Temperature Crystallization

Solvent winterization of seed oil and free fatty acids (FFAs) was employed to obtain γ-linolenic acid (GLA; 18:3ω6) concentrates from seed oils of two Boraginaceae species, Echium fastuosum and Borago officinalis. Different solutions of seed oils and FFAs from these two oils at 10%, 20% and 40% (w/w) were crystallized at 4°C, −24°C and −70°C, respectively, using hexane, acetone, diethyl ether, isobutanol and ethanol as solvents. Best results were obtained for B. officinalis FFAs in hexane, reaching a maximum GLA concentration of 58.8% in the liquid fraction (LF). In E. fastuosum, the highest GLA concentration (39.9%) was also achieved with FFAs in hexane.