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Peltate Glandular Trichomes of Leonotis leonurus Leaves: Ultrastructure and Histochemical Characterization of Secretions

The histochemical characterization of the oleoresin produced by peltate trichomes of Leonotis leonurus revealed terpenoids and flavonoid aglycones. At the onset of secretion, glandular cells were more densely cytoplasmic than the other trichome cells. The lateral stalk wall underwent cutinization, and the cuticle over the glandular cells became thicker. During the active secretory stage, the most striking ultrastructural features of glandular cells were the hypertrophy of the leucoplastidome and the extensive proliferation of ER. The high development of these two cellular compartments was related with the biosynthesis and transport of the secretory product. A granulocrine secretion mechanism may operate alone or concurrently with an eccrine process. In the glandular head, the loosening of the outer wall fibrillar matrix, and the accumulation of secretion in the newly formed interfibrillar spaces led to a secretory cavity development by detachment of the cuticle and the outermost pectic layer of cell wall. Successive accumulation of secretion in the secretory cavity conferred the spherical shape, characteristic of a peltate gland, to the trichome. The interfibrillar spaces, initially small and elongated, enlarged, became roundish, and appeared as vesicles delimited by an electron-dense layer. These vesicle-like structures are interpreted as lipophilic secretion globules in a hydrophilic phase. It is suggested that pectic polysaccharide wall constituents may be the main components of this hydrophilic matrix. The dense layer around the vesicles may represent an interphase between lipophilic and hydrophilic compounds. The secretion seemed to remain trapped in the secretory cavity, since no cuticular disruption was observed.

Glandular Trichomes of Artemisia campestris (ssp. Maritima): Ontogeny and Histochemistry of the Secretory Product

The glandular trichomes of Artemisia campestris (ssp. maritima) originate from a single protodermal cell and develop through an anticlinal and several periclinal divisions. These multicellular and biseriate trichomes have a secretory head with three pairs of cells, two stalk cells, and two basal cells. The number of glandular trichomes is established early during leaf differentiation; thus, gland density decreases with leaf development. Oleoresin production begins as soon as the glandular trichomes are fully developed. The main components of the oleoresin are terpenoids (mono- and sesquiterpenes and probably steroids), fatty acids, flavonoids as free aglycones, hydroxycoumarins, and alkaloids. The oleoresin accumulates in the subcuticular space and is released over the leaf blade by rupture of the cuticle.

Plectranthus madagascariensis: Morphology of the Glandular Trichomes, Essential Oil Composition, and Its Biological Activity

Glandular and nonglandular trichomes are spread over the vegetative and reproductive organs of Plectranthus madagascariensis. Two morphologically distinct types of glandular trichomes (capitate and peltate) are described. Capitate trichomes have an ovoid unicellular head and a short stalk cell or an elongated two- to three-celled stalk slightly enlarged at the base. Peltate trichomes, which show in vivo a characteristic orange-to-reddish color, are large, flattened-to-depressed in the center, and often appear as a ring of minute gems. They are composed of a large head with eight glandular cells arranged in a single layer. An unusual kind of capitate trichomes, reported for the first time in Lamiaceae, occurs typically restricted to the calyx. These trichomes possess a two- or three-celled stalk and a long, unicellular conical, glandular head. The several types of trichomes differ in the secretion process The essential oils of P. madagascariensis, isolated by hydrodistillation and by distillation-extraction separately from flowers and from leaves collected during the flowering and the vegetative phases of the plant were analyzed by gas chromatography and gas chromatography-mass spectrometry (GC and GC-MS). The main component of the oils was a diterpene, 6,7-dehydroroyleanone, isolated as orange-to-reddish crystals, which represented 28%, 87%, and 41% of the oils from the flowers and from the leaves collected during the flowering and vegetative phases, respectively. The essential oils of P. madagascariensis showed bactericidal activity against Bacillus subtilis, Micrococcus sp., Staphylococcus aureus, and Yersinia enterocolitica, and revealed a modest antioxidant activity.

Comparative structure of the labellum in Ophrys fusca and O. lutea (Orchidaceae)

The morphology and anatomy of the labellar epidermal cells and the way in which they are arranged are described in an attempt to locate and characterize the osmophore in Ophrys fusca and O. lutea. The micromorphology of the labellum of these two species is similar. Four types of epidermal cells are present on the adaxial surface of the labellum. Long unicellular trichomes with straight tips cover the basal region of the labellum, whereas short unicellular trichomes with polygonal flattened bases form the reflective median speculum. The apical region of the labellum possesses a villous indumentum of long acuminate trichomes with bent or sinuate tips. Large smooth-walled, dome-shaped papillae occur on the margins and on the distal region of the abaxial surface of the labellum. These remarkable papillae have high polarity; the protoplasm at the apex of each cell contains several small vacuoles, while a prominent nucleus surrounded by numerous hypertrophied amyloplasts occurs at the opposite end of the cell. Positive reactions to Vogels staining test and to Sudan black B enabled us to conclude that the osmophores of both species are composed of these peculiar secretory epidermal cells and by two or three subsecretory layers of parenchyma cells.

Grasses of different C4 subtypes reveal leaf traits related to drought tolerance in their natural habitats: Changes in structure, water potential, and amino acid content

Three grasses (Poaceae) of different C(4) subtypes, Paspalum dilatatum (NADP-malic enzyme [ME]), Cynodon dactylon (NAD-ME) and Zoysia japonica (phosphoenolpyruvate carboxykinase), occur in natural habitats that differ in annual rainfall. Their leaf characteristics were studied to identify traits related to drought tolerance. Plants were grown in pots, and water deficit was gradually induced by withholding water. Leaves of Z. japonica had the greatest and P. dilatatum the lowest relative dry matter content. Transverse sections of leaves that developed during the water deficit showed little change compared to control leaves, consistent with low phenotypic plasticity. Anatomical features distinguished the three species, with xeromorphic characteristics most strongly represented in Z. japonica. The leaf relative water content (RWC) decreased with the soil water content similarly for the three grasses. However, at 80% RWC, the leaf water potential was -3.1 MPa for Z. japonica and only -1.3 MPa for P. dilatatum and C. dactylon. Soluble amino acids, especially proline, increased as RWC decreased in leaves of C. dactylon and Z. japonica. Phenylalanine, valine, leucine, and isoleucine increased more in Z. japonica than in the other two species. The results provide evidence that C. dactylon and, especially, Z. japonica have evolved leaf traits better suited to arid habitats.

Adventitious shoot mass production of hop (Humulus lupulus L.) var. Eroica in liquid medium from organogenic nodule cultures

Two efficient methods with high potential for automation are described for mass propagation of Humulus lupulus L. var. Eroica in liquid medium. Suspensions were initiated by culturing petioles directly in a Murashige and Skoog’s modified liquid medium (MS%) containing 0.8 mg:l indole-3-acetic acid (IAA) and 0.02 mg:l kinetin or by inoculating petiole-derived callus into MS% liquid medium supplemented with 0.01 mg:l IAA or indole-3-butyric acid (IBA) and 1 mg:l 6-benzylaminopurine (BAP). In the last case, transfer of calli to liquid medium proved essential for inducing morphogenesis. Organogenic nodules forming green organogenic nodular clusters (GONCs) were the basic regenerative structures producing large amounts of adventitious shoots. Both methods induced the formation of GONCs within 2‐3 months. Establishment of GONCs cultures in hormone-free liquid medium favoured nodule multiplication and continuous shoot emergence and development. However, GONCs should only be separated from the suspensions when reaching a high level of differentiation. Histological studies revealed that nodules arise through a complex process that includes four developmental stages: (1) organization centers derived from neoformed xylemic cells; (2) independent nodules displaying a consistent internal cell:tissue differentiation with at least three cell types (meristematic cells, parenchymatous cells and vascular elements) and two cell regions (epidermal and cortex:vascular); (3) ‘polycenter’ nodules; and (4) organogenic nodules.

Polymeric nanoparticles modified with fatty acids encapsulating betamethasone for anti-inflammatory treatment.

Topical glucocorticosteroids were incorporated into nanocarrier-based formulations, to overcome side effects of conventional formulations and to achieve maximum skin deposition. Nanoparticulate carriers have the potential to prolong the anti-inflammatory effect and provide higher local concentration of drugs, offering a better solution for treating dermatological conditions and improving patient compliance. Nanoparticles were formulated with poly-ϵ-caprolactone as the polymeric core along with stearic acid as the fatty acid, for incorporation of betamethasone-21-acetate. Oleic acid was applied as the coating fatty acid. Improvement of the drug efficacy, and reduction in drug degradation with time in the encapsulated form was examined, while administering it locally through controlled release. Nanoparticles were spherical with mean size of 300 nm and negatively charged surface. Encapsulation efficiency was 90%. Physicochemical stability in aqueous media of the empty and loaded nanoparticles was evaluated for six months. Drug degradation was reduced compared to free drug, after encapsulation into nanoparticles, avoiding the potency decline and promoting a controlled drug release over one month. Fourier transform infrared spectroscopy and thermal analysis confirmed drug entrapment, while cytotoxicity studies performed in vitro on human keratinocytes, Saccharomyces cerevisiae models and Artemia salina, showed a dose-response relationship for nanoparticles and free drug. In all models, drug loaded nanoparticles had a greater inhibitory effect. Nanoparticles increased drug permeation into lipid membranes in vitro. Preliminary safety and permeation studies conducted on rats, showed betamethasone-21-acetate in serum after 48 h application of a gel containing nanoparticles. No skin reactions were observed. In conclusion, the developed nanoparticles may be applied as topical treatment, after encapsulation of betamethasone-21-acetate, as nanoparticles promote prolonged drug release, increase drug stability in aqueous media, reducing drug degradation, and increase drug permeability through lipid membranes.

Interaction between Plectranthus barbatus herbal tea components and acetylcholinesterase: binding and activity studies

Plectranthus barbatus water extracts, have been used as herbal teas, for the treatment of various diseases. In a previous study it was demonstrated that antioxidant and anti-acetylcholinesterase active extract constituents and their metabolites were found in the plasma of rats after P. barbatus tea intraperitoneal administration. Consequently, a decrease in brain acetylcholinesterase activity occurred. The aim of the present research is to elucidate how P. barbatus extract components interact with acetylcholinesterase. The estimated thermodynamic parameters suggest that the main intermolecular interaction is hydrophobic association, although hydrogen bonds between flavonoids and the active gorge of the acetylcholinesterase molecule seem to occur and have a great impact on acetylcholinesterase inhibition. The hydroxyl positions in flavonoids seem to be of utmost importance for enzyme inhibition, as they interact with specific amino acid residues in the active gorge. FTIR analysis showed that the plant extract components do not interfere with the secondary structure of the enzyme, but decreases the rate of hydrogen-deuterium exchange, possibly by decreasing solvent accessibility in the acetylcholinesterase active gorge. The spectroscopic data complements docking studies of acetylcholinesterase inhibition by plant phenolic compounds, clarifying the dominant interactions between enzyme and inhibitor and the most important structural features of the inhibitor molecules.

Development and Evaluation of a Novel Topical Treatment for Acne with Azelaic Acid-Loaded Nanoparticles

Azelaic acid (AzA) is used in the treatment of acne. However, side effects and low compliance have been associated with several topical treatments with AzA. Nanotechnology presents a strategy that can overcome these problems. Polymeric nanoparticles can control drug release and targeting and reduce local drug toxicity. The aim of this study was to produce and evaluate an innovative topical treatment for acne with AzA-loaded poly-DL-lactide/glycolide copolymer nanoparticles. A soft white powder of nanoparticles was prepared. The mean size of loaded nanoparticles was < 400 nm and zeta potential was negative. Spherical nanoparticles were observed by scanning electron microscopy. Encapsulation efficiency was around 80% and a strong interaction between the polymer and the drug was confirmed by differential scanning calorimetric analysis. In vitro drug release studies suggested a controlled and pulsatile release profile. System efficacy tests suggested similar results between the loaded nanoparticles and the nonencapsulated drug against the most common bacteria associated with acne. Cytotoxicity of AzA-loaded nanoparticles was concentration dependent, although not pronounced. The occluded patch test seemed to indicate that the formulation excipients were safe and thus AzA-loaded nanoparticles appear to be an efficient and safe treatment for acne.

Structure of the Osmophore and Labellum Micromorphology in the Sexually Deceptive Orchids Ophrys Bombyliflora and Ophrys tenthredinifera (Orchidaceae)

Premise of research. The insect-like flowers of the Ophrys orchids are adapted to sexually deceptive pollination through pseudocopulation, providing chemical, visual, and tactile stimuli for male insects. Although the chemical composition of the odor bouquet of several species has long been identified, the precise site of fragrance production in the labellum remains unknown for most species, and little attention has been given to the visual and tactile signals provided by the labellum for pollinators. Here, the occurrence of an osmophore is investigated and the labellum micromorphology is characterized in detail for Ophrys bombyliflora and Ophrys tenthredinifera, two closely related species pollinated by Eucera bees. Methodology. Labella of flowers before and at anthesis were studied with scanning electron microscopy, light microscopy, and histochemistry. Pivotal results. The labellum of O. bombyliflora presents a distinctive hidden apical appendix that forms a concavity with a multicellular protuberance and a tuft of trichomes at the tip. An osmophore occurs in the apical region of the labellum in both species; in O. bombyliflora it is confined mostly to the adaxial surface of the appendix, and in O. tenthredinifera it comprises the labellum margin and the abaxial surface of both the appendix and the adjacent region of the labellum. A terpene-rich lipophilic secretion likely containing a phenolic fraction was found. The two species have a great diversity of epidermal cell types in the adaxial surface of the labellum and differ mainly in the micromorphology of the basal field, speculum, labellum margins, and appendix. Conclusions. This study demonstrates for the first time that a specialized secretory structure (osmophore) occurs in the labellum of both species and synthesizes a secretion that probably includes highly volatile long-range attractants for pollinators. This finding seems to suggest that two sources of potential semiochemicals have evolved in the Ophrys labellum for pollinator attraction.