Sclareol is a diterpene natural product of high value for the fragrance industry. Its labdane carbon skeleton and its two hydroxyl groups also make it a valued starting material for semisynthesis of numerous commercial substances, including production of Ambrox® and related ambergris substitutes used in the formulation of high end perfumes. Most of the commercially-produced sclareol is derived from cultivated clary sage (Salvia sclarea) and extraction of the plant material. In clary sage, sclareol mainly accumulates in essential oil-producing trichomes that densely cover flower calices. Manool also is a minor diterpene of this species and the main diterpene of related Salvia species.
Based on previous general knowledge of diterpene biosynthesis in angiosperms, and based on mining of our recently published transcriptome database obtained by deep 454-sequencing of cDNA from clary sage calices, we cloned and functionally characterized two new diterpene synthase (diTPS) enzymes for the complete biosynthesis of sclareol in clary sage. A class II diTPS (SsLPPS) produced labda-13-en-8-ol diphosphate as major product from geranylgeranyl diphosphate (GGPP) with some minor quantities of its non-hydroxylated analogue, (9 S, 10 S)-copalyl diphosphate. A class I diTPS (SsSS) then transformed these intermediates into sclareol and manool, respectively. The production of sclareol was reconstructed in vitro by combining the two recombinant diTPS enzymes with the GGPP starting substrate and in vivo by co-expression of the two proteins in yeast (Saccharomyces cerevisiae). Tobacco-based transient expression assays of green fluorescent protein-fusion constructs revealed that both enzymes possess an N-terminal signal sequence that actively targets SsLPPS and SsSS to the chloroplast, a major site of GGPP and diterpene production in plants.
SsLPPS and SsSS are two monofunctional diTPSs which, together, produce the diterpenoid specialized metabolite sclareol in a two-step process. They represent two of the first characterized hydroxylating diTPSs in angiosperms and generate the dihydroxylated labdane sclareol without requirement for additional enzymatic oxidation by activities such as cytochrome P450 monoxygenases. Yeast-based production of sclareol by co-expresssion of SsLPPS and SsSS was efficient enough to warrant the development and use of such technology for the biotechnological production of scareol and other oxygenated diterpenes.
Diterpene; Sage; Salvia sclarea; Sclareol; Terpene synthase
Sclareol is a natural product initially isolated form Salvia sclarea which possesses immune-regulation and anti-inflammatory activities. However, the anti-osteoarthritic properties of sclareol have not been investigated. The present study is aimed at evaluating the potential effects of sclareol in interleukin-1β (IL-1β)-induced rabbit chondrocytes as well as an experimental rabbit knee osteoarthritis model induced by anterior cruciate ligament transection (ACLT). Cultured rabbit chondrocytes were pretreated with 1, 5 and 10 μg/mL sclareol for 1 h and followed by stimulation of IL-1β (10 ng/mL) for 24 h. Gene expression of matrix metalloproteinase-1 (MMP-1), MMP-3, MMP-13, tissue inhibitors of metalloproteinase-1 (TIMP-1), inducible nitric oxide synthase (iNOS) and cyclooxygenase (COX)-2 was determined by quantitative real-time polymerase chain reaction (qRT-PCR). MMP-3, TIMP-1, iNOS and COX-2 proteins were measured by Western blotting. Enzyme-linked immunosorbent assay (ELISA) was applied for nitric oxide (NO) and prostaglandin E2 (PGE2) assessment. For the in vivo study, rabbits received six weekly 0.3 mL sclareol (10 μg/mL) intra-articular injections in the knees four weeks after ACLT surgery. Cartilage was harvested for measurement of MMP-1, MMP-3, MMP-13, TIMP-1, iNOS and COX-2 by qRT-PCR, while femoral condyles were used for histological evaluation. The in vitro results we obtained showed that sclareol inhibited the MMPs, iNOS and COX-2 expression on mRNA and protein levels, while increased the TIMP-1 expression. And over-production of NO and PGE2 was also suppressed. For the in vivo study, both qRT-PCR results and histological evaluation confirmed that sclareol ameliorated cartilage degradation. Hence, we speculated that sclareol may be an ideal approach for treating osteoarthritis.
Osteoarthritis; sclareol; matrix metalloproteinase; nitric oxide; ACLT
Although Salvia sclarea (clary sage) is widely used in aromatherapy and has antioxidant and antimicrobial properties, its mechanisms of action remain poorly understood. We therefore assessed whether clary sage is effective in treating endothelial dysfunction induced by chronic immobilization stress in rats.
Rats were intraperitoneally injected with almond oil, clary sage oil (5%, 10% or 20%), or nifedipine once daily, followed by immobilization stress (2 h/day) for 14 days. Systolic blood pressure (SBP) and heart rate (HR) were measured, as were serum concentrations of corticosterone (CORT); a biomarker of chronic stress, malondialdehyde (MDA); a biomarker of oxidative stress. Nitric oxide production was assessed by nitrite assays, and eNOS level, a biomarker of endothelial dysfunction, was measured by western blotting. Endothelial dysfunction was also assayed by measuring the effect of clary sage on the contraction of rat aortae.
Treatment with 5% (p = 0.029), 10% (p = 0.008), and 20% (p = 0.008) clary sage significantly reduced SBP and treatment with 20% clary sage significantly reduced HR (p = 0.039) compared with the chronic immobilization stress group. Clary sage decreased CORT serum concentration (10%, p = 0.026; 20%, p = 0.012) and MDA (10%, p = 0.007; 20%, p = 0.027), findings similar to those observed with nifedipine. In addition, 20% clary sage significantly increased nitric oxide production (p <0.001) and eNOS expression level (p <0.001) and relaxed aortic rings in rats subjected to chronic immobilization stress.
Clary sage treatment of rats subjected to immobilization stress contributed to their recovery from endothelial dysfunction by increasing NO production and eNOS level as well as by decreasing oxidative stress. Appropriate concentration of clary sage may result in recovery from endothelial dysfunction. These findings indicate that clary sage oil may be effective in the prevention and treatment of stress-induced cardiovascular diseases.
Salvia sclarea; Chronic immobilization stress; Endothelial dysfunction; Oxidative stress
The spreading of bacterial antibiotic resistance among clinical strains of pathogenic bacteria has made investigators to search for other active antibacterial agents which could provide a valuable complement to the existing therapies.
To determine the antibacterial activity of clary sage oil (Salvia sclarea L.) against Staphylococcus clinical strains which were isolated from patients with wound infections.
Material and methods
A comprehensive evaluation of Staphylococcus clinical strain resistance to antibiotics was performed. The constituents of clary sage oil were assayed by GC-FID-MS analysis. The minimal inhibitory concentration (MIC) of the tested essential oil against staphylococci by the micro-dilution broth method was determined.
The clary sage oil was active against Staphylococcus aureus, S. epidermidis and S. xylosus with MIC values ranging from 3.75 to 7.00 µl/ml.
The results of the in vitro tests encourage to use formulations containing sage oil as the active natural antimicrobial agent. Because of its antimicrobial properties clary sage oil may be applied to treat wounds and skin infections.
clary sage oil; minimal inhibitory concentration; Staphylococcus; wounds
The aim of this study was to investigate the effect of inhalation of Salvia sclarea (clary sage; clary) or Lavandula angustifolia (lavender) essential oil vapors on autonomic nervous system activity in female patients with urinary incontinence undergoing urodynamic assessment.
Study design, location, and subjects
This study was a double-blind, randomized, controlled trial carried out in 34 female patients with urinary incontinence.
The subjects were randomized to inhale lavender, clary, or almond (control) oil at concentrations of 5% (vol/vol) each. Systolic blood pressure, diastolic blood pressure, pulse rate, respiratory rate, and salivary cortisol were measured before and after inhalation of these odors for 60 minutes.
The clary oil group experienced a significant decrease in systolic blood pressure compared with the control (p=0.048) and lavender oil (p=0.026) groups, a significant decrease in diastolic blood pressure compared with the lavender oil group (p=0.034) and a significant decrease in respiratory rate compared with the control group (p<0.001). In contrast, the lavender oil group tended to increase systolic and diastolic blood pressure compared with the control group. Compared with the control group, inhalation of lavender oil (p=0.045) and clary oil (p<0.001) resulted in statistically significant reductions in respiratory rate.
These results suggest that lavender oil inhalation may be inappropriate in lowering stress during urodynamic examinations, despite its antistress effects, while clary oil inhalation may be useful in inducing relaxation in female urinary incontinence patients undergoing urodynamic assessments.
• Background and Aims
Salvia divinorum produces several closely related neoclerodane diterpenes. The most abundant of these, salvinorin A, is responsible for the psychoactive properties of the plant. To determine where these compounds occur in the plant, various organs, tissues and glandular secretions were chemically analysed. A microscopic survey of the S. divinorum plant was performed to examine the various types of trichomes present and to determine their distribution.
• Methods Chemical analyses were performed using thin layer chromatographic and histochemical techniques. Trichomes were examined using conventional light microscopy and scanning electron microscopy.
• Key Results It was found that neoclerodane diterpenes are secreted as components of a resin that accumulates in peltate glandular trichomes, specifically in the subcuticular space that exists between the trichome head cells and the cuticle that encloses them. Four main types of trichomes were observed: peltate glandular trichomes, short‐stalked capitate glandular trichomes, long‐stalked capitate glandular trichomes and non‐glandular trichomes. Their morphology and distribution is described. Peltate glandular trichomes were only found on the abaxial surfaces of the leaves, stems, rachises, bracts, pedicles and calyces. This was consistent with chemical analyses, which showed the presence of neoclerodane diterpenes in these organs, but not in parts of the plant where peltate glandular trichomes are absent.
• Conclusions Salvinorin A and related compounds are secreted as components of a complex resin that accumulates in the subcuticular space of peltate glandular trichomes.
Salvia divinorum; Labiatae; diviner’s sage; salvinorin A; salvinorins; neoclerodane diterpenes; trichomes; thin layer chromatography; histochemistry; morphology
Carnosic acid (CA) is a phenolic diterpene with anti-tumour, anti-diabetic, antibacterial and neuroprotective properties that is produced by a number of species from several genera of the Lamiaceae family, including Salvia fruticosa (Cretan sage) and Rosmarinus officinalis (Rosemary). To elucidate CA biosynthesis, glandular trichome transcriptome data of S. fruticosa were mined for terpene synthase genes. Two putative diterpene synthase genes, namely SfCPS and SfKSL, showing similarities to copalyl diphosphate synthase and kaurene synthase-like genes, respectively, were isolated and functionally characterized. Recombinant expression in Escherichia coli followed by in vitro enzyme activity assays confirmed that SfCPS is a copalyl diphosphate synthase. Coupling of SfCPS with SfKSL, both in vitro and in yeast, resulted in the synthesis miltiradiene, as confirmed by 1D and 2D NMR analyses (1H, 13C, DEPT, COSY H-H, HMQC and HMBC). Coupled transient in vivo assays of SfCPS and SfKSL in Nicotiana benthamiana further confirmed production of miltiradiene in planta. To elucidate the subsequent biosynthetic step, RNA-Seq data of S. fruticosa and R. officinalis were searched for cytochrome P450 (CYP) encoding genes potentially involved in the synthesis of the first phenolic compound in the CA pathway, ferruginol. Three candidate genes were selected, SfFS, RoFS1 and RoFS2. Using yeast and N. benthamiana expression systems, all three where confirmed to be coding for ferruginol synthases, thus revealing the enzymatic activities responsible for the first three steps leading to CA in two Lamiaceae genera.
Terpenoids (isoprenoids) have numerous applications in flavors, fragrances, drugs and biofuels. The number of microbially produced terpenoids is increasing as new biosynthetic pathways are being elucidated. However, efforts to improve terpenoid production in yeast have mostly taken advantage of existing knowledge of the sterol biosynthetic pathway, while many additional factors may affect the output of the engineered system.
Aiming to develop a yeast strain that can support high titers of sclareol, a diterpene of great importance for the perfume industry, we sought to identify gene deletions that improved carotenoid, and thus potentially sclareol, production. Using a carotenogenic screen, the best 100 deletion mutants, out of 4,700 mutant strains, were selected to create a subset for further analysis. To identify combinations of deletions that cooperate to further boost production, iterative carotenogenic screens were applied, and each time the top performing gene deletions were further ranked according to the number of genetic and physical interactions known for each specific gene. The gene selected in each round was deleted and the resulting strain was employed in a new round of selection. This approach led to the development of an EG60 derived haploid strain combining six deletions (rox1, dos2, yer134c, vba5, ynr063w and ygr259c) and exhibiting a 40-fold increase in carotenoid and 12-fold increase in sclareol titers, reaching 750 mg/L sclareol in shake flask cultivation.
Using an iterative approach, we identified novel combinations of yeast gene deletions that improve carotenoid and sclareol production titers without compromising strain growth and viability. Most of the identified deletions have not previously been implicated in sterol pathway control. Applying the same approach using a different starting point could yield alternative sets of deletions with similar or improved outcome.
Electronic supplementary material
The online version of this article (doi:10.1186/s12934-015-0246-0) contains supplementary material, which is available to authorized users.
Terpenoids; Saccharomyces cerevisiae; Sclareol; Carotenoid; Ergosterol biosynthesis
Four new labdane-type diterpenoid glycosides, laevissiosides A-D (1–4) were isolated from the 95% ethanol extract of Diplopterygium laevissimum (Christ) Nakai, along with two known analogues, 18- β-D-glucopyranosyl ester-sclareol (5) and 18-hydroxy-sclareol (6). The structures of compounds 1–4 were elucidated by extensive 1D and 2D NMR spectroscopy as well as high-resolution MS analyses. All isolated compounds were evaluated for their cytotoxic effects.
Diplopterygium laevissimum; labdane-type diterpenoid glycosides; laevissiosides
The crude extracts of plants from Asteraceae and Lamiaceae family and essential oils from Salvia officinalis and Salvia sclarea were studied for their antibacterial as well as antibiotic resistance modifying activity. Using disc diffusion and broth microdilution assays we determined higher antibacterial effect of three Salvia spp. and by evaluating the leakage of 260 nm absorbing material we detected effect of extracts and, namely, of essential oils on the disruption of cytoplasmic membrane. The evaluation of in vitro interactions between plant extracts and oxacillin described in terms of fractional inhibitory concentration (FIC) indices revealed synergistic or additive effects of plant extracts and clearly synergistic effects of essential oil from Salvia officinalis with oxacillin in methicillin-resistant Staphylococcus epidermidis.
The plants of the genus Salvia synthesize several types of secondary metabolites with antimicrobial, cytotoxic, and radical scavenging activities and are used in the folk medicine of different countries. Eleven Salvia species including S. aegyptiaca, S. aethiopis, S. atropatana, S. eremophila, S. hypoleuca, S. limbata, S. nemorosa, S. santolinifolia, S. sclarea, S. syriaca, and S. xanthocheila were collected from different localities in Iran and screened for their cytotoxic activity using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) colorimetric assay. The antioxidant potential and total phenol contents of the plant extracts were assessed by 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging assay and Folin- Ciocalteu reagent respectively and finally antimicrobial activity of the above extracts were determined by using agar disc diffusion (ADD) and nutrient broth micro-dilution (NBMD) bioassays. Cytotoxic activity of methanol, 80% methanol and dichloromethane extracts of these plants were assessed on 3 human cancer cell lines. All of the extracts of S. eremophila and S. santolinifolia were active at IC50 values of 10.5-75.2 μg extract/mL, while the methanol and dichloromethane extracts of S. limbata, S. hypoleuca and S. aethiopis showed considerable cytotoxic activity against the tested cell lines. Among the tested plants for their antioxidant activity, S. nemorosa, S. atropatana, S. santolinifolia, and S. eremophila were the most active radical scavengers with higher total phenol contents while, S. limbata, S. xanthocheila and S. aegyptiaca were the weakest ones. The methanol extracts of S. santolinifolia, S. eremophila, S. sclarea and S. limbata inhibited the growth of all tested bacterial strains.
Salvia; Phenolics; DPPH; Radical-scavenging; Cytotoxic activity; Antibacterial activity
Using special lotions and repellent sprays on skin is one of the effective methods to prevent Arthropods biting which was verified in this study.
Essential oils of four plants (Satureja khuzestanica, Salvia sclarea, Lavendula officinalis and Myrtus communis) were separately extracted by Clevenger used hydro distillation method. Then separated solutions with 10%, 20% and 40% concentrations of essential oils of plants in 99.6 % ethanol were prepared. WHO guidelines for efficacy testing of mosquito repellents for human skin were used on different concentrations of essential oils of plants, traditional repellents (DEET, 50% and 33%) as positive control, and ethanol 99.6% and naked hands as negative controls.
In negative control groups, the number of bits were comparable (P= 0.42) and had decreasing time trends (naked hands P= 0.011, ethanol P< 0.001). In all time points, minimum bites were observed in traditional repellents and it was significantly less than the other groups (P< 0.001). The time trend in the number of bites in the other groups was positive and showed minimum number of bites in time zero in all groups. We also found that the concentration of repellents had association with the number of bites. The maximum and minimum numbers of bites were observed with 10% and 40% concentrations respectively in all groups.
Essential oils of Salvia sclarea, Lavendula officinalis and Myrtus communis have repellency effect, even with 10% concentration of essential oils.
Malaria; repellents; Anopheles stephensi; essential oil; plants
The family Lamiaceae (Labiatae) has included some medicinal plants. some monoterpene synthases, including linalool and limonene synthases, have been cloned and functionally characterized from several plants of Labiatae family.
Materials and Methods:
In this study, presence of linalool and limonene synthases, in four species of Labiatae family including Nepeta cataria, Lavandula angustifolia, Hyssopus officinalis and Salvia sclarea has been determined by molecular biological techniques together with the Head space SPME – GC-MS analysis of the aroma profile of these species.
Indicated that none of the plant species produced distinguishable bands with primer pairs related to d-limonene synthase. Distinguishable bands around 1800 bp in cDNA samples of L. angustifolia, H. officinalis and S. sclarea were observed regarding to the presence of linalool synthase. Head space SPME-GC-MS analysis of the aroma profiles of the above-mentioned plants showed that linalool (31.0%), linalyl acetate (18.2%), were found as the major compounds of L. angustifolia, while geraniol (5.5%), nerol (34.0%) and α- citral (52.0%) were identified as the main compounds of the N. cataria. The major components of H. officinalis and S. sclarea oils were determined as cis-pinocamphone (57.3%), and linalool (19.0%), linalyl acetate (51.5%), respectively.
H. officinalis was rich of cyclic monoterpenes, L. angustifolia, N. cataria and S. sclarea showed considerable amount of linear monoterpenes. The aroma profile of the above-mentioned plants contained low concentration of sesquiterpenes except N. cataria, which indicated no sesquiterpene. The profiles of the main components of these plants are in agreement with molecular assays.
Essential oil; headspace solid phase microextraction; labiatae; monoterpene synthase
Brucellosis, a zoonosis caused by four species of brucella, has a high morbidity. Brucella melitensis is the main causative agent of brucellosis in both human and small ruminants. As an alternative to conventional antibiotics, medicinal plants are valuable resources for new agents against antibiotic-resistant strains. The aim of this study was to investigate the usage of native plants for brucellosis treatment. For this purpose, the anti-brucella activities of ethanolic and methanolic extracts of Salvia sclarea, Oliveria decumbens, Ferulago angulata, Vitex pseudo-negundo, Teucrium polium, Plantago ovata, Cordia myxa, and Crocus sativus were assessed. The activity against a resistant Br. melitensis strain was determined by disc diffusion method at various concentrations from 50–400 mg/ml. Antibiotic discs were also used as a control. Among the evaluated herbs, six plant (Salvia sclarea, Oliveria decumbens, Ferulago angulata, Vitex pseudo-negundo, Teucrium polium, and Crocus sativus) showed anti-brucella activity. Oliveria decumbens was chosen as the most effective plant for further studies. A tested isolate exhibited resistance to tetracycline, nafcillin, oxacillin, methicillin, and colistin. Minimal inhibitory concentration (MIC) and minimal bactericidal concentration (MBC) values for Oliveria decumbens against resistant Br. melitensis were the same (5 mg/ml), and for gentamicin they were both 2 mg/ml. Time-kill kinetics for a methanolic extract of Oliveria decumbens was 7 h whereas for an ethanolic extract it was 28 h. Also, Oliveria decumbens extracts showed a synergistic effect in combination with doxycycline and tetracycline. In general, the similar values of MIC and MBC for Oliveria decumbens suggest that these extracts could act as bactericidal agents against Br. melitensis. In addition to Oliveria decumbens, Crocus sativus and Salvia sclarea also had good anti-brucella activity and these should be considered for further study.
Brucellosis; Antibiotic resistance; Brucella melitensis; Medicinal plant; Oliveria decumbens
The genus Salvia, with nearly 900 species, is one of the largest members of Lamiaceae family. In the Flora of Iran, the genus Salvia is represented by 58 species of which 17 species are endemic. Salvia hypoleuca Benth., is one of these species growing wildly in northern and central parts of Iran. Salvia species are well known in folk medicine and widely used for therapeutic purposes. Literature review shows that there is no report on phytochemical investigation of the roots of S. hypoleuca.
The separation and purification process were carried out using various chromatographic methods. Structural elucidation was on the basis of NMR and MS data, in comparison with those reported in the literature. The isolated compounds were identified as sitosteryl oleate (1), β-sitosterol (2), stigmasterol (3), manool (4), 7α-acetoxy royleanone (5), ursolic acid (6), oleanolic acid (7), 3-epicorosolic acid (8), 3-epimaslinic acid (9) and coleonolic acid (10).
In the present study, three sterols, two diterpenes and five triterpenes were isolated from the ethyl acetate extract of the roots of S. hypoleuca. As the chemotaxonomic significance, some of the isolated compounds (1–7, 9) have not been previously reported from the species S. hypoleuca, while the triterpenes 8 and 10 are now documented from Salvia genus for the first time.
Salvia hypoleuca; Coleonolic acid; 7α-acetoxyroyleanone; 3-epimaslinic acid; 3-epicorosolic acid; Manool
The morphology, ultrastructure, density and distribution of trichomes on leaves of Betula pendula, B. pubescens ssp. pubescens, B. pubescens ssp. czerepanovii and B. nana were examined by means of light, scanning and transmission electron microscopy. The composition of flavonoids in ethanolic leaf surface extracts was analysed by high pressure liquid chromatography. All taxa examined contained both glandular and non‐glandular trichomes (short and/or long hairs) but differed from each other in trichome ultrastructure, density and location on the leaf. Leaves of B. pubescens were more hairy than those of B. pendula, but the latter species had a higher density of glandular trichomes. Of the two subspecies of B. pubescens, leaves of ssp. pubescens had more short hairs on the leaf surface and four times the density of glandular trichomes of leaves of ssp. czerepanovii, whereas, in the latter subspecies, short hairs occurred largely on leaf veins, as in B. nana. The glandular trichomes were peltate glands, consisting of medullar and cortical cells, which differed structurally. Cortical cells possessed numerous small, poorly developed plastids and small vacuoles, whereas medullar cells had several large plastids with well‐developed thylakoid systems and fewer vacuoles. In B. pubescens subspecies, vacuoles of the glandular cells contained osmiophilic deposits, which were probably phenolic, whereas in B. pendula, vacuoles of glandular trichomes were characterized by the presence of numerous myelin‐like membranes. The composition of epicuticular flavonoids also differed among species. The two subspecies of B. pubescens and B. nana shared the same 12 compounds, but five of these occurred only in trace amounts in B. nana. Leaf surface extracts of B. pendula contained just six flavonoids, three of which occurred only in this species. In summary, the structure, density and distribution of leaf trichomes and the composition of epicuticular flavonoids represent good taxonomic markers for Finnish birch species.
Betula pendula; silver birch; Betula pubescens ssp. pubescens; white birch; Betula pubescens ssp. czerepanovii; mountain birch; Betula nana; dwarf birch; non‐glandular trichomes; glandular trichomes; ultrastructure; flavonoids
• Background and Aims Changes in number of trichomes and in composition and concentrations of their exudates throughout leaf development may have important consequences for plant adaptation to abiotic and biotic factors. In the present study, seasonal changes in leaf trichomes and epicuticular flavonoid aglycones in three Finnish birch taxa (Betula pendula, B. pubescens ssp. pubescens, and B. pubescens ssp. czerepanovii) were followed.
• Methods Trichome number and ultrastructure were studied by means of light, scanning and transmission electron microscopy, while flavonoid aglycones in ethanolic leaf surface extracts were analysed by high-pressure liquid chromatography.
• Key Results Density of both glandular and non-glandular trichomes decreased drastically with leaf expansion while the total number of trichomes per leaf remained constant, indicating that the final number of trichomes is established early in leaf development. Cells of glandular trichomes differentiate before those of the epidermis and produce secreted material only during the relatively short period (around 1–2 weeks) of leaf unfolding and expansion. In fully expanded leaves, glandular trichomes appeared to be at the post-secretory phase and function mainly as storage organs; they contained lipid droplets and osmiophilic material (probably phenolics). Concentrations (mg g−1 d. wt) of surface flavonoids decreased with leaf age in all taxa. However, the changes in total amount (µg per leaf) of flavonoids during leaf development were taxon-specific: no changes in B. pubescens ssp. czerepanovii, increase in B. pendula and in B. pubescens ssp. pubescens followed by the decline in the latter taxon. Concentrations of most of the individual leaf surface flavonoids correlated positively with the density of glandular trichomes within species, suggesting the participation of glandular trichomes in production of surface flavonoids.
• Conclusions Rapid decline in the density of leaf trichomes and in the concentrations of flavonoid aglycones with leaf age suggests that the functional role of trichomes is likely to be most important at the early stages of birch leaf development.
Birch; Betula pendula; Betula pubescens ssp.; pubescens; Betula pubescens ssp.; czerepanovii; glandular trichomes; non-glandular trichomes; flavonoid aglycones; leaf development
Two flavones, ladanein and 6-hydroxy-5,7,4′-trimethoxyflavone and one labdane-type diterpene, ent-13-epi-manoyloxide, were isolated from an ethyl acetate-methanol extract of the aerial parts of Salvia sharifii. The compounds were purified using several chromatographic methods. Structural elucidation of the compounds was performed using their 1H and 13C-NMR data, EI mass and UV spectral data. The compounds have been subjected to antimicrobial, antioxidant and cytotoxic activity. The diterpene showed higher cytotoxic activity than the flavones while the later compounds were better antioxidants compared with the isolated diterpene.
TLadanein; 6-hydroxy-5; 7; 4′-trimethoxyflavone; Ent-13-epi-manoyloxide; Antioxidant; Antimicrobial; Cytotoxic
Aqueous extracts from leaves of well known species of the Lamiaceae family were examined for their potency to inhibit infection by human immunodeficiency virus type 1 (HIV-1).
Extracts from lemon balm (Melissa officinalis L.), peppermint (Mentha × piperita L.), and sage (Salvia officinalis L.) exhibited a high and concentration-dependent activity against the infection of HIV-1 in T-cell lines, primary macrophages, and in ex vivo tonsil histocultures with 50% inhibitory concentrations as low as 0.004%. The aqueous Lamiaceae extracts did not or only at very high concentrations interfere with cell viability. Mechanistically, extract exposure of free virions potently and rapidly inhibited infection, while exposure of surface-bound virions or target cells alone had virtually no antiviral effect. In line with this observation, a virion-fusion assay demonstrated that HIV-1 entry was drastically impaired following treatment of particles with Lamiaceae extracts, and the magnitude of this effect at the early stage of infection correlated with the inhibitory potency on HIV-1 replication. Extracts were active against virions carrying diverse envelopes (X4 and R5 HIV-1, vesicular stomatitis virus, ecotropic murine leukemia virus), but not against a non-enveloped adenovirus. Following exposure to Lamiaceae extracts, the stability of virions as well as virion-associated levels of envelope glycoprotein and processed Gag protein were unaffected, while, surprisingly, sucrose-density equilibrium gradient analyses disclosed a marked increase of virion density.
Aqueous extracts from Lamiaceae can drastically and rapidly reduce the infectivity of HIV-1 virions at non-cytotoxic concentrations. An extract-induced enhancement of the virion's density prior to its surface engagement appears to be the most likely mode of action. By harbouring also a strong activity against herpes simplex virus type 2, these extracts may provide a basis for the development of novel virucidal topical microbicides.
In the present study we aimed to evaluate the effects of methanolic extracts of the bulbs of Garlic (Allium sativum L., Alliaceae) and Persian shallot (Allium ascalonicum L., Alliaceae ) and leaves of Sage (Salvia officinalis L., Lamiaceae ), ASE, AAE and SOE respectively, on the antioxidant enzymes such as superoxide dismutase (SOD), glutathione peroxidase (GPX) and catalase (CAT) activities and on the levels of plasma lipids profiles such as triglycerides (TG), total cholesterol (TC), high-density lipoproteins (HDL), low-density lipoproteins (LDL) and very low-density lipoproteins (VLDL) in Alloxan diabetic Wistar rats.
In comparison with diabetic control rats in diabetic treated rats, AAE increases the activities of SOD (65%), GPX (43%) and CAT (55%). ASE and SOE increase SOD activity by 60% and 33% respectively. ASE reduces TC (34%), SOE decreases TG (40%) and LDL (30%) and AAE reduces VLDL (24%). Metformin exhibits mild antioxidant and hypolipidemic properties. Results of quantitative phytochemical analysis show that the methanolic garlic and Persian shallot bulbs extracts contain secondary metabolites including alkaloids (3.490% and 3.430%), glycosides (18.023% and 13.301%) and saponins (0.812% and 0.752%). Methanolic sage leaves extract contains flavonoids (1.014%), glycosides (23.142%) and saponins (2.096%). The total phenolic contents of ASE, AAE and SOE were in order 4.273, 3.621 and 6.548 mg GAE/g dry weight (DW).
These results suggest that Allium sativum, Allium ascalonicum and Salvia officinalis are beneficial in the control of diabetes by noticeable antioxidant and hypolipidemic properties.
Allium ascalonicum; Allium sativum; Salvia officinalis; Antioxidant enzymes; Plasma lipids
Garlic (Allium sativum L., Alliaceae), Persian shallot (Allium ascalonicum L., Alliaceae ) and Sage (Salvia officinalis L., Lamiaceae) are believed to have hypoglycemic properties and have been used traditionally as antidiabetic herbal medicines in Iran. In this study, diabetes was induced by subcutaneous injection of alloxan monohydrate (100 mg kg−1) to male Wistar rats. Antidiabetic effects of methanolic extracts of the above mentioned three plants on alloxan-diabetic rats was investigated in comparison with the effects of antidiabetic drugs such as acarbose, glibenclamide and metformin by measuring postprandial blood glucose (PBG), oral glucose tolerance test (OGTT), inhibition of rat intestinal α-glucosidase enzymes activities and pancreatic Insulin and cardiac Glut-4 mRNAs expression. In short term period, hypoglycemic effects of A. sativum and A. ascalonicum showed significant reduction of PBG similar to glibenclamide (5 mg kg−1 bw) while S. officinalis significantly reduced PBG similar to acarbose (20 mg kg−1 bw). After 3 weeks of treatment by methanolic plant extracts, significant chronic decrease in the PBG was observed similar to metformin (100 mg kg−1 bw). For OGTT, S. officinalis reduced PBG in a similar way as acarbose (20 mg kg−1 bw). Intestinal sucrase and maltase activities were inhibited significantly by A. sativum, A. ascalonicum and S. officinalis. In addition, we observed increased expression of Insulin and Glut-4 genes in diabetic rats treated with these plants extracts. Up regulation of Insulin and Glut-4 genes expression and inhibition of α-glucosidaseactivities are the two mechanisms that play a considerable role in hypoglycemic action of garlic, shallot and sage.
Diabetes; Glut-4; Insulin; OGTT; PBG
The family Cistaceae (Angiosperm, Malvales) consists of 8 genera and 180 species, with 5 genera native to the Mediterranean area (Cistus, Fumara, Halimium, Helianthemum, and Tuberaria). Traditionally, a number of Cistus species have been used in Mediterranean folk medicine as herbal tea infusions for healing digestive problems and colds, as extracts for the treatment of diseases, and as fragrances. The resin, ladano, secreted by the glandular trichomes of certain Cistus species contains a number of phytochemicals with antioxidant, antibacterial, antifungal, and anticancer properties. Furthermore, total leaf aqueous extracts possess anti-influenza virus activity. All these properties have been attributed to phytochemicals such as terpenoids, including diterpenes, labdane-type diterpenes and clerodanes, phenylpropanoids, including flavonoids and ellagitannins, several groups of alkaloids and other types of secondary metabolites. In the past 20 years, research on Cistus involved chemical, biological and phylogenetic analyses but recent investigations have involved genomic and molecular approaches. Our lab is exploring the biosynthetic machinery that generates terpenoids and phenylpropanoids, with a goal to harness their numerous properties that have applications in the pharmaceutical, chemical and aromatic industries. This review focuses on the systematics, botanical characteristics, geographic distribution, chemical analyses, biological function and biosynthesis of major compounds, as well as genomic analyses and biotechnological approaches of the main Cistus species found in the Mediterranean basin, namely C. albidus, C. creticus, C. crispus, C. parviflorus, C. monspeliensis, C. populifolius, C. salviifolius, C. ladanifer, C. laurifolius, and C. clusii.
Cistus; biosynthesis; labdane-type diterpenes; phenylpropanoids; biological action; genomic approaches
The mint family (Lamiaceae) produces a wide variety of constituents with medicinal properties. Several family members have been reported to have antiviral activity, including lemon balm (Melissa officinalis L.), sage (Salvia spp.), peppermint (Mentha × piperita L.), hyssop (Hyssopus officinalis L.), basil (Ocimum spp.) and self-heal (Prunella vulgaris L.). To further characterize the anti-lentiviral activities of Prunella vulgaris, water and ethanol extracts were tested for their ability to inhibit HIV-1 infection.
Aqueous extracts contained more anti-viral activity than did ethanol extracts, displaying potent antiviral activity against HIV-1 at sub μg/mL concentrations with little to no cellular cytotoxicity at concentrations more than 100-fold higher. Time-of-addition studies demonstrated that aqueous extracts were effective when added during the first five hours following initiation of infection, suggesting that the botanical constituents were targeting entry events. Further analysis revealed that extracts inhibited both virus/cell interactions and post-binding events. While only 40% inhibition was maximally achieved in our virus/cell interaction studies, extract effectively blocked post-binding events at concentrations similar to those that blocked infection, suggesting that it was targeting of these latter steps that was most important for mediating inhibition of virus infectivity.
We demonstrate that aqueous P. vulgaris extracts inhibited HIV-1 infectivity. Our studies suggest that inhibition occurs primarily by interference of early, post-virion binding events. The ability of aqueous extracts to inhibit early events within the HIV life cycle suggests that these extracts, or purified constituents responsible for the antiviral activity, are promising microbicides and/or antivirals against HIV-1.
human immunodeficiency virus; HIV; antiviral; microbicide; plant extract; self-heal
The purpose of this study was to assess the safety and efficacy of the ClariVein® system that employs mechanochemical ablation of the great saphenous vein (GSV).
Patients eligible for ablation of the GSV underwent micropuncture access with only local anaesthesia to insert a 4 or 5 Fr sheath. The ClariVein® catheter was placed through the sheath, the wire was extruded, and the distal tip of the wire positioned 2 cm from the saphenofemoral junction under ultrasound guidance. Catheter wire rotation was then activated for 2–3 seconds at approximately 3500 rpm. With the wire rotating, infusion of the sclerosant was started simultaneously with catheter pullback. The sclerosant used was 1.5% liquid sodium tetradecyl sulphate (Sotradecol©, Bioniche Pharma Group, Geneva, Switzerland).
Thirty GSVs in 29 patients were treated. All patients have reached six-month follow-up; the average number of postoperative days is 260. No adverse events have been reported. The Primary Closure Rate is 96.7%.
Mechanochemical ablation appears to be safe and efficacious. The ClariVein® technique eliminates the need for tumescent anaesthesia. The great majority of incompetent GSVs can be treated with this technique.
chronic venous disease; endovenous techniques; ultrasound-guided sclerotherapy; varicose veins; venous insufficiency
To determine whether the spheroid culture can be used to evaluate drug efficacy, we have evaluated the toxicity of free or carrier-associated doxorubicin as a single drug or in combination with other antineoplastic agents using the spheroid cultures of drug-resistant cancer cells. Paclitaxel, cisplatin, dexamethasone, mitoxantrone, sclareol or methotrexate were used in combination with doxorubicin. The effect of the treatment protocols on free, micellar and liposomal doxorubicin accumulation in spheroids and on resulting toxicity was evaluated by fluorescence and lactate dehydrogenase release, respectively. Enhanced doxorubicin accumulation and toxicity were observed after spheroid pretreatment with mitoxantrone or paclitaxel. Effects of the drug combination with doxorubicin were sequence dependent, use of doxorubicin as the first drug being the least inducer of toxicity. Finally, spheroids were recognized by a cancer cell-specific antibody. Our results suggest the usefulness of spheroids to evaluate chemotherapy combinations.
cancer cell spheroids; doxorubicin; paclitaxel; mitoxantrone; combination