A comparative study of phytoconstituents and antibacterial activity of in vitro derived materials of four Passiflora species.

Passiflora species are well known for their common use in popular medicine for the treatment of several diseases, such as insomnia, anxiety, and hysteria, in addition to their anti-inflammatory, antioxidant, analgesic and antibacterial potential. However, few data about the chemical composition and the medicinal potential of in vitro derived materials are available. Therefore, the goal of this work was to compare, for the first time, the phytoconstituents of in vitro derived materials of four Passiflora species, and evaluate the antibacterial potential of their extracts against 20 Gram-positive and negative strains. Chromatographic analysis indicated the presence of saponins in roots extracts from all studied species, whereas leaf extracts presented both saponins and flavonoids. Extracts from leaves and roots of P. alata and P. foetida exhibited a selective inhibitory activity against B. thuringiensis and S. pyogenes, which might be related to the presence of a high concentration of secondary metabolites, including flavonoids and saponins.

Histological characterization of Passiflora pohlii Mast. root tips cryopreserved using the V-Cryo-plate technique.

Cryopreservation stands out as the main strategy to ensure safe and cost efficient long-term conservation of plant germplasm, especially for biotechnological materials. However, the injuries associated with the procedure may result in structural damage and low recovery rates after cooling. Histological analysis provides useful information on the effects of osmotic dehydration, LN exposure, and recovery conditions on cellular integrity and tissue organization, allowing the determination of the critical steps of the cryopreservation protocol and, thus, the use of optimized treatments. Passiflora pohlii Mast. (Passifloraceae) is a native species from Brazil with potential agronomic interest. Recent studies showed the presence of saponins in its roots, which presented antioxidant activity. The goal of this work was to develop a cryopreservation technique for root tips of in vitro-derived plants of P. pohlii using the V-Cryo-plate technique and to characterize the anatomical alterations that occurred during the successive steps of the protocol. Root tips were excised from in vitro plants and precultured before adhesion to cryo-plates and then treated for different periods with the plant vitrification solutions PVS2 or PVS3. Treatment with PVS2 for 45 min resulted in higher recovery (79%) when compared with PVS3 (43%). The greatest number of adventitious roots per cryopreserved explant was also observed after a 45-min exposure to PVS2. Plasmolysis levels were higher in cortical cells of cryopreserved explants treated with PVS2, while pericycle and central cylinder cells were not damaged after this treatment. Thirty days after rewarming, no plasmolysis could be detected, regardless of the experimental conditions.

Antifungal activities of the essential oil and its fractions rich in sesquiterpenes from leaves of Casearia sylvestris Sw.

Casearia genus (Salicaceae) is found in sub-tropical and tropical regions of the world and comprises about 160-200 species. It is a medicinal plant used in South America, also known as "guaçatonga", "erva-de-tiú", "cafezinho-do-mato". In Brazil, there are about 48 species and 12 are registered in the State of Rio de Janeiro, including Casearia sylvestris Sw. There are many studies related to the chemical profile and cytotoxic activities of extracts from these plants, although few studies about the antifungal potential of the essential oil have been reported. In this work, we have studied the antifungal properties of the essential oil of C. sylvestris leaves, as well as of their fractions, against four yeasts (Saccharomyces cerevisae, Candida albicans, C. glabrata and C. krusei) for the first time. The chemical analysis of the essential oil revealed a very diversified (n = 21 compounds) volatile fraction composed mainly of non-oxygenated sesquiterpenes (72.1%). These sesquiterpenes included α-humulene (17.8%) and α-copaene (8.5%) and the oxygenated sesquiterpene spathulenol (11.8%) were also identified. Monoterpenes were not identified. The fractions are mainly composed of oxygenated sesquiterpenes, and the most active fraction is rich in the sesquiterpene 14-hydroxy -9-epi-ß-caryophyllene. This fraction was the most effective in inhibiting the growth of three yeast strains.

Antifungal activities of the essential oil and its fractions rich in sesquiterpenes from leaves of Casearia sylvestris Sw

ABSTRACT Casearia genus (Salicaceae) is found in sub-tropical and tropical regions of the world and comprises about 160-200 species. It is a medicinal plant used in South America, also known as "guaçatonga", "erva-de-tiú", "cafezinho-do-mato". In Brazil, there are about 48 species and 12 are registered in the State of Rio de Janeiro, including Casearia sylvestris Sw. There are many studies related to the chemical profile and cytotoxic activities of extracts from these plants, although few studies about the antifungal potential of the essential oil have been reported. In this work, we have studied the antifungal properties of the essential oil of C. sylvestris leaves, as well as of their fractions, against four yeasts (Saccharomyces cerevisae, Candida albicans, C. glabrata and C. krusei) for the first time. The chemical analysis of the essential oil revealed a very diversified (n = 21 compounds) volatile fraction composed mainly of non-oxygenated sesquiterpenes (72.1%). These sesquiterpenes included α-humulene (17.8%) and α-copaene (8.5%) and the oxygenated sesquiterpene spathulenol (11.8%) were also identified. Monoterpenes were not identified. The fractions are mainly composed of oxygenated sesquiterpenes, and the most active fraction is rich in the sesquiterpene 14-hydroxy -9-epi-β-caryophyllene. This fraction was the most effective in inhibiting the growth of three yeast strains.

Cytotoxic effects of the essential oil from leaves of Casearia sylvestris Sw. (Salicaceae) and its nanoemulsion on A549 tumor cell line / Efectos citotóxicos del aceite esencial de las hojas de Casearia sylvestris Sw. (Salicaceae) y su nanoemulsión sobre líneas celulares tumorales A549

Extracts from leaves of C. sylvestris have cytotoxic effect in different tumor cell lines, possibly due to clerodane type diterpenes (casearins). On the other hand, there are few studies related to the antitumor activity of the essential oils from this species. This work evaluated for the first time the cytotoxicity effects of the pure essential oil and its nanoemulsion against A549 tumor cell line (human lung carcinoma). The essential oil was obtained from fresh leaves by hydrodistillation in a Clevenger-type apparatus and analyzed by GC/MS and GC/FID. Cytotoxicity evaluation was performed using the WST-1 test. The chemical analysis of the essential oil revealed a volatile fraction composed mainly of non-oxygenated sesquiterpenes (72.1%). The essential oil and its nanoemulsion exhibited cytotoxic activity against A549 tumor cells with EC50 of 4.0 µg/mL and EC50 of 1.0 µg/mL, respectively. Both samples displayed a dose dependent pattern (r = -0.79, p = 0.03) as determined by linear regression test.

Los extractos de las hojas de Casearia sylvestris tienen efectos citotóxicos en diferentes líneas celulares tumorales, posiblemente debido a los diterpenos tipo clerodane (casearinas). Por otra parte, hay muy pocos estudios relacionados con la actividad antitumoral del aceite esencial de estas especies. Este trabajo evalúa por primera vez el efecto citotóxico del aceite esencial puro y su nanoemulsión contra la línea de células tumorales A549 (carcinoma humano de pulmón). El aceite esencial fue obtenido de hojas frescas por hidrodestilación en un aparato tipo Clevenger y analizado por GC/MS y GC/FID. La evaluación de citotoxicidad fue realizada usando la prueba WST-1. El análisis químico del aceite esencial reveló una fracción volátil compuesta principalmente por sesquiterpenos no oxigenados (72,1%). El aceite esencial y su nanoemulsiónexhibió actividad citotóxica contra las células tumorales A549 con una EC50 de 4,0 µg/mL y una EC50 de 1,0 µg/mL, respectivamente. Ambas muestras exhibieron un patrón dosis-dependiente (r = -0,79, p = 0,03) determinado por análisis de regresión lineal.

Anatomical aspects, chemical analysis and cytotoxic effect of the essential oil from leaves of Casearia arborea (Salicaceae) / Aspectos anatómicos, análisis químico y efecto citotóxico del aceite esencial de Casearia arborea (Salicaceae)

The genus Casearia (Salicaceae) is found in sub-tropical and tropical regions of the world and comprises about 200 species. In Brazil, there are about 48 species and 12 are registered in the State of Rio de Janeiro; including Casearia arborea (Rich.) Urb. Essential oil was obtained from the fresh leaves by hydrodistillation and analyzed by GC-MS and GC-FID. The cytotoxic effect was determined by WST-1 assay. Chemical analysis of the essential oil revealed a very diversified (n = 37 compounds) volatile fraction composed mainly of non-oxygenated sesquiterpenes (90.2%). These sesquiterpenes included byciclogermacrene (18.7%), germacrene D (12.1%) and α-humulene (11.5%). In addition, the essential oil demonstrated cytotoxic effects against A549 tumor cells in the concentration of 4 µg/mL (EC50) (p < 0.05).

El género Casearia (Salicáceas) se encuentra en las regiones tropicales y sub-tropicales del planeta y comprende alrededor de 200 especies. En Brasil existen 48 especies, 12 de las cuales fueron registradas en el Estado de Río de Janeiro incluyendo Casearia arborea (Rich.) Urb. El aceite esencial fue extraído de hojas frescas por hidrodestilación y analizado por GC-MS y GC-FID. El efecto citotóxico fue determinado por ensayo WST-1. Las cavidades secretorias fueron ocasionalmente encontradas tanto en la lámina foliar como en el pecíolo. El análisis químico del aceite esencial reveló una muy diversa fracción volátil (n = 37 compuestos) formada principalmente por sesquiterpenos no oxigenados (90,2%). Estos sesquiterpenos incluyen biciclogermacreno (18,7%), germacreno D (12,1%) y α-humuleno (11,5%). Además, el aceite esencial demostró efectos citotóxicos contra las células tumorales A549 en una concentración de 4µg/mL (EC50) (p < 0.05).

Optimized Extraction of Resveratrol from Arachis repens Handro by Ultrasound and Microwave: A Correlation Study with the Antioxidant Properties and Phenol Contents.

The vegetal species Arachis repens, commonly known as peanut grass, was studied and, for the first time, we detected the presence of the bioactive compound trans-resveratrol (t-RSV). We compared the efficiency of three different methodologies (conventional maceration [CM], ultrasound-assisted extractions [UAE], and microwave-assisted extractions [MAE]) concerning total phenolics (TP) and resveratrol (t-RSV) content, followed by antioxidant activity (AA) evaluation. By CM, at 1 h, the highest RSV content (1.024 ± 0.036 mg/L) and, correspondingly, the highest DPPH capture (23.90 ± 0.04%) were found. The TP contents, at 1 h, presented the highest value (27.26 ± 0.26 mg/g GAE). By the UAE, the maximum yields of TP (357.18 mg/g GAE) and RSV (2.14 mg/L), as well as, the highest AA (70.95%), were obtained by 5 min after a maceration pretreatment, on the solid-solvent ratio 1 : 40 w/v. For MAE, a central composite rotatable design (CCRD) was applied followed by the FFD design in order to evaluate the statistical effects of four independent variables on the extraction of RSV. The optimal conditions established for obtaining the highest recovery (2.516 mg/g) were 20 min; 90% MeOH aq.; 120 rpm; 60°C; and solid-solvent ratio: 1 : 35 w/v. Relevant correlations were established considering the TP and RSV contents, as well as the AA, corroborating obvious advantages of such techniques in terms of high extraction efficiency in shorter times.

Somatic embryogenesis and pant regeneration from callus cultures of Cleome rosea Vahl

This paper describes a protocol for the efficient vegetative propagation of Cleome rosea by somatic embryogenesis. Leaf and stem explants from nursery-grown seedlings of C. rosea were cultivated on Murashige and Skoog (MS) medium supplemented with indole-3-acetic acid (IAA), a -naphthaleneacetic acid (NAA), 4-amino-3,5,6-trichloropicolinic acid (picloram) or 2,4-dichlorophenoxyacetic acid (2,4-D). Nodular calli were produced from both explant types in the presence of 4.5 and 9.0 µM 2,4-D. Embryo development and maturation were achieved when calli from stem explants were transferred to media containing a ten-fold reduction of 2,4-D concentration initially used (0.45 and 0.90 µM). Leaf-derived calli did not form embryos with the same treatments. The highest frequency of embryogenic callus formation (85 percent) and number of embryo per callus (13.45 ± 2.8) were achieved during the first subculture on medium supplemented with 0.90 µM 2,4-D. Embryo conversion into plantlets was achieved following transfer to growth regulator-free MS medium solidified with 2 g.L-1 phytagel. An acclimatization rate of 53 percent was found three months after transfer to ex vitro conditions and the recovered plants presented a normal phenotypic aspect.

O trabalho descreve um protocolo para a propagação in vitro de Cleome rosea por embriogênese somática. Explantes foliares e caulinares, obtidos de plantas germinadas sob condições in vivo, foram cultivados em meio de Murashige and Skoog (MS) suplementado com ácido 3-indolacético (AIA), ácido naftalenoacético (ANA), ácido 4-amino-3,5,6-tricloropicolínico (picloram) ou ácido 2,4-diclorofenoxiacético (2,4-D). Calos de aspecto nodular foram produzidos a partir de ambos os tipos de explante na presença de 4,5 e 9,0 μM de 2,4-D. O desenvolvimento e a maturação de embriões somáticos foram alcançados quando calos obtidos de explantes caulinares foram transferidos para meio de cultura suplementado com uma concentração de 2,4-D dez vezes menor do que aquelas utilizadas na indução do processo de calogênese (0,45 e 0,90 μM). Calos derivados de explantes foliares não produziram embriões ao serem submetidos a estes mesmos tratamentos. Os maiores valores de freqüência de calos embriogênicos (85 por cento) e número médio de embriões por calo (13,45±2,8) foram alcançados durante a primeira subcultura em meio suplementado com 0,90 μM de 2,4-D. O processo de conversão dos embriões somáticos em plantas foi observado após transferência dos embriões para meio MS sem suplementação hormonal solidificado com 2 g.L-1 de fitagel. Três meses após a transferência para condições ex vitro a taxa de aclimatização alcançada foi de 53 por cento e as plantas apresentavam um aspecto fenotípico normal.

Anthocyanin production in callus cultures of Cleome rosea: modulation by culture conditions and characterization of pigments by means of HPLC-DAD/ESIMS.

Leaf and stem explants of Cleome rosea formed calluses when cultured on MS medium supplemented with different concentrations of 2,4-dichlorophenoxyacetic acid (2,4-D) or 4-amino-3,5,6-trichloropicolinic acid (PIC). The highest biomass accumulation was obtained in the callus cultures initiated from stem explants on medium supplemented with 0.90 microM 2,4-D. Reddish-pink regions were observed on callus surface after 6-7 months in culture and these pigments were identified as anthocyanins. Anthocyanins production was enhanced by reducing temperature and increasing light irradiation. Pigmented calluses transferred to MS1/2 with a 1:4 ratio NH(4)(+)/NO(3)(-), 70 g L(-1) sucrose and supplementation with 0.90 microM 2,4-D maintained a high biomass accumulation and showed an increase of 150% on anthocyanin production as compared with the initial culture conditions. Qualitative analysis of calluses was performed by high performance liquid chromatography coupled to diode array detector and electrospray ionization mass spectrometry (HPLC-DAD/ESIMS). Eleven anthocyanins were characterized and the majority of them were identified as acylated cyanidins, although two peonidins were also detected. The major peak was composed by two anthocyanins, whose proposed identity were cyanidin 3-(p-coumaroyl) diglucoside-5-glucoside and cyanidin 3-(feruloyl) diglucoside-5-glucoside.