Sandra Beatriz Rech

Supercritical fluid extraction and high performance liquid chromatographic determination of benzopyrans and phloroglucinol derivative in Hypericum polyanthemum

The aerial parts of Hypericum polyanthemum Klotzsch ex Reichardt (Guttiferae) were successively extracted with supercritical carbon dioxide (SC CO(2)) under pressures of 90, 120, 150 and 200 bar at different temperatures (40, 50 and 60 degrees C), and compared with the n-hexane extract obtained by ultrasound-assisted extraction. The samples obtained were examined regarding extraction yield and HPLC quantification of the main secondary metabolites, the benzopyrans HP1 (6-isobutyryl-5,7-dimethoxy-2,2-dimethylbenzopyran), HP2 (7-hydroxy-6-isobutyryl-5-methoxy-2,2-dimethyl-benzopyran) and HP3 (5-hydroxy-6-isobutyryl-7-methoxy-2,2-dimethyl) and the phloroglucinol derivative, uliginosin B. SFE presented higher selectivity than the n-hexane maceration, and the best condition to extract the target metabolites has been determined to be at 50 degrees C and for the high molecular-weight compound, uliginosin B, higher pressures were required.

Cell cultures of Rauwolfia sellowii: growth and alkaloid production

Callus and cell suspension cultures of Rauwolfia sellowii were established in Gamborg B5 medium supplemented with 1 mg l-1 2,4-dichlorophenoxyacetic acid, 0.2 mg l-1 kinetin and 30 g l-1 sucrose. The growth cycle of suspension cultures was completed in ca. 22 days and the maximum specific growth rate was 0.0098 h-1 with a doubling time of 71 h. The cultures accumulated the same major alkaloids as in the leaves of the parent plant, such as sellowiine, 19α,20α-epoxyakuammicine, vomilenine, picrinine and 12-demethoxytabernulosine. The alkaloid contents of leaves, callus and cell suspension cultures were quantitatively compared by HPLC.

ANTIOXIDANT ACTIVITY OF FLAVONOIDS ISOLATED FROM HYPERICUM TERNUM

In the present work we have studied the scavenging activity of several flavonoids isolated from Hypericum ternum A. St. Hil. The evaluation of the free radical scavenging capacity was based on the 2,2-diphenyl-l-picrylhydrazyl radical (DPPH) consumption elicited by their addition. Also, we have attempted to evaluate their capacity to delay pyrogallol red consumption promoted by peroxyl radicals. The compounds isolated and characterized were 13,118-biapigenin and five quercetin derivatives (quercetin 3-methyl ether, quercetin 3,7-dimethyl ether, hyperoside, isoquercitrin and guaijaverin). All compounds were able to scavenge DPPH radicals. The order in scavenging capacity (from highest to lowest) was: guaijaverin > hyperoside ≈ isoquercitrin > quercetin-3-methyl-ether. No protection of pyrogallol red was evidenced for all flavonoids derivatives at relatively high (100 µM) concentrations. This lack of protection contrasts with the efficient protection afforded by 10 µM quercetin, indicating that substitution at the 3-position in quercetin strongly reduces the capacity of the molecule to scavenge peroxyl radicals

Phenolic compounds profiles during ex vitro acclimatization of micropropagated Hypericum polyanthemum.

Accumulation of benzopyrans and total phenolic compounds were assessed in acclimatized field grown plants of Hypericum polyanthemum, an endemic species of southern Brazil, harvested at different developmental stages. The HPLC analysis of bioactive compounds 6-isobutyryl-5,7-dimethoxy-2,2-dimethylbenzopyran (HP1), 7-hydroxy-6-isobutyryl-5-methoxy-2,2-dimethyl-benzopyran (HP2) and 5-hydroxy-6-isobutyryl-7-methoxy-2,2-dimethyl-benzopyran (HP3) revealed that the three benzopyrans are accumulated both in the vegetative and reproductive parts with maximum contents observed after 18 weeks (in the former) and 20 weeks (in the later) of plant growth (1.92+/-0.085 g % DW and 2.62+/-0.13 g % DW in the vegetative and reproductive parts, respectively). Highest contents of HP1 (1.56+/-0.12 g % DW) and HP2 (0.19+/-0.01 g % DW) were quantified in the green floral buds of the plants, whereas HP3 reached the highest level (1.02+/-0.08 g % DW) in the overblown flowers. The evaluation of total phenolic compounds showed that the vegetative parts accumulated the highest levels of the metabolites (51.93+/-0.67 mg QE (g DW)(-1)) after 16 weeks of plant growth. Considering the reproductive parts, the open flowers accumulated the greatest levels of the bioactive compounds (75.99+/-0.95 mg QE (g DW)(-1)). The results show that H. polyanthemum can be efficiently propagated and acclimatized to produce benzopyrans and other phenolic compounds.

VALEPOTRIATES ACCUMULATION IN CALLUS, SUSPENDED CELLS AND UNTRANSFORMED ROOT CULTURES OF VALERIANA GLECHOMIFOLIA

SummaryValeriana glechomifolia is an endemic species of southern Brazil, capable of accumulating, in all of its organs, the terpene derivatives known as valepotriates, the presumed sedative components of the roots of pharmaceutically used species of Valeriana. In vitro cultures of the plant were established and the accumulation of acevaltrate, didrovaltrate, and valtrate in callus, cell suspension, and untransformed root cultures was studied. Leaves of in natura plants and roots of micropropagated plantlets were used as the explants for callus induction and root culture establishment, respectively, on Gamborg B5 basal medium supplemented with 2,4-dichlorophenoxyacetic acid (2,4-D) alone or with kinetin (KIN). Culture growth and secondary metabolite yields were enhanced with 2,4-D (4.52μM) and KIN (0.93μM). Maximum valepotriate contents, quantified by HPLC, of acevaltrate (ACE) 2.6mg g−1 DW, valtrate (VAL) 10.2mgg−1 DW, and didrovaltrate (DID) 2.9mg g−1 DW were observed in root cultures after 7–8wk of culture.

Valeriana glechomifolia: in vitro propagation and production of valepotriates

A practical method for the multiplication of Valeriana glechomifolia Meyer was developed and valepotriates synthesis in the plantlets was studied. Axillary buds and shoot tips were cultured in 0.3/ Murashige and Skoog basal medium supplemented with either 0.4 mg l 1 of BAP or without plant growth regulators. The cultured segments grew on both media and produced roots after 3/4 weeks. The dienic valepotriates valtrate, DIA-valtrate, acevaltrate and 1-b-acevaltrate and the monoenic valepotriate didrovaltrate have each been quantitated in the leaves, stems and roots of the micropropagated plantlets and compared with the field-grown plants by reversed-phase HPLC. Plantlets grown on growth regulator-free medium displayed higher valepotriate contents (ranging from 3.54% in stems to 0.98% in leaves) than with the addition of BAP (from 1.22% in leaves to 0.36% in stems) and field-grown plants (from 0.89% in stems to 0.10% in inflorescences). In most cases, acevaltrate was the most abundant compound. # 2002 Elsevier Science Ireland Ltd. All rights reserved.

ANTIOXIDANT ACTIVITY IN SOUTHERN BRAZIL HYPERICUM SPECIES

The present study was conducted to assess the antioxidant activity of Hypericum species endemic to South Brazil, H. caprifoliatum, H. carinatum, H. myrianthum and H. polyanthemum. The free radical scavenging properties of plant extracts were evaluated employing different methodologies, including the bleaching of a stable free radical (2,2-diphenyl-1-picrylhydrazyl, DPPH) and the peroxyl reactivity indexes TRAP (Total Reactive Antioxidant Potential) and ORAC-pyrogallol red (Oxygen Radicals Absorbance Capacity). A fair correlation was found between total phenol content determined by Folin-Ciocalteau and DPPH consumption, both in crude methanol and n-hexane extracts. In particular, H. myrianthum and H. caprifoliatum showed the highest TRAP and ORACpyrogallol red values, respectively. This would imply that H. myrianthum contains a larger amount of antioxidants of lower reactivity.

A Valepotriate Fraction of Valeriana glechomifolia Shows Sedative and Anxiolytic Properties and Impairs Recognition But Not Aversive Memory in Mice

Plants of the genus Valeriana (Valerianaceae) are used in traditional medicine as a mild sedative, antispasmodic and tranquilizer in many countries. This study was undertaken to explore the neurobehavioral effects of systemic administration of a valepotriate extract fraction of known quantitative composition of Valeriana glechomifolia (endemic of southern Brazil) in mice. Adult animals were treated with a single intraperitoneal injection of valepotriate fraction (VF) in the concentrations of 1, 3 or 10 mg kg−1, or with vehicle in the pre-training period before each behavioral test. During the exploration of an open field, mice treated with 10 mg kg−1 of VF showed reduced locomotion and exploratory behavior. Although overall habituation sessions for locomotion and exploratory behavior among vehicle control and doses of VF were not affected, comparison between open-field and habituation sessions within each treatment showed that VF administration at 1 and 10 mg kg−1 impaired habituation. In the elevated plus-maze test, mice treated with VF (10 mg kg−1) showed a significant increase in the percentage of time spent in the open arms without significant effects in the number of total arm entries. VF at 3 mg kg−1 produced an impairment of novel-object recognition memory. In contrast, VF did not affect fear-related memory assessed in an inhibitory avoidance task. The results indicate that VF can have sedative effects and affect behavioral parameters related to recognition memory.

Phenolic compounds accumulation in Hypericum ternum propagated in vitro and during plant development acclimatization

The phenolic compound content of Hypericum ternum was investigated after micropropagation establishment and during acclimatization over the phenological development of the plant. Plantlets cultured in vitro on full Murashige and Skoog medium without growth regulators displayed higher phenolic compound yields, were acclimatized, and field grown. Production of total phenolic compounds as well as hyperoside, chlorogenic acid, quercitrin, guaijaverin, isoquercitrin, and uliginosin B were quantified at vegetative, flowering and fructification stages, and different plant organs (roots, stems, leaves and reproductive parts) showing that reproductive parts at flowering stage and the leaves at fructification stage were the main repository site of secondary metabolites, except for uliginosin B. The stems were the least accumulative organ, while the roots accumulated only hyperoside and uliginosin B. Moreover, the accumulation of most of the flavonoids and uliginosin B in acclimatized plants surpassed the levels found in the wild plant, warranting further research with the species.