Effects of diene valepotriates from Valeriana glechomifolia on Na+/K+-ATPase activity in the cortex and hippocampus of mice.

Diene valepotriates obtained from Valeriana glechomifolia present antidepressant-like activity, mediated by dopaminergic and noradrenergic neurotransmissions. Also, previous studies have shown inhibitory activity of diene valepotriates towards Na(+)/K(+)-ATPase from the rat brain in vitro. Nevertheless, in vivo studies regarding the action of diene valepotriates on this enzyme are still lacking. Considering that Na(+)/K(+)-ATPase cerebral activity is involved in depressive disorders, the aim of this study was to investigate the effects of acute (5 mg/kg, p. o.) and repeated (5 mg/kg, p. o., once a day for three days) diene valepotriate administration on Na(+)/K(+)-ATPase activity in the cortex and hippocampus of mice submitted or not submitted to the forced swimming test. In addition, the protein expression of Na(+)/K(+)-ATPase α1, α2, and α3 isoforms in the cortex of mice repeatedly treated with diene valepotriates (and submitted or not submitted to the forced swimming test) was investigated. Diene valepotriates significantly decreased mice immobility time in the forced swimming test when compared to the control group. Only the animals repeatedly treated with diene valepotriates presented increased Na(+)/K(+)-ATPase activity in the cerebral cortex, and the exposure to the forced swimming test counteracted the effects of the diene valepotriates. No alterations in the hippocampal Na(+)/K(+)-ATPase activity were observed. Repeated diene valepotriate administration increased the cortical content of the α2 isoform, but the α3 isoform protein expression was augmented only in mice repeatedly treated with diene valepotriates and forced to swim. Mice treated with the vehicle and submitted to the forced swimming test also presented an increase in the content of the α2 isoform, but no alterations in Na(+)/K(+)-ATPase activity. These results suggest that cortical Na(+)/K(+)-ATPase may represent a molecular target of the diene valepotriates in vivo and long-term regulatory mechanisms are involved in this effect. Also, the forced swimming test per se influences the protein expression of Na(+)/K(+)-ATPase isoforms and counteracts the effects of the diene valepotriates on cortical Na(+)/K(+)-ATPase.

Antidepressant-like effect of Valeriana glechomifolia Meyer (Valerianaceae) in mice.

The antidepressant-like effect of a supercritical CO2 (SCCO2) Valeriana glechomifolia extract enriched in valepotriates was investigated in a mice tail suspension test (TST) and forced swimming test (FST). The SCCO2 extract decreased mice immobility in the FST (0.5-20 mg/kg p.o.) and elicited a biphasic dose-response relationship in the TST (1-20 mg/kg p.o.) with no alterations in locomotor activity and motor coordination (assessed in the open-field and rota-rod tests, respectively). The anti-immobility effect of the SCCO2 extract (5 mg/kg, p.o.) was prevented by mice pre-treatment with yohimbine (1 mg/kg, i.p., an α2 adrenoceptor antagonist), SCH 23390 (15 µg/kg, s.c., D1 dopamine receptor antagonist) and sulpiride (50 mg/kg, i.p., D2 dopamine receptor antagonist). However, mice pre-treatments with prazosin (1 mg/kg, i.p., α1 adrenoceptor antagonist) and p-chlorophenilalanine methyl ester (4×100 mg/kg/day, i.p., a serotonin synthesis inhibitor) were not able to block the anti-immobility effect of the SCCO2 extract. Administration (p.o.) of the SCCO2 extract (0.25 mg/kg) and imipramine (10 mg/kg), desipramine (5 mg/kg) and bupropion (3 mg/kg) at sub-effective doses significantly reduced mice immobility time in the FST. These data provide the first evidence of the antidepressant-like activity of V. glechomifolia valepotriates, which is due to an interaction with dopaminergic and noradrenergic neurotransmission.

Effect of storage time and conditions on the diene valepotriates content of the extract of Valeriana glechomifolia obtained by supercritical carbon dioxide.

INTRODUCTION: Valepotriates (epoxy iridoid esters) represent an important group of constituents that contribute to pharmacological effects for the genus Valeriana. Storage and extraction of valepotriates is a demanding task, as these compounds are thermolabile and unstable: even when decomposition products are not formed, isovaleric acid liberation from the iridoid nucleus originate compounds with less complex substituents. OBJECTIVE: To study the influence of time and storage conditions on the diene valepotriates (valtrate, isovaltrate, acevaltrate, 1-ß-acevaltrate, 1-ß-aceacevaltrate) content of the Valeriana glechomifolia (native to southern Brazil), extract was obtained by supercritical fluid extraction using CO2 as the fluid (SF-CO2). METHODOLOGY: Above-ground and below-ground material of V. glechomifolia was extracted by SF-CO2 (40 °C, 90 bar). The extract was stored under nitrogen atmosphere or solubilised in methanol. Valepotriates stability was accessed during storage at -20 °C over 8 months through reverse-phase HPLC (mobile phase acetonitrile:water 50:50 (v/v); 254 nm). RESULTS: A gradual increase in valtrate levels and decrease in acevaltrate, 1-ß-acevaltrate and 1-ß-aceacevaltrate, concentration were observed from the first month of storage for the dry extract. However, for the methanol solubilised extract these changes occurred only after the third month and were accompanied by reduction in isovaltrate levels and formation of decomposition products. CONCLUSION: SF-CO2 showed high selectivity for valepotriates extraction. This is the first report on valepotriates molecular conversion, which was less accelerated when the extract was stored in methanol, but under this condition degradation products are also present, probably baldrinals, that are not observed in the dry extract.

In vitro effect of valepotriates isolated from Valeriana glechomifolia on rat P-type ATPases.

Valepotriates are iridoids found in variable amounts in Valerianaceae and might be among the bioactive compounds which confer anxiolytic properties to the Valeriana species. On the other hand, unspecific cytotoxicity has also been described. Presently, however, no particular molecular target has been defined for these compounds. Here we studied the effect of valtrate, acevaltrate, and 1- ß-acevaltrate isolated from Valeriana glechomifolia on the enzymatic activity of rat P-type ATPases. Valepotriates did not affect rat skeletal muscle sarco/endoplasmic reticulum Ca²âº-ATPase (SERCA) activity at the highest concentration used (100 µM). In contrast, the same concentration inhibited roughly half of the total H⁺/K⁺-ATPase activity from rat gastric epithelium (valtrate 54.6 ± 3.2 %, acevaltrate 60.7 ± 7.3 %, 1- ß-acevaltrate 50.2 ± 3.1 %; mean ± SEM, n = 3-5). Finally, these substances showed the highest inhibitory potency toward Na⁺/K⁺-ATPase, and the inhibition curves obtained provided a similar IC50 (in µM) for rat kidney α1 isoform (valtrate 21.2, acevaltrate 22.8, 1- ß-acevaltrate 24.4) and brain hemispheres α2/ α3 isoforms (valtrate 19.4, acevaltrate 42.3, 1- ß-acevaltrate 38.3). Our results suggest that P-type ATPases are differentially inhibited by valepotriates and that Na⁺/K⁺-ATPase might be one of their molecular targets in vivo.

Pilocarpine accumulation on Pilocarpus pennatifolius tissue culture

Callus and cell suspension cultures were established from young leaves of Pilocarpus pennatifolius on Murashige & Skoog (MS) medium supplemented with 5.0 mg/L 2,4-dichlorophenoxyacetic acid (2,4-D) and 1.0 mg/L kinetine. The pilocarpine contents of callus and cell suspension cultures were quantitatively compared by HPLC