Passiflora incarnata L. (Passionflower) extracts elicit GABA currents in hippocampal neurons in vitro, and show anxiogenic and anticonvulsant effects in vivo, varying with extraction method

Abstract

Potential mechanisms of Passiflora incarnata extracts and the effect of extraction methods on ingredients and biological effects were explored. Using the same batch of plant material, total flavonoid yields as measured by high-performance liquid chromatography coupled to diode array detection (HPLC-DAD) increased substantially with hot versus cold extraction methods. Whole Passiflora extract induced prominent, dose-dependent direct GABA(A) currents in hippocampal slices, but the expected modulation of synaptic GABA(A) currents was not seen. GABA was found to be a prominent ingredient of Passiflora extract, and GABA currents were absent when amino acids were removed from the extract. Five different extracts, prepared from a single batch of Passiflora incarnata, were administered to CF-1 mice for 1 week in their drinking water prior to evaluation of their behavioral effects. Anticonvulsant effects against PTZ-induced seizures were seen in mice that received 2 of the 5 Passiflora extracts. Instead of the anxiolytic effects described by others, anxiogenic effects in the elevated plus maze were seen in mice receiving any of the 5 Passiflora extracts.

Fig. 1

HPLC analysis of (A) the flavonoid standard vitexin, and (B) Passiflora incarnata extract (PAS1) using UV detection at 330 nm.

Fig. 2

LC-MS analysis of Passiflora incarnata extract (PAS1). Numbers above the peaks indicate the designated peak number. The likely identification of the numbered peaks is given in Table 2.

Fig. 3

TLC chromatogram (A) and semiquantification by densitometry (B) of amino acids in the five different Passiflora extracts.

Fig. 4

Hippocampal pyramidal cell responses to Passiflora extract. Each specimen trace is from a different cell. The scale bar is the same for all traces. A, Traces of inhibitory currents elicited by Passiflora extract. Inhibition by GABAzine was tested in 2 cells at 0.128 mg/ml, 3 cells at 0.256 mg/ml and 2 cells at 0.512 mg/ml Passiflora extract. In all 7 cases, the block by GABAzine (10 μM) was 100%. B, No currents were seen with amino acid reduced Passiflora extract. C, Dose response curve with points representing 0 mg/ml, 0.128 mg/ml (n=6), 0.256 mg/ml (n=5) and 0.512 mg/ml (n=4). The amino acid reduced extract (red dot) was tested at 0.138 mg/ml (n=8). D, TLC comparison of whole Passiflora extract (Whole PAS), amino acid-reduced PAS extract (AA-reduced PAS), and amino acids removed from PAS extract (Extracted AA) with standard amino acids including GABA.

Fig. 5

Effects of Passiflora extracts on pentylenetetrazol (PTZ) induced seizures in CF-1 mice. A, the average number of stage 2 seizures (face and forelimb clonus). B, the average number of stage 4 seizures (fatal tonic hindlimb extension). Means and standard errors are shown, and statistically significant differences versus control (**) as well as trends toward significance (*) are noted (**p < 0.05, *p < 0.08, one-way ANOVA followed by Tukey’s LSD, two-tailed).

Fig. 6

Effect of Passiflora extracts on CF-1 mice in the elevated plus maze. A, percent time spent in the open arms (time spent in open arms/(time spent in open + closed arms). B, Extending over edges of the open arms. C, ratio of entries into open arms over entries into open + closed arms. D, distance moved in the open arms. Means and standard errors are shown, *p < 0.05 versus control, **p < 0.01 versus control, ***p < 0.001 versus control, one-way ANOVA followed by Dunnet’s posthoc test, two-tailed. All extracts show anxiogenic effects in one or more measures of anxiety.

Fig. 7

Effect of 5 extracts on sensorimotor activity in the rotarod (A, average of 3 trials) and total distance moved in the plus maze (B). None of the extracts resulted in a reduction of locomotor activity.