sentation of a row of letters and numbers that appeared on a screen for 4 seconds followed by 10 seconds of black screen. This was followed by a multiple choice test with four possible answers.
All recordings were repeated 0.5, 1.5, 3 and 4 hours after the administration of three tablets following the reference pre-drug period (overview in Figure 1). Between assessments, subjects spent their time in the facility’s break room. All experiments took place at the same time of the day (starting at 8 a.m.).
The EEG was recorded bipolary from 17 surface electrodes according to the international 10/20 system with Cz as physical reference electrode for calculation of the common average reference  (Computer aided topographical electroencephalometry: CATEEM®—from Mewicon GmbH, 4164Schwarzenberg, Austria) using an electrocap. The raw signals were amplified, digitalized (2048 Hz/12 bit) and transmitted to the computer via fibre optical devices. The automatic artefact rejection of the CATEEM®-System, which eradicates EEG-alterations caused by eye blinks, swallowing, respiration etc. during the recording was visually controlled and individually adjusted by the investigator. Electrocardiogram (ECG) and electrooculogram (EOG) were recorded on one channel each in order to facilitate detection of those signals superposed onto the EEG. The artefact rejection set-up was observed for about 5 minutes prior to the start of the recording to ensure that all artefacts were correctly recognized and eliminated from further evaluation. For safety purposes, the original raw data were saved on optical disk and separate hard disks in order to allow reevaluation of the artefact rejection mode if necessary. In these cases, the experimental session was re-examined off-line with a newly adapted rejection mode. The amount of rejected data was determined automatically and given as a percentage of total recording time. In addition, the entire recording and the computer-based automatic artefact rejection were continuously supervised and adjusted by a trained technician . Logarithm of data was taken for discriminant analysis in order to approach normal distribution of values, which underwent frequency analysis for quantitative evaluation as first proposed nearly 80 years ago .
In this study, the EEG was computed not in the potential mode measured as voltage, but in a surface charge mode obtained by Laplacian estimates also known as current source density (CSD) analysis . The charge is the 2nd derivative of the potential and gives the spatial curvature of the potential. All calculations are based on the standard set-up of the 10/20 system of recording. Under the condition of using a homogenous, steadily
Figure 1. Timeline of experimental setup.
conducting medium, the surface charge mode provides the source density of the electrical flow on the cortex surface. Whereas the EEG in the potential mode tends to produce a more extensive and diffuse picture of changes, the Laplacian estimate acts as a spatial filter emphasizing local sources over distant sources. Others  were able to demonstrate that spectral parameters obtained from the CSD showed higher correlations with computer tomography data than those calculated from the potential mode of the EEG. We therefore used this methodology in order to describe the focal changes in brain activity. The signals from all 17 electrode positions underwent the Fast Fourier Transformation (FFT) based on 4-second sweeps of data epochs (Hanning window). Data were analysed from 0.86 to 35 Hz using the CATEEM® software. In this software, the resulting frequency spectra are divided into six frequency bands: delta (1.25 - 4.50 Hz), theta (4.75 - 6.75 Hz), alpha1 (7.00 - 9.50 Hz), alpha2 (9.75 - 12.50 Hz), beta1 (12.75 - 18.50 Hz) and beta2 (18.75 - 35.00 Hz). This frequency analysis is based on absolute spectral power values. Band powers are first compared between placebo and verum during relaxation, and the results of verum and placebo then compared under different mental loads.
EEG data from the first recording session before drug intake are given as absolute figures (μV2). By setting the absolute electric power of this first pre-drug recording to 100%, changes produced by the preparations are given as percentage of this pre-drug condition. Except for generating brain maps, only those particular electrode positions were used for the numeric comparison of verum to placebo during mental load, which have been found to change during the particular mental challenge. For explorative statistical evaluation, the nonparametric sign test was used. For mathematical classification of drug effects, linear discriminant analysis according to Fischer was used. Results from the first three discriminant functions were projected into space (x, y and z coordinates), whereas results from the fourth to sixth discriminant functions were coded into red, green and blue respectively. Values on the 4th to 6th linear discriminant functions respectively determine the amount of red, green and blue in additive colour mixtures analogous to the RGB mode of TV. Reference preparations used for comparison were tested in our laboratory earlier under identical conditions. A linear projection was defined by taking these reference data. Data sets from other preparations to be analysed are processed according to the originally defined mapping function of reference compounds.
Psychometric performance was evaluated by the mathematical product of “quantity × quality”, where quantity was defined as the number of correct answers and quality as the number of correct answers divided by the number of tasks tackled.
Pascoflair® 425 mg is a coated tablet containing dry extract (5 - 7:1) of Passiflora incarnata L. (Passionflower) 425 mg, extractant: ethanol 50% (V/V). Pascoflair® 425 mg is registered in different countries for the treatment of nervous restlessness and anxiety and also as an aid to sleep (in cases of restlessness or insomnia due to mental stress).
Pascoflair® 425 mg is marketed by Pascoe pharmazeutische Präparate GmbH, Giessen, Germany. The verum preparation consisted of the maximum daily dosage of 3 coated tablets of Pascoflair® 425 mg.
3.1. Analysis of Single Frequency Ranges during Relaxation
Source density analysis of the primary recording of the reference EEG before drug administration revealed similar absolute spectral power values for the day of placebo administration (Pl) compared with the day of active drug administration (Pas) for all electrode positions as documented in Table 1. These data were taken as the baseline; all other data after drug intake refer to this reference recording of the particular experimental day and are expressed as % of these pre-drug data. After ingestion of 3 coated tablets of Pascoflair® 425 mg, some differences were recognized with respect to spectral power values after Fast Fourier Transformation in comparison to the pre-drug values, when the median was calculated for all electrode positions. The greatest differences between verum and placebo were seen with respect of the diurnal increases of delta and additionally to a lesser degree of theta spectral power. In the presence of verum, the increase in delta power as normally observed was completely attenuated as documented in Figure 2. This difference was highly statistically significant (p < 0.02) at 3 and 4 hours after administration. Theta spectral power also was attenuated at 3 and 4 hours after ingestion. Statistical significance, however, was less pronounced at 3 hours (p < 0.08). In addition, there was some increase of alpha2 spectral power which did not reach statistical significance.
3.2. Analysis of Spectral Power during Mental Performance
Changes of electric power within all frequency bands at single electrode positions during mental performance are shown in Figure 3. Obviously, fronto-temporal beta power
Table 1. Absolute EEG spectral power.
increased to some degree during all three mental challenges. The greatest differences between placebo and Pascoflair® 425 mg were observed during the performance of the memory test. Here not only beta but also delta and theta activity was enhanced. There are some more statistically significant changes to spectral power with regard to single frequency changes in comparison to placebo, but statistical analysis of the different locations is not Bonferroni-corrected for multiple comparisons. With regard to median values of all electrode positions, EEG analysis revealed a statistically significant attenuation of delta and theta power. During performance of the d2-test, significant attenuation of delta power took place 3 and 4 hours after ingestion (Figure 4). Attenuation of theta power was significant at 1.5 and 4 hours after ingestion, alpha2 power at 3 hours. During the concentration-performance test (CPT), delta and to some degree also theta power was attenuated with regard to the medians of all electrode positions. This effect was strongest at 3 and 4 hours after intake of the active preparation as shown in Figure 5. Attenuation of the other frequencies was not statistically significant (not shown). However, there was a highly significant enhancement of alpha2 power after only 30 minutes (p < 0.02). During performance of the memory test, less theta and alpha1 spectral power was produced in the presence of verum in comparison to placebo with regard to the median of all electrode positions. Statistical significance was reached for theta at 4 hours after intake and for alpha1 after 3 hours (not shown). Changes with respect to the other frequencies were not consistent and only reached statistical significance for beta1 at 3 hours after administration (not shown).
Performance of psychometric tasks did not reveal any
Figure 2. Time course of spectral frequency changes in comparison to pre-drug values, which are set to 100%. Comparison of verum and placebo. Statistics: **correspond to p < 0.8; ***correspond to p < 0.02.
Figure 3. Spectral frequency changes at 17 electrode positions during mental performance (CPT = concentration performance test with calculations). Red: delta, orange: theta, yellow: alpha1, green: alpha2, light blue: beta1 and dark blue: beta2. F = frontal, C = central, P = parietal, T = temporal, 0 = occipital. Even numbers represent the right hemisphere, uneven number represent the left hemisphere. Statistics: **correspond to p < 0.8; ***correspond to p < 0.02. Data are documented for 3 h after intake.
Figure 4. Time course of spectral frequency changes in comparison to pre-drug values, which are set to 100%. Comparison of verum and placebo. Statistics: **correspond to p < 0.8; ***correspond to p < 0.02.
Figure 5. Depiction of the result of linear discriminant analysis of EEG source density data for all four recording periods after administration of verum and placebo. Result from the first three discriminant functions is displayed by means of space coordinates x, y and z. Values of the 4th to 6th linear discriminant functions respectively determine the amount of red, green and blue in additive colour mixtures analogous to the RGB mode of TV.
difference between placebo and verum (Table 2).
This experimental series showed that the preparation Pascoflair® 425 mg already modulated electric brain activity after a single acute intake compared to placebo. Spectral power in the delta and theta range was attenuated in comparison with the time-dependent increase normally observed due to circadian rhythm. The median value calculated from all electrode positions indicated a statistically significant attenuation of spectral power 3 and 4 hours after administration. The meaning of this preparation-dependent effect is not absolutely clear, especially since it was observed under nearly every recording condition. It can possibly be interpreted as a kind of stabilization of electric activity which might make brain activity more resistant to external disturbances. Focal pathological increases of delta and theta power are regularly observed in patients suffering from epilepsy. In fact, there is one report in the literature describing anticonvulsive effects of the aerial parts of Passiflora incarnata extract in mice . It is possible that the use of Pascoflair® 425 mg might eventually be used in an addon therapy for epileptic patients. But this interpretation must remain speculative until supported by data from further clinical tests.
Since it is quite difficult to evaluate the data from 17 electrode positions in the presence of 6 frequency ranges (a total of 102 parameters), the mathematical tool of discriminant analysis was used to describe the action of the active drug compared to placebo for the recording condition “eyes open”. This method permits the time-dependent evaluation of all changes with respect to the predrug condition. As can be seen in Figure 5, all placebo values (labelled PL05 to PL4)—representing circadian rhythm dependent changes—are clustered. Depiction of the result of the effect of verum at 0.5, 1.5, 3 and 4 hours after administration in comparison to the effect of other synthetic reference drugs or plant-derived preparations tested earlier revealed clear effects for the active drug after only 30 minutes (labelled Pas05, Pas15, Pas3 and Pas4 in Figure 5). This early effect of Pascoflair® 425 mg is in line with a report on another Passiflora incarnata
Table 2. Result of psychometric procedures during d2-test.
extract tested in 60 ambulatory surgery patients. In a placebo-controlled, double-blind study, preoperative anxiety scores after only 30 minutes were significantly lower in the Passiflora group than in the control group .
In the past, discriminant analysis of spectral power was successfully used to differentiate drug profiles from each other in rats according to their clinical indication . Drugs with a similar clinical indication clustered together whereas drugs with different indications were widely diverse in space and colour. The same reasoning can be followed in showing that Pascoflair® 425 mg took position near some other drugs with well-known clinical indications. One of the closest neighbours in space (representing the result of the first to third discriminant function)—but with a different colour due to the result of the fourth to sixth discriminant function—is L-Theanine. Interestingly, L-Theanine is regarded as a compound with relaxing properties and is reported to reduce psychologycal and physiological stress responses . At the same time, possible neuroprotective and cognitive enhancing properties are also reported . Another neighbour not too far away is a preparation consisting of Gingko and Ginseng extract .
Interestingly, there was obviously no negative influence on mental activity, since psychometric performance remained stable throughout the whole experimental period. On the contrary, during performance of the memory test, increases of delta, theta and beta power at frontal electrode positions indicative of good mental fitness were observed. This frontal increase of delta and theta power during mental load was not observed in demented people  and subjects suffering from mild cognitive impairment . Presumably, the great scatter of psychometric results from only 16 volunteers prevented more insight into effects on memory function in the presence of Pascoflair® 425 mg.
In summary, Pascoflair® 425 mg was shown to produce a significant change to the pattern of electric activity in the brain as a surrogate parameter for its central efficacy. The increase of delta, theta and beta power at frontal electrode positions during performance of the memory test is indicative of the preservation of good mental fitness. Spectral changes of electric activity resemble those seen in the presence of L-Theanine and Ginkgo/Ginseng. The results from this study document the effectiveness of the preparation after only 30 minutes. In contrast with reports in the literature on sedative and cognition attenuating effects of various synthetic preparations such as benzodiazepines, this study did not reveal any such negative side effects for Pascoflair® 425 mg.
Petra Werling is gratefully acknowledged for taking the EEG recordings. We thank Leonie Schombert for her help in documenting the results. We appreciate the help of Bianka Krick in critically reviewing the manuscript. Ingrid Keplinger-Dimpfel performed the logistics and quality control of the study.
7. Authors’ Contributions
W.D. provided the electrophysiological technology, supervised the performance of the experiments, gave interpretation of the results and wrote the manuscript. K.K. performed the medical examinations and evaluated unexpected events. G.W. initiated the study and made major contributions to the design. She also provided important information on the pharmacology of the preparation’s constituents.
*Competing interests: The authors declare that they have no competing interests.