Twenty-four healthy male and female subjects aged 18 - 40 years were recruited by advertisements in newspapers. Using an anxiety questionnaire (Prüfungsangstfragebogen PAF-S from Hogrefe Verlag, Göttingen, Germany [8] ) only subjects having score values above 60 were selected to participate in the study. Score value 60 is the cut off for being healthy according to evaluation criteria of the test. Subjects must have been capable of giving informed consent. Subjects had to meet following exclusion criteria:

Ÿ Acute or chronic disease with an impact on the study, which becomes obvious by case history or clinical examination (i.e. also Hamilton depression scale).

Ÿ Clinically relevant pathological findings from clinical and laboratory findings.

Ÿ Clinically relevant allergic symptoms.

Ÿ Detection of alcohol at the time of initial examination (screening day SC) or on study day A (positive alcohol test) or by case history. Detection of drugs (positive drug test) at the time of initial examination (screening day SC).

Ÿ Consumption of clinically relevant medication during last fourteen days before and during the active study period based on the notification of the subject or his case history.

Ÿ Consumption of medication with primarily central action (i.e. psychotropic drugs or centrally acting antihypertensives). Known intolerance/hypersensitivity (allergy) to plant derived extracts (Cimicifuga, Cocculos, Passiflora, Valeriana etc.) or any of the ingredients of the investigational product (anamnestic). Presence of a rare genetic disease such as fructose intolerance, glucose-galactose malabsorption or sucrase-isomaltase deficiency (anamnestic).

Ÿ BMI (Body Mass Index) <18 or >32.

Ÿ Consumption of unusual quantities or misuse of coffee (more than 4 cups a day), tea (more than 4 cups a day) or tobacco (more than 20 cigarettes per day).

Ÿ Smoking on study day A.

Ÿ Participation in another clinical trial within the last 60 days.

Ÿ Positive pregnancy test (study day A).

Ÿ Lactation.

Ÿ Cancellation of informed consent.

Ingredients of Calmvalera Hevert tablets and placebo tablets are listed in Table 1 and Table 2.

Each subject was asked for intake of 6 tablets (daily dosage) at a time. The subjects selected for the study had a normal physical examination, electrocardiogram (ECG) and blood count for safety reasons. Laboratory tests for safety are listed in Table 3. Average age of the subjects was 24.88 ± 4.21 years.

EnkephaloVision is the term for the entirely new combination of quantitative EEG (Neurocode-Tracking) and Eye-Tracking.

In this study a 17-channel EEG recording was combined with Eye-Tracking. Details

Other ingredients: lactose-monohydrate (35 mg), magnesium stearate (Ph. Eur.), corn starch.

of the EEG recording have been published earlier [4] but a new modified software for analysis of shorter time epochs of 364 ms duration was used (neo-CATEEM®, supplied by MEWICON CATEEM-Tec GmbH, 4164 Schwarzenberg am Böhmerwald, Austria). A manuscript containing details and validation of the software package―now called “Neurocode-Tracking”―has been published [5] . In short: For the new approach EEG frequency ranges to be analyzed had to be adjusted slightly to give the following exact ranges. Delta: 1.375 - 4.125 Hz; Theta: 4.125 - 6.875 Hz; Alpha1: 6.875 - 9.625 Hz; Alpha2: 9.625 - 12.375 Hz; Beta1: 12.375 - 17.875 Hz; Beta2: 17.875 - 34.375 Hz. This adjustment is a precondition for the new fast dynamic frequency analysis (Neurocode- Tracking) because under this condition each frequency range from delta up to alpha2 contains only one frequency (middle frequency within the particular frequency band). Data were analyzed in the current source density mode. All other features remained identical to the classic analysis used now for nearly 20 years. Maps were constructed by transforming spectral power values into spectral colors followed by additive color mixture (RGB-mode like in TV).

EEG recording was performed in the presence of a video clip, which contained several different cognitive and emotional challenges in series. At the beginning, after a gong for synchronization purposes, one minute was recorded under relaxation (eyes open) as reference, followed by presentation of a fixation cross. After this several cognitive and emotional challenges were presented: The total video was presented before and after intake of 6 tablets with some changes of cognitive tasks for prevention of memorized results. Between baseline recording and intake of Calmvalera Hevert or placebo tablets subjects had a pause for 90 minutes in the restroom. The time line of consecutive scenes is documented in the following Table 4.

Eye-Tracking (equipment and software from Tobii Technology GmbH, Frankfurt, Germany), was performed concomitantly with Neurocode-Tracking (fast dynamic quantitative EEG recording). All mental and emotional challenges were presented as a single video clip. Single challenges were first constructed as a power point file and then converted into a final video clip (by Adobe Captivate software). For offline analysis and synchronization with the eye-track data a screen grabber (Adobe Captivate software) was used to produce a video containing all successively calculated EEG maps of 364 ms duration. A second video was obtained from the eye-tracker software running on a separate computer. It is called a “gaze overlay” movie depicting the presented pictures, tasks or video film. In this gaze overlay video the momentary gaze of the subject is documented by a red spot. Data from the whole group are depicted as so-called heat maps. The presentation always started with an audio signal (gong). This audio signal was registered by screen capture of the EEG computer and was used for synchronization of both videos by means of a video cut software (Adobe Premiere Pro). Due to the processing time of the brain (300 to 400 ms) plus that of the computer (depends individually on the type and number of active processors!) the gaze overlay video is shifted in our case (quad core, 3.4 Giga-Hz) for one second in order to obtain synchronized images between gaze and the particular EEG epoch of 364 ms. For detailed offline documentation a movie was exported and analyzed image by image. Single representative images containing the gaze and corresponding EEG map were cut from the screen by a software tool available on all computers.

On the experimental day (Day A), subjects started with a standardized breakfast. After this two questionnaires had to be filled out: Hamilton depression scale (HAMD) [9] and Hamilton anxiety scale (HAMA) [10] before randomization took place. Then, Enkephalo Vision session was started. A time line is given in Figure 1.

EEG data from the first recording session before intake of the capsules are given as ab-

solute numbers (µV²). For explorative statistical evaluation the nonparametric Wilcoxon test was used. For mathematical differentiation of the different mental loads the 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 color, respectively, followed by an additive color mixture (so-called RGB-mode). In order to document statistically the different electric reaction of the brain to various cognitive and emotional loads, data from each part of the presentation were divided by the data obtained during recording of a relaxed state with eyes open (1 minute) at the beginning. Comparison of Calmvalera Hevert tablets versus placebo was accomplished by evaluation of the second recording of the day 90 minutes after intake. Data from the first recording (baseline) were set to 100% and electrophysiological changes produced by placebo or Calmvalera Hevert tablets are depicted as %-changes thereof. Sample size was defined according to several earlier studies performed under identical conditions.

The study was conducted according to the protocol after approval by the BfArM (Bonn, Germany) and Ethikkommission (Landesärztekammer Frankfurt, Germany) (PP_5014 _HEVERT_FINAL V1.0_23042015) and “Amendment 1 for PP_5014_HEVERT_FIN AL V1.0 _23042015”.

Twenty-four subjects were recruited for this study on the base of filling out a questionnaire dealing with examination anxiety. Concomitant presentation of a series of mental and emotional challenges during recording of the EEG was performed before and 90 minutes after intake of 6 Calmvalera Hevert tablets or placebo. The absolute starting values of spectral power in the six frequency ranges (delta to beta2) are given in Table 5. These values do not differ from each other in a statistically significant way suggesting that both groups placebo and verum have a similar starting position. This allows for a pre-post comparison of drug action. These absolute values are set to 100% and drug induced changes are documented in % of these starting values.

In order to relate the result from the anxiety questionnaire “HAMA” with EEG parameters a correlation was searched based on the first recordings before administration of the drug. The result indicates that absolute spectral alpha power from all 24 subjects during the recording condition “eyes open” in a relaxed state before drug administration correlated with the score values of the questionnaire. Figure 2 and Figure 3 document the results of this analysis showing a correlation coefficient r= −0.418 (p < 0.017) for alpha1 and r = −0.329 (p < 0.084) for alpha 2 spectral power.

Next step in the analysis was to find out if the different cognitive and visually presented

emotional challenges lead to consistent changes of spectral power. For this purpose, data from each challenge were averaged over the presentation time in order to follow each of the brain regions with respect to the pattern of spectral power changes with respect to each frequency. In order to evaluate challenge specific changes of spectral power each challenge was calculated against the “eyes open” condition in order to eliminate unspecific responses. As expected, highly statistically significant increases of spectral power in the delta, theta and beta range were observed during all challenges. An overview on the statistical significance of the regional distribution of spectral power enhancement in comparison to the recording condition “eyes open” is documented in Figure 4(a) and Figure 4(b) for cognitive challenges and Figure 5(a) and Figure 5(b) for emotional challenges.

There is wide believe that the two brain hemispheres can function independently from each other. We therefore have analyzed the frequency changes in the presence of all challenges for each hemisphere in separate. The result is documented in Table 6 and Table 7. In general, the challenges led to statistically significant increases of delta (occasionally also theta) and beta2 spectral power in comparison to the recording condition “eyes open”. As one can see, there are some differences like the extent of delta and beta2 spectral power under the recording condition “eyes open”. During presentation of emotional pictures higher beta2 power is seen in the left hemisphere. Watching the examination video clip also leads to stronger enhancement of beta2 power in the left hemisphere. The largest difference with respect to beta2 power between the hemispheres was observed during the horror video. Regarding now the effects of all challenges with respect to single hemispheres strongest involvement of delta waves is recognized in both hemispheres.

First impressions of the effectiveness of Calmvalera Hevert tablets are obtained during single scenes of the presented challenges like emotional images (example: Figure 6), video clips (example: Figure 7). In order to uncover the influence of Calmvalera Hevert tablets under the different recording conditions identical single scenes were docu-

mented before and after intake of the drug. The spectral power of all regions was looked at in terms of possible changes. A first impression was that alpha1 as well as alpha2 spectral power had increased in regions like the temporal lobe (yellow bars in Figure 6). An example recorded during watching the video “the dentist” is documented in Figure 7. Here stronger alpha1 activity is seen at central and temporal regions.

Quantitative analysis of frequency changes induced by Calmvalera Hevert tablets with respect to different brain regions revealed a statistically significant increase of apha1

and alpha2 spectral power at electrode positions F₃, C₃, P₃, T₃ and O₁ already under the recording condition “eyes open” as depicted in Figure 8. Remarkably, these electrode positions represent all areas on the left hemisphere (uneven numbers). There are also minor increases on the right hemisphere (even numbers) but none became statistically significant. Colors of the maps shift into a more yellow green direction as consequence of increased alpha activity.

When subjects were concentrating their attention on a fixation cross for one minute again alpha1 and alpha2 spectral power increases emerged nearly at the same electrode positions plus at F₄ and F₈ on the right hemisphere in a significant manner in comparison to placebo. An overview is given in Figure 9. Again colors of the maps shift into a more yellow green direction as consequence of increased alpha activity stronger within the left hemisphere (right side).

A very similar pattern was observed when watching the video “at the dentist”. Highest increases of alpha spectral power appeared at electrode position C₃, followed by F₃ and O₁. Under this recording condition also P_z and T₆ showed very slight, but statistically significant increases as documented in Figure 10. Due to increases of alpha activity dominant prevalence of yellow-green colors is observed within the left hemisphere (map right side of the image).

Analysis of the spectral power changes during watching the video dealing with an emergency surgery in the bush (Horror-OP) again revealed largest increases of alpha2 power at electrode positions C₃ and O₁. Minor but still significant increases were seen at C₄, O₂ and T₆. An overview on changes with respect to all brain areas is given in Figure 11. Please note yellow-green spot in the map due to stronger focal alpha activity.

During performance of a cognitive task like the “Picture Comparison” again electrode positions C₃ and O₁ showed high statistically significant increases of alpha power. In addition, some increase of beta power emerged at electrode positions C₃, T₃ and O₁. Data are documented in Figure 12. With respect to the map increased alpha activity is

observed in frontal parts of the brain at interpolated regions indicated by predominating yellow and/or green color (more in the left hemisphere).

Since right and left hemisphere reacted in a somewhat quantitatively different way to several challenges, they were analyzed separately with respect to an action of Calmvalera Hevert tablets in comparison to placebo. Regarding spectral delta and theta spectral activity virtually no effects were seen except for a tiny increase of theta power when watching emergency surgery (Horror-OP). Strongest statistically significant changes were observed in the alpha frequencies during several challenges as depicted in Figure 13 for the left hemisphere. However, there were also some minor but statistically sig-

nificant increases of beta1 power and one result in beta2 power. Statistically significant spectral changes within the right hemisphere were only observed in the alpha1 range during watching the fix cross (p < 0.1), in the alpha2 range during watching the fix cross (p < 0.05) and the horror video (p < 0.05). Finally, beta1 spectral power had increased again during looking at the fix cross (p < 0.1).

Finally, all 102 parameters (17 electrode positions × 6 frequency ranges) were fed into a linear discriminant analysis for comparison with other drugs. As documented in Figure 14 the effect of Calmvalera Hevert tablets (verum) was projected at a considerable distance to the effect of its placebo (Figure 13) proving its fast effectiveness within 90 min

after intake. Interestingly, another homeopathic drug (Neurexan®) and a plant derived preparation (Neuravena®), both prescribed for the same indication as Calmvalera Hevert tablets, are projected into close vicinity of Calmvalera Hevert tablets (verum in Figure 14).