The endemic South African succulent plant Sceletium tortuosum (L.) N.E. Br., family Mesembryathemaceae, is known as kanna in Nama, kougoed in Afrikaans, and sceletium in English. This plant has one of the oldest documented histories of use in South Africa, with the earliest unequivocal written record dated to 1685 [1] . Kolben declared that sceletium was “the greatest cheerer of the spirits, and the noblest restorative in the world” [2] . The plant has been used as a tea and as a masticatory for millennia by indigenous San hunter-gatherers and Nama pastoralists for endurance and well-being. An 1873 ethnobotanical record (the entry is for kaauwgoed the old Dutch name for sceletium) reports that the plant “gives strength to their limbs, and takes away pain, and makes their memory strong” (Manuscript MSS BC151 006, Manuscript and Archives Department of the University of Cape Town) [3] .

Over the last two decades manufactured products containing sceletium raw material and extracts have become widely available as supplements, health teas and beverages sold through farm stalls, health food shops, pharmacies and via internet and by multi-level marketing. The products are typically used for promoting a sense of calm, well-being, relieving stress, elevating mood, reducing anxiety and enhancing focus and concentration.

A proprietary standardized and characterized extract of a cultivated selection of Sceletium tortuosum (Zembrin^®) was shown in-vitro to be a dual 5-HT uptake inhibitor and PDE4 inhibitor [4] . In an in-vivo study in rats, field potential recordings were made for three doses of this extract and for a saline control [5] . Zembrin^® dose dependently attenuated spectral power in all electroencephalogram frequency ranges to varying degrees. Comparison of the electropharmacograms of Zembrin^® with the electropharmacograms of selected botanical extracts and pharmaceuticals demonstrated that this extract Sceletium tortuosum produces a similar EEG signature to Ginkgo biloba, and to the EEG signature of the synthetic PDE4-inhibitor rolipram, supporting potential health and wellness applications for improving mood and cognitive function.

Extract Sceletium tortuosum (Zembrin^®) has been shown to be safe and well tolerated in pre-clinical [6] and clinical studies [7] . The acute effects of extract Sceletium tortuosum (Zembrin^®) administration in 16 healthy young adults was studied in a pharmaco-fMRI study focused on anxiety-related activity in the amygdala and its connected neuro-circuitry. Amygdala reactivity to fearful faces under low perceptual load conditions was attenuated after a single 25.0 mg dose of Zembrin^®. Follow-up connectivity analysis on the emotion matching task demonstrated that amygdale-hypothalamus coupling was also reduced. These results demonstrated for the first time the attenuating effects of Sceletium tortuosum on the threat circuitry of the human brain and provide supporting evidence that the dual 5-HT reuptake inhibition and PDE4 inhibition of Zembrin^® might have anxiolytic potential by attenuating the sub-cortical threat response [8] .

The neurocognitive effects of extract Sceletium tortuosum (Zembrin) was tested for the first time in a repeat dose crossover study in a group of adults (n = 21, mean age 54.6 years) using the CNS Vital Signs^® battery of tests. Zembrin^® 25.0 mg daily dosage taken for 3 weeks significantly improved executive function and cognitive set flexibility compared with the placebo group. These promising cognitive enhancing effects lend support to PDE4 inhibition as a possible mechanism of action for Zembrin^®, operating through the PDE4-cAMP-PCREB cascade [9] .

Drugs typically exert their action within the organism by interaction with defined large protein molecular targets, including receptors, enzymes, ion channels, and transporters. With respect to the central nervous system, neurotransmitter receptors represent a major drug target. Interaction of drugs with these molecules induces a signaling cascade, which finally ends up with the control of ion channel conductance. Since the electric activity of single neurons depends on the set of momentarily active ion channels, communication between neurons is governed by channel activity. From here, it is obvious that electrical field potentials contain the net information of larger local networks of electrically active neurons, reflecting the interaction of drugs with their targets within the concert of neurotransmission.

Frequency analysis of the field potentials in the presence of drugs can be depicted as the so-called electropharmacogram, which has been widely used to characterize drug actions on rat [10] and human brains [11] . Interpretation of the results is made with respect to the likely underlying neurotransmitter activity responsible for changes in the selected frequency ranges, and aims at advancing an understanding of possible clinical applications in humans. The relationship between EEG delta waves and cholinergic neurotransmission was first recognized in 2005 [12] , and it was recognized that theta waves are influenced by drugs acting at the biochemically defined norepinephrine alpha2 receptor [13] . Presynaptic interaction with this receptor leads to drowsiness and sleep, and increases of theta waves have been used as part of a formula describing depth of sleep in humans. Dopaminergic activity is reflected by changes in alpha2 frequencies [14] .

Many herbal preparations have been tested using neurophysiological techniques [15] [16] . In order to characterize the psychophysiological effect of Zembrin^®, a new clinical study design was used, named “EnkephaloVision”. This new approach consists of the combination of quantitative EEG recording during cognitive and emotional challenges with conventional Eye-Tracking, providing very high time resolution of epochs of 364 ms (Neurocode-Tracking) [17] .

In the present study 60 subjects, randomized to receive a single dose of 25.0 mg or 50.0 mg Zembrin^®, or placebo, were exposed visually to 5 cognitive (Stroop-Test, Brain Teaser, Memory-Test, Picture Comparison, CPT-Test) and 4 emotional challenges (Emotion Pictures, Animal Videos and Horror Videos), by asking for task performance and watching video clips of a short duration. Regional quantitative EEG changes in the presence of these challenges during baseline recording have been documented in a preceding publication [18] .

Sixty healthy male and female subjects (30/30) aged between 40 and 75 years were recruited by announcements in the newspaper. Mean age was 51.53 ± 7.78 (male) and 51.93 ± 7.34 (female).

Inclusion criteria:

・ Healthy male and female subjects.

・ Age between 40 and 75 years (both included).

・ Subjects fluent in the German language.

・ Right handed.

・ BMI (body mass index) > 18 or <32.

・ Subject must be capable of giving informed consent.

・ Acceptance of written consent to participate in the study after education in written and oral form (informed consent).

・ No psychiatric or neurologic disease including epilepsy, cerebrovascular disturbance or traumatic injury.

・ No major or untreated disease including severe uncontrolled diabetes, ischemia, infarct, unstable angina pectoris or uncontrolled high blood pressure.

・ No presence of clinically relevant pathological EEG features or artifact-free portion of the screening EEG <30%.

・ No clinically relevant allergic symptoms.

・ No detection of alcohol at the time of initial examination (day SC) or on study day A (negative alcohol test) or by case history.

・ No detection of drugs (positive drug test) at the time of initial examination (day SC) or drug abuse within the last 6 months.

・ No 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.

・ No consumption of medication with primarily central action (i.e. psychotropic drugs or centrally acting antihypertensive).

・ No known intolerance/hypersensitivity (allergy) to plant-derived extracts or any of the ingredients of the investigational product (anamnestic).

・ No presence of a rare genetic disease such as fructose intolerance, glucose-galactose malabsorption or sucrase-isomaltase deficiency (anamnestic).

・ No 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).

・ No smoking on day A.

・ No participation in another clinical trial within the last 30 days.

・ No positive pregnancy test (day A).

・ No lactation.

The active ingredient of the investigational product is a special extract of the above ground material of a cultivated traditionally used selection of Sceletium tortuosum, Zembrin^®. This is standardized and characterized after aqueous ethanolic extraction (purified water 30% V/V and ethanol 70% V/V, spray-dried onto a maltodextrin carrier), and was manufactured to GMP by Polifenoles Naturales S.L., Las Palmas, Gran Canaria, Spain. UPLC analysis against validated analytical reference standards confirmed that the total alkaloid content of the extract conformed to the specification of 0.4% total alkaloids, and the relative concentrations of mesembrenol and mesembrenone greater than 60% by weight, and mesembrine less than 20% by weight. Active capsules contained 25.0 mg and 50.0 mg of the extract, together with mannitol as excipient, while identical-looking placebo capsules contained only mannitol. Placebo and active capsules were manufactured according to GMP by Hubertus Apotheke, Berlin, Germany.

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

In this study a 17-channel EEG recording was combined with Eye-Tracking. Details of the EEG recording method have been published earlier [19] . 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, Austria). A manuscript containing details and validation of the software package―now called “Neurocode-Track- ing”―has been published [20] . In short: For the present new approach, the 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 [21] . All other features remained identical to the classic analysis currently in use for nearly 20 years [17] . Maps were constructed by transforming spectral power values into spectral colors followed by additive color mixture (RGB-mode as in TV).

The recording was performed in the presence of a video clip (s. methods), which contained several different cognitive and emotional challenges in series (Table 1). The total video was presented twice: a) during baseline

recording and b) 120 min after intake of one capsule Zembrin^® 25.0 mg or 50.0 mg or one placebo capsule. Representative scenes from the total video are given in Figures 3-11. The time line of consecutive scenes is documented in the following Table 1.

Eye-Tracking (equipment and software from Tobii AB, S-18217 Danderyd Sweden), 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) 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 films. In this gaze overlay video the momentary gaze of the subject is documented by a red spot. 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 (called an Enkephaloglyph [21] ) were cut from the screen by a video editing software tool available on all computers.

EEG data from the first recording session before intake of the capsules are given as absolute numbers (µV²). For exploratory statistical evaluation the non-parametric Wilcoxon test was used. For mathematical differentiation of the different mental loads 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 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 fixation of the cross on the monitor (1 minute) at the beginning. Comparison of the effect of 25.0 mg, 50.0 mg Zembrin^® capsules versus placebo was accomplished by evaluation of the second EEG recording of the day 120 minutes after intake. Data from the first recording (baseline) were set to 100% and electrophysiological changes produced by placebo or Zembrin^® 25.0 mg or 50.0 mg capsules are depicted as %-changes thereof. Estimation of the number of subjects to be included into the study was performed by considering data from earlier experimental results obtained under a similar experimental design. Level of statistical significance in comparison to placebo was defined as ^*p < 0.10, ^**p < 0.05 and ^***p < 0.01. The study was conducted according to the Protocol after approval by the Ethikkommission [ethics committee] of the “Landesärztekammer Frankfurt, Germany on 14.07.2015 (PP_2615_HG & H_FINAL V1.0_14072015). The study was conducted from September to December 2015.