Eight adult male Fisher 344 rats (8 months of age and day-night converted, weight about 400 g, obtained from Charles River Laboratories, Sulzfeld, Germany) were implanted with 4 bipolar concentric steel electrodes within a stereotactic surgical procedure. The principles of laboratory animal care were followed in all trials and the local authorities responsible for animal care allowed the performance according to German Health Guidelines.

For our investigations we used herbal extracts of Herba Sideritis scardica or euboa produced by Finzelberg GmbH & Co KG, Andernach/Germany under GMP conditions. Raw material was sourced from cultivations in Bulgaria for Sideritis scardica and from Greece for Sideritis euboa. Extraction was done by exhaustive percolation with Ethanol 20% V/V at 40˚C (during 12 hours). The Drug-to-Extract-Ratio (DER) native was 6:1 for each. The extracts was gently dried on a vacuum belt with 30% drying excipient (Maltodextrin) and finally milled to a homogene powder (95% under 50% mesh).

Reference extract from Humuli cones was supported by Martin Bauer Group GmbH & Co. KG, Vestenbergsgreuth, Germany (extraction solvent Methanol 40% V/V). Tebonin® tablets (containing standardized Ginkgo biloba extract acc. Ph. Eur.) and Memantine as single compound was sourced from local pharmacies.

Hippocampus slices were obtained from 25 adult male Sprague-Dawley rats (Charles River Wiga, Sulzbach, Germany). The methodology used was identical to that reported earlier [12] . Rats were kept under a reversed day/night cycle for 2 weeks prior start of the experiments, to allow recording of in vitro activity from slices during the active phase of their circadian rhythm [13] [14] . All slices were pre-incubated for at least 1 h in Carbogen saturated ACSF (pH 7.4) in a pre-chamber before use [15] . Immersion chamber (List Electronics, Darmstadt, Germany) according to Haas [16] at 35˚C [17] . Recording of extracellular field potentials from the pyramidal cell layer of CA1 [15] was performed according to conventional electrophysiological methods [18] .

Statistics were calculated according to Wilcoxon, Mann and Whitney. Finally, data were compared to a database containing about 200 preparations tested under identical conditions. Comparison also included use of linear discriminant analysis according to Fischer for positioning of the preparation-induced electric pattern within a matrix of reference drugs with known clinical indication.

Effects of vehicle (0.9% NaCl)

The electropharmacogram was recorded continuously for 5 h after administration of the test item, in this case saline. Changes of spectral power within the four brain regions following administration could not be detected. The pattern remained stable for 5 hours as documented in Figure 1.

Effects of Sideritis euboa Extract UB2010-57

Spectral frequency changes in the presence of 100 mg/kg of Sideritis euboa Extract were dominantly seen in the frontal cortex (a lower dosage of 50 mg/kg did not reveal changes of spectral power). In difference to the results of Sideritis scardica increases of power within all frequencies are seen which diminish with time (Figure 2).

Effects of Sideritis scardica Extract UB2010-55

Spectral frequency decreases in the presence of 50 mg/kg of Sideritis scardica UB2010-55 were dominantly seen during the first hour after administration (Figure 3). All brain areas are involved. Within the hippocampus decreases of spectral beta power are seen throughout the whole recording period.

When comparing the pattern of frequency changes of both Sideritis extracts to changes

induced by other preparations a close similarity of Sideritis scardica extract to those of Gingko biloba extract (Tebonin®) and Memantine emerges, whereas the change induced by Sideritis euboa extract approached that observed in the presence of Humulus cone extract (Figure 4).

No significant difference was detected with respect to motion. The extracts of Sideritis scardica and euboa showed a tendency of increased motion within one hour after administration, followed by a decrease on output level. However, due to high variation of values no safe conclusion can be drawn. Data are documented in Table 1.

In order to compare the complicated pattern of frequency changes of the preparations with each other, the procedure of linear discriminant analysis (LDA) was used. Projection of the result of this calculation into space and colour (six-dimensional) led to an obvious separation of the action of some reference compounds with known

clinical indication. Using the three spatial coordinates for the first three discriminant axes and green, red and blue colour for the depiction of the result of the second three discriminant functions, indication specific activity changes can be visualized in a more interpretable manner by comparison to the reference compounds. Figure 5 documents the result for the extracts from 2 different Sideritis species. Firstly, three-dimensional space gives evidence of similarity or dissimilarity of drug action. Additionally, equal or similar colours suggest equal or similar mechanism of action. Reference drugs were mostly injected i.p. and were analysed for their action during the first half hour after administration. The orally administered extract from Sideritis scardica is analysed for the time period first sixty minutes after administration because of slower uptake by oral administration. Result from Sideritis scardica extract groups in the neighbourhood of Ginkgo extract (Ph. Eur. quality) and Memantine. The projection of the Sideritis euboa data reveals a different projection into space and colour and thus indicates a different mode of action. But remark that Sideritis euboa develops its main action not before the second hour after administration. Its position is found at a completely different position in the discriminant projection not far away from Humulus cone extract.

Electric activation of the pyramidal cells can be achieved by single stimuli or a by using a burst pattern of impulses for only one second. The result of this activation is recorded as a so-called population spike. Its amplitude reflects the number of pyramidal cells

recruited. Responses following single stimuli are stable for hours. Examples for responses to single stimuli or theta burst stimulation are documented in Figure 6 for a single

slice. Under basic conditions the amplitude of the population spike following a constant current impulse amounts to about 1 mV. As can be seen from Figure 6 there is a profound difference with respect to the activation by the two extracts which could be confirmed in all other slices.

The presence of the two basic Sideritis extracts led to a different concentration dependent effect on the amplitude of the population spike during single shock stimulation in the range of 0 to 100 mg/L. Whereas Sideritis scardica induced an increase of the spike amplitude, extract from Sideritis euboa produced either no response or at higher concentrations an attenuation of the spike. Maximum increase by Sideritis scardica was achieved by 50 mg/L. Decreases induced by Sideritis euboa started with 50 mg/L and continuously seemed to induce further decrease of population spike amplitude after 100 mg/L. Effects of the blend point into the direction of an increase of the potential but clearly less at a concentration of up to 50 mg/L in comparison to the effect of Sideritis scardica alone. Data for average values (n = 4) of the two extracts and a blend there of are documented in Figure 7 for the recording condition “single shock stimulation, SS”.

The presence of the two different Sideritis extracts led to a different concentration dependent effect on the population spike amplitudes also during theta burst stimulation in the range of 0 to 100 mg/L. As already seen after single shock stimulation Sideritis scardica produced a clear concentration dependent increase of the amplitude of the population spike, whereas Sideritis euboa led to effects in the opposite direction at least at higher concentrations. Again no effect was seen in the case of Sideritis euboa up to

50 mg/L. After this, a concentration dependent decrease could be documented. A similar picture as seen after single shock stimulation also emerged in the presence of the 1:1 blend. Up to 50 mg/kg effects―as seen after Sideritis scardica―were attenuated. Data for mean values (n = 4) of the two extracts and the 1:1 blend thereof are documented in Figure 8 for the recording condition “theta burst stimulation, TBS”.

Finally, results were compared to two preparations prescribed for improvement of cognitive performance as tested earlier in this model, namely Ginkgo biloba extract (Ph. Eur. Quality) and Memantine (Figure 9). Sideritis scardica extract produced a stronger enhancement of the population spike than Ginkgo biloba extract (Ph. Eur. Quality) under same stimulation conditions.