USE OF EXTRACTS OF THE LEAVES OF LEMON VERBENA (ALOYSIA CITRIODORA) FOR INCREASING THE NEURONAL, CEREBRAL AVAILABILITY OF NEUROTRANSMITTERS SELECTED FROM THE GROUP OF SEROTONIN, DOPAMINE, NORADRENALINE

Abstract

The aim of the invention is to provide preparations and extracts of lemon verbena (Aloysia citriodora, or its synonyms) for the prophylaxis of mental stress, for increasing cognitive performance and for treating ADHS. For this purpose, preparations and extracts of lemon verbena (Aloysia citriodora, or its synonyms) are used, particularly in the form of hydroalcoholic extracts. These extracts can be used in food products, food supplements, supplementary balanced diets or pharmaceutical preparations.

Claims

1. A method of treating and/or preventing a neurological disorder, the method comprising: providing a formulation to a person to treat a neurological disorder and/or a tendency toward a neurological discorder, wherein the neurological disorder is selected from the group consisting of mental stress, stress-related exhaustion conditions stress disorders, somatoform diseases, acute and/or posttraumatic stress disorders, chronic fatigue syndrome, attention deficit hyperactivity disorder (ADHD), a cognitive performance issue, a neurodegenerative disease associated with accumulated proteins/peptides, mental stress, or stress symptoms, and a combination thereof, wherein the formulation comprises an extract prepared by alcoholic-aqueous extraction from leaves of lemon verbena (Aloysia citriodora) using an extractant having an alcohol concentration of 10-60% v/v, wherein said extract has a content of essential oil of less than 0.05% by weight, based on the extract without additives.

2. (canceled)

3. The method according to claim 1, wherein said somatoform diseases are the burnout syndrome.

4-5. (canceled)

6. The method according to claim 1, wherein said extract is obtained by extracting lemon verbena with mixtures of water and ethanol.

7. The method according to claim 1, wherein said extract is subjected to a secondary purification by removing lipophilic components by a liquid/liquid treatment with suitable solvents not miscible with water; or a liquid/solid treatment with suitable adsorber resins/adsorbents.

8. The method according to claim 1, wherein the formulation comprises fractions of the extract in a liquid or dried form.

9. The method according to claim 1, wherein the formulation further comprises other plants in the form of plant parts and/or extracts and/or chemical-synthetic substances.

10. The method according to claim 1, wherein said formulation is taken in orally.

11. The method according to claim 1, wherein said extract has a content of verbascoside of at least 2% by weight, based on the extract without additives.

12. The method according to claim 1, wherein said extract has a content of flavonoids of at least 0.05% by weight, based on the extract without additives.

13. The method according to claim 1, wherein said extract has a content of essential oil of less than 0.01% by weight, based on the extract without additives.

14. A process for preparing an extract from the leaves of lemon verbena (Aloysia citriodora), comprising the steps: primary extraction with an extractant consisting of mixtures of alcohol and water, the alcohol having a concentration of 10-60% (v/v); separating off a plant residue; and at least partially removing the extractant wherein said extract has a content of essential oil of less than 0.05% by weight, based on the extract without additives.

15. (canceled)

16. An ingestible formulation for treating and/or preventing a neurological disorder comprising an effective amount of a composition comprising an extract formed using alcoholic-aqueous extraction of leaves of lemon verbena (Aloysia citriodora) with an alcohol concentration of 10-60% v/v, wherein the extract has a content of essential oil of less than 0.05% by weight, based on the extract without additives.

17. The method according to claim 6, wherein the ethanol has a concentration of 20-50 v/v.

18. The method according to claim 10, wherein said formulation comprises a medicament or a food supplement, and wherein the formulation comprises a beverage formulation, a tablet, a capsule, a lozenge, a chewing formulation, a wafer, or a fused tablet.

19. The method according to claim 1, wherein said extract has a content of verbascoside of at least 5% by weight, based on the extract without additives.

20. The process according to claim 14, wherein after at least partially removing the extractant further comprising the steps: removing lipophilic components; and drying a comprised liquid viscous spissum extract to a dry extract.

21. The method according to claim 1 further comprising identifying the neurological disorder and/or the tendency toward a neurological disorder prior to providing the formulation.

22. The process according to claim 20, wherein after at least partially removing the extractant further comprising enzymatic treatment, distillation, liquid-liquid purification, liquid-liquid extraction, and/or liquid-solid extraction.

23. A method for inhibiting the biological response of a neurotransmitter comprising oral administration of a formulation comprising an extract from leaves of lemon verbena (Aloysia citriodora), wherein said extract has a content of essential oil of less than 0.05% by weight and a content of verbascoside of at least 5% by weight, based on the extract without additives.

24. The method of claim 23, wherein the neurotransmitter comprises dopamine, serotonin, and/or noradrenaline and wherein the normalized IC.sub.50 of serotonin is less than <40 μg/ml, the normalized IC.sub.50 of noradrenaline is less than <6.5 μg/ml, and/or the normalized IC.sub.50 of dopamine is less than <15 μg/ml, wherein the normalized IC.sub.50 is calculated as the IC.sub.50 divided by the drug to extract ratio.

Description

[0066] The following Examples of a preferred embodiment explain the invention.

[0067] FIG. 1 shows a thin-layer fingerprint comparison on flavonoid substances (yellow) and CQA compounds (light blue) using as examples extract fractions from lemon verbena vs. primary extract 50% v/v EtOH. Identical native fractions of the extracts were applied.

[0068] FIG. 2a shows a Morris water maze behavior test in vivo.

[0069] FIG. 2b shows the test set-up of the Morris water maze behavior test.

[0070] FIG. 2c shows the effect of lemon verbena extracts (20% v/v and 50% v/v EtOH) on the memory performance in a cognitive test model in mice (n=6).

[0071] FIG. 3a shows a tele-stereo in vivo EEG determination; n=7 rats when using the lemon verbena extract according to the invention (50% v/v EtOH); 150 mg/kg of body weight, and a fast CNS activity within 65 min FIG. 3b shows a tele-stereo in vivo EEG determination in a control group in rats with the administration of water at 1 ml/kg of body weight.

[0072] FIG. 3c shows a tele-stereo in vivo EEG determination when using the lemon verbena extract according to the invention (50% v/v EtOH)—batch UB2010-98 on healthy rats (n=8) as compared to synthetic active substances and ginkgo extract of Ph. Eur. grade.

PREPARATION AND CHARACTERIZATION OF EXTRACTS FROM LEMON VERBENA LEAVES

Example 1: Extraction with Water

[0073] 1500 g of lemon verbena leaves (folia Aloysia citriodora) is admixed with 15 liters of osmosis water twice at 80° C. and extracted for 8 hours with occasional stirring (moved maceration). The mixture is allowed to stand over night for cooling. The eluate is separated from the extracted starting material and filtered clear through a cellulose filter. The filtrate is evaporated on a plate evaporator to form a viscous spissum extract. The evaporation process is repeated twice after the addition of water in order to remove all volatile components. To 622 g of a spissum extract evaporated to 71.4% solids, 190 g of the drying additive maltodextrin is added. This 70% native extract formulation is spray-dried to form a beige powder (temperature at the spray head: 185° C., outlet temperature from the spray tower: 105° C.). The resulting extract formulation is a beige-colored powder characterized by a fraction of essential oil of <0.01%, a content of 0.06% flavonoids, and 1.8% verbascoside.

Example 2: Extraction with 20% by Volume Ethanol in Water

[0074] 1700 g of lemon verbena leaves (folia Aloysia citriodora) is admixed with 10 liters of 20% (v/v) ethanol twice at 50° C. and percolated for 8 hours (moved extraction). The mixture is separated from the extracted starting material and filtered clear through a 40 μm cellulose filter. The filtrate is freed from its ethanol fraction in a Sambay evaporator and evaporated to form a homogeneous spissum extract. By repeatedly adding water and evaporating it again under a reduced pressure, the extract is freed from volatile components. To 611 g of a spissum extract evaporated to 69.5% solids, 182 g of drying additives is added. This 70% native extract formulation comprises 28% maltodextrin and 2% Aerosil (fumed silica), and is dried in a vacuum drying oven at 45° C. The extract is subsequently ground in a sieve mill with a 0.315 mm sieve to form a homogeneous beige-brown powder. The resulting extract formulation is characterized by a fraction of essential oil of <0.01%, a content of 0.06% flavonoids, and 4.3% verbascoside.

Example 3: Extraction with 50% by Volume Ethanol in Water

[0075] 1700 g of lemon verbena leaves (folia Aloysia citriodora) is admixed with 10 liters of 50% (v/v) ethanol twice at 50° C. and percolated for 8 hours (moved extraction). The mixture is separated from the extracted starting material and filtered clear through a 40 μm cellulose filter. The filtrate is pre-evaporated on a plate evaporator and finally evaporated on a rotary evaporator under vacuum at 45° C. to form a solvent-free spissum extract. By adding water, the evaporation process is repeated twice in order to remove all volatile components. To 750 g of a spissum extract evaporated to 64.3% solids, 206 g of drying additives is added. This 70% native extract formulation comprises 28% maltodextrin and 2% Aerosil (fumed silica), and is dried in a vacuum drying oven at 45° C. The extract is subsequently ground in a sieve mill with a 0.315 mm sieve to form a homogeneous brown powder. The resulting extract formulation is characterized by a fraction of essential oil of <0.01%, a content of 0.51% flavonoids, and 11.6% verbascoside.

Example 4: Extraction with 96% by Volume Ethanol

[0076] 1500 g of lemon verbena leaves (folia Aloysia citriodora) is admixed with 15 liters of 96% (v/v) ethanol twice at room temperature, 20° C., and extracted for 8 hours with occasional stirring (moved maceration). The mixture is separated from the dry extract and filtered clear through a 40 μm cellulose filter. The clear greenish filtrate is evaporated on a plate evaporator to form a spissum extract. By adding water, the evaporation process is repeated twice in order to remove all volatile components. To 190 g of a spissum extract evaporated to 65.8% solids, 54 g of drying additives is added. This 70% native extract formulation comprises 20% maltodextrin and 10% Aerosil. The increasing silica content is necessary for binding and drying the increased chlorophyll fractions with this lipophilic extractant. Finally, the material is dried in a vacuum drying oven at 45° C., and ground in a ball mill to form a powder. The resulting extract formulation is a green-brown powder characterized by a fraction of essential oil of <0.1%, a content of 1.70% flavonoids, and 19.6% verbascoside.

Characterization of the Extracts

[0077]

TABLE-US-00001 TABLE 1 Extract characterization as a function of the extraction solvent Yield of Appearance of native Drug-to- 70% native Extract extracted extract Character of extract No. Extractant material ratio extractant formulation 1 water 29.6% 3.4:1 polar, highly beige powder hydrophilic; water- soluble 2 EtOH 25.0% 4.0:1 polar, hydrophilic; beige-brown 20% (v/v) water-soluble powder 3 EtOH 28.4% 3.5:1 polar, partially brown grain 50% (v/v) hydrophilic; partially water-soluble 4 EtOH 8.3% 12.0:1  non-polar, lipophilic green-brown 96% (v/v) maximum; not water- grain soluble

[0078] The drug-to-extract ratio represents the concentration factor; arithmetically, it is the reciprocal of the yield of native extracted material.

[0079] All comparable mixtures were generated from one and the same batch of lemon verbena leaves. This batch corresponded to the Ph. Eur. grade (content of essential oil: 0.5%; content of acteoside: 5.6%). All preparation variants resulted in extract formulations free of essential oils having different solution properties, appearances, and different contents of polar components, such as the flavonoids or verbascoside (phenylethanoid). The determination of flavonoids was performed as a group determination by UV spectroscopy by analogy with the Ph. Eur. method with birch leaves with the reference substance hyperoside. Verbascoside is a synonym of acteoside, whose determination was effected by analogy with the acteoside HPLC method for lemon verbena leaves by analogy with Ph. Eur. 1834.

Example 5: Determination of the Properties of Influencing the Neurotransmitters Noradrenaline, Dopamine and Serotonin by Extracts from Lemon Verbena Leaves

[0080] Freshly isolated synaptosomes from rats were used according to Perovic and Müller [Perovic S, Müller W E, 1995, Arzneimittelforschung 45; 1145-1148] for a dopamine reuptake test, and a serotonin reuptake test, and a noradrenaline reuptake test. [.sup.3H]-dopamine, [.sup.3H]-noradrenaline and [.sup.3H]-serotonin, respectively, served as the ligands. The test substance was incubated with the synaptosomes and the corresponding ligand for 15 min at 37° C. in the dark. The samples were then transferred to GF/C filter plates, and washed cold twice, and dried, before the filter-bound radioactivity was measured with a microtitration plate counter (Microbeta, Wallac, Finland). For determining the 100% reuptake, the experiment was performed without test substances. The measured value for incubation with corresponding inhibitors (GBR12909 in the dopamine reuptake inhibition, protriptyline in the noradrenaline reuptake inhibition, and imipramine hydrochloride for the serotonin reuptake inhibition) was considered as a 0% uptake. For a first screening for the suitability of the extracts for ADHD, Sommer et al. (Sommer et al.: Ein pflanzliches Arzneimittel im Vergleich zu Methylphenidat: Ein alternativer Weg in der zukünftigen Behandlung von ADHS?, GPT poster contribution to the GPT Phytokongress in September 2017 in Münster, Germany, Zeitschrift für Phytotherapie Issue S 01 ⋅Volume 38⋅DOI: 10.1055/s-007-34868) recommend a mean reuptake inhibition within a range of <50 μg/ml for dopamine or noradrenaline for a combination medicament with about 20 μg/ml as a potent example. Markowitz et al. found a significantly weaker effect for serotonin and confirmed the effects for noradrenaline and dopamine (Moskowitz et al.: A Comprehensive In Vitro Screening of d-, l-, and dl-threo-Methylphenidate, Journal of child and adolescent Psychopharmacology 16, 2006, 687-698).

TABLE-US-00002 TABLE 2 Neurotransmitter reuptake inhibition by extracts from lemon verbena leaves as compared to methylphenidate DER Serotonin Noradrenaline Dopamine Sample name native (IC.sub.50 μg/ml) (IC.sub.50 μg/ml) (IC.sub.50 μg/ml) Extract No. 1 (water) 3.4:1 308 23 82 Extract No. 2 (20% EtOH) 4.0:1 16 4 6 Extract No. 3 (50% EtOH) 3.5:1 128 21 30 Extract No. 4 (96% EtOH) 12.0:1  43 12 20 Methylphenidate not 0.050 (Sommer 0.114 (Sommer (positive control) determined et al.) et al.) 4.310 (Markowitz 0.052 (Markowitz 0.260 (Markowitz et al.) et al.) et al.)

[0081] Surprisingly, a very good potential for inhibiting the reuptake of the three neurotransmitters was found for all four tested lemon verbena extracts. The highest potential is found for noradrenaline, where all four extracts show a very potent reuptake inhibition of <25 μg/ml. Also, all four extracts are capable of inhibiting the reuptake of dopamine in a dose-dependent way. The three hydroethanolic extracts show good IC50 values of max. 30 μg/ml; the aqueous extract still shows a good effect but falls behind the hydroethanolic extracts by a factor of at least 2.6. A similar picture is seen for the influencing of serotonin. Here too, an aqueous extract shows the least activity. The hydroethanolic extracts show a clear dependence on the extractant from a good (extract 4) and very good (extract 2) to medium activity (extract 3). All in all, however, the extracts show a significantly higher activity in serotonin reuptake inhibition as compared to methylphenidate. Thus, the activity ratio of serotonin to noradrenaline is a factor of 86 for methylphenidate, while extract 2 or extract 4 have factors of about 4.1 in this case. Thus, surprisingly, a basically similar activity profile for the influencing of noradrenaline and dopamine is found for the extracts from lemon verbena leaves, and additionally an improved influencing of serotonin. Considering the three most important neurotransmitters tested, the extracts are very suitable candidates for the treatment of ADHD.

[0082] Further, if the mentioned results are taken in relation to the concentration factors (native DER value) and to the galenic properties of the extracts, a preferred range is obtained for medium polar extracts, such as extract No. 2 or 3. Here, as compared to pure water as an extractant, there is a significant improvement of the pharmacological properties while the enrichment is almost identical and the galenic properties are similar. If this is compared to extract No. 4, a highly lipophilic extractant with 96% v/v EtOH, the degree of enrichment is higher by a factor of 3 for extract 4 vs. extract 2, with a DER of 12:1. If the IC.sub.50 values obtained for the three neurotransmitters are divided by the additional enrichment factor 3, the IC.sub.50 values are obtained for an arithmetically similar total enrichment from the dried leaves, here a native DER of 4:1.

TABLE-US-00003 IC.sub.50 for extract 2 IC.sub.50 for extract 4 calculated for Neurotransmitter (DER 4:1) a DER of 4:1 Serotonin 16 14.3 (43/3)  Noradrenaline 4 .sup. 4 (12/3) Dopamine 6 6.7 (20/3)

[0083] This numerical comparison shows that lipophilic extracts with pure ethanol (extract 4) include the effectiveness-codetermining components of the starting material on the same level as a medium polar-aqueous-ethanolic extract (extract 2), if they are based on the same extract yields (here DER=4:1). There are virtually identical results for all three neurotransmitters considering a relative gain error of 5-10%.

[0084] This is not an advantage even if the economic efficiency with a higher extraction price (a higher material usage is necessary: 12 times vs. 4 times) is considered. Further, worse galenic properties with respect to appearance (green-brown), handling (hygroscopic dry extract) and solubility (virtually insoluble in aqueous medium) result for extract No. 4 as compared to extracts Nos. 2 and 3. This is caused by the transfers of cuticular wax and chlorophyll fractions from leaves, which is favored by the extractant 96% v/v EtOH. These problems can be avoided when using maximally 50% or 60% (v/v) ethanol.

Further Purification

[0085] Proceeding from a primary extract obtained with 50% v/v ethanol, the further separation into strongly lipophilic, non-polar and polar hydrophilic components was performed in terms of production technology. Thus, a total of six subfractions were obtained using known separation techniques, such as liquid-liquid partition, liquid-solid phase separation, and reprecipitation in ethanol. These were examined for their recoveries (=enrichment factors), typical components and their fingerprint by thin-layer chromatography (see FIG. 1).

Example 6: Liquid-Liquid Extraction

[0086] A primary extract from 50% v/v ethanol, prepared by analogy with Example 3, was redissolved in demineralized water to 10% dry matter, and permanently stirred. Thus, tree additions with one third each of the volume of the primary solution of n-butanol were performed, followed by intense stirring. Subsequently, phase separation in a separating funnel was performed. The three butanol phases combined were evaporated on a rotary evaporator under vacuum until free of solvent. Subsequently, this extract phase of 70% native/30% drying additives (28% maltodextrin and 2% silicas) was dried in a vacuum drying oven at 45° C., and then ground to a homogeneous powder. Similarly, the remaining aqueous phase was processed and dried as 70% native extract powder.

Example 7: Reprecipitation in Ethanol

[0087] A primary extract from 50% v/v ethanol, prepared by analogy with Example 3, was added in portions to 96% v/v ethanol until a total of 10% by weight dry matter was present in dissolved form. After 60 min of stirring, the solution was left to stand in a cold place at 6° C. for 24 hours. Hydrophilic substances precipitated.

[0088] After separating the upper ethanol phase by decanting, the precipitate was taken up in water. The mixture was evaporated on a rotary evaporator under vacuum until free of solvent. Subsequently, this extract phase of 70% native/30% drying additives (28% maltodextrin and 2% silicas) was dried in a vacuum drying oven at 45° C., and then ground to a homogeneous powder. The ethanolic supernatant was also evaporated on a rotary evaporator under vacuum until free of solvent, and also dried as 70% native extract powder.

Example 8: Liquid-Solid Distribution

[0089] A primary extract from 50% v/v ethanol, prepared by analogy with Example 3, was redissolved in demineralized water to 10% dry matter, and stirred for 30 min. The solution was left to stand in a cool place over night for 12 hours. A small fraction of the extract precipitated as an insoluble solid. It was filtered off through a folded filter and discarded. The clear eluate was placed on a glass column filled with an adsorber of the type XAD-7HP. The charged amount of dissolved native extract was 25% by weight of the dry weight of the adsorber. The adsorber is so selective that it binds polar medium-sized substances like flavonoids, phenols etc., and allows smaller molecules like sugars, salts and amino acids to pass. The filtering effect of the adsorber retains substances in the interior of the adsorber. These were washed off by elution with two bed volumes of 96% v/v ethanol. The ethanolic eluate obtained was evaporated on a rotary evaporator under vacuum until free of solvent. Subsequently, this extract phase of 70% native/30% drying additives (28% maltodextrin and 2% silica) was dried in a vacuum drying oven at 45° C., and then ground to a homogeneous powder. Also, the previously collected aqueous phase, which had passed the adsorber, was processed and dried to a dry extract powder with 70% native fraction and 30% additives.

TABLE-US-00004 TABLE 3 Extract characterization of six extract fractions of lemon verbena Primary extract: Extr. Aloysiae citriodor. e herb. spir. spiss. (50% v/v EtOH), enrichment factor = 4:1 (DER native) (5.88% verbascoside/0.54% CQA compounds/4.65% flavonoids) Liquid-liquid partition Reprecipitation in EtOH Separation on adsorber resin Example 6 Example 7 Example 8 Water n-butanol Water EtOH Water EtOH phase phase phase phase phase phase Extract No. 5 Extract No. 6 Extract No. 7 Extract No. 8 Extract No. 9 Extract No. 10 66%   34% .sup. 50% .sup. 50% .sup. 69%   31% rel. quant. distribution 6:1 11.8:1 8:1 8:1 5.8:1 12.9:1 enrichment 1.24% 11.64% 1.61% 9.35% 0.01% 14.01% flavonoids 1.52% 17.01% 2.07% 11.92%  0.01% 20.02% Verbascoside 0.31%  1.20% 0.54% 0.82% 0.02%  1.42% CQA compounds Thin-layer caffeic caffeic caffeic all caffeic chromatography acids + acids + acids + distinctive acids + caffeic acids + flavonoids flavonoids flavonoids zones are flavonoids flavonoids enriched enriched enriched lacking! enriched enriched

TABLE-US-00005 TABLE 4 Neurotransmitter reuptake inhibition by extract fractions from lemon verbena leaves as compared to the 50% v/v EtOH primary extract Serotonin Noradrenaline Dopamine (IC.sub.50 (IC.sub.50 (IC.sub.50 Sample name μg/ml) μg/ml) μg/ml) Primary extract No. 3 128 21 30 Extract No. 5 (water phase) 234 6 82 Extract No. 6 (butanol phase) 17 1 14 Extract No. 7 (water phase) 54 7 43 Extract No. 8 (ethanol phase) 26 8 15 Extract No. 9 (water phase) 363 25 191 Extract No. 10 (ethanol phase) 44 5 9

[0090] Also considering the analytical data relating to the components, the fractionations demonstrate that an enrichment towards medium polar to polar-lipophilic substances can enhance the activity effects. With the enrichment of the substance groups flavonoids, caffeoylquinic acids (CQA compounds) and verbascoside, the reuptake inhibitions increase for all three neurotransmitters tested.

[0091] This can be seen particularly well in the extracts Nos. 9 and 10, which resulted from the adsorber resin separation. The adsorber selectively separated the target substances from the water phase (extract No. 9), whereby its activity was clearly reduced as compared to the primary extract for serotonin and dopamine. The fact that noradrenaline was affected at about the same order of magnitude also shows that the overall extract determines the activity, rather than only individual marker substances/groups of substances. By implication, the ethanolic extract phase from the adsorber (extract No. 10) could achieve a concentration by a factor of 3.2 merely in terms of production technology (12.9 for fractionation vs. 4.0 for primary extract). The overall influence on the neurotransmitters improved precisely by about this order of magnitude.

[0092] Taking the extracts Nos. 7 and 8 (reprecipitation in ethanol) as examples, the fractionations also show that if the relative quantitative distribution is at 50% each and no difference in the degree of enrichment can be seen, then also approximately equal pharmacological activities result. Here too, despite the different distribution of some analytical marker substances (flavonoids, verbascoside), it is found that although these contribute to the effect, they are by no means the sole active substances. It all depends on the method of extract preparation. Also as compared to the primary extract, this Example again supports the influencing of all three neurotransmitters tested herein by lemon verbena extracts.

[0093] In view of these positive in vitro data available, the question arises to what extent this can be also demonstrated in vivo. A suitable model for this is the nematode C. elegans. Caenorhabditis elegans is a nematode that is being explored as a model organism mainly in developmental biology and genetics. An important reason for this is that, despite only having almost 1000 somatic cells, it has more than 20,000 genes (human: almost 40,000), many of which have similar functions as in mammals. Almost half of all proteins encoded in the worm's genome have homologues in Homo sapiens, including a wide variety of known human disease genes. Thus, it is a representative model organism with regard to metabolization, targeted gene expression, and neuronal behavior. The complete nervous system of the nematodes consists of about 300 neurons and is constituted of 2 independent parts: a larger somatic, and a smaller pharyngeal, nervous system. The nematodes use acetylcholine, glutamate, GABA (γ-amino-butyric acid), and biogenic amines, such as serotonin and dopamine, as neurotransmitters.

[0094] C. elegans lives in the soil of moderate climate zones, where it feeds on bacteria. The animals reach lengths of about 1 mm and have a transparent cuticula. C. elegans has a short lifespan of about 15 to 20 days (depending on the temperature and amount of feed). They can be cultivated in a well reproducible manner by placing Escherichia coli bacteria as their feed on agar plates. Depending on the problem, transgenic worms whose conversion products with defined substances can be detected directly through fluorescence measurements or by behavioral tests are selectively employed.

Example 9: Protection of Dopaminergic Neurons from 6-OHDA-Induced Degeneration

[0095] 6-Hydroxydopamine (6-OHDA) damages dopaminergic neurons, which is used in different animal models. The transgenic C. elegans strain BZ555 expresses green fluorescent protein in dopaminergic neurons. Thus, the 6-OHDA-induced neurodegeneration or the reduction thereof can be quantified by fluorescence microscopy. The worms were exposed to 50 mM 6-OHDA, which reduces the fluorescence intensity of the neurons because of the degeneration thereof. Bupropione, a selective noradrenaline and dopamine reuptake inhibitor (NDRI), serves as the positive control. Through the inhibition of the noradrenaline (NA) or dopamine (DA) transporters (reuptake), bupropion prevents the neurotoxin 6-OHDA from getting into and damaging the neurons. Treatment with 600 μg/ml of a lemon verbena extract from 50% ethanol according to Example 3 resulted in an about 20% higher fluorescence intensity (protective function) as compared to the negative control.

TABLE-US-00006 TABLE 5 Influence of lemon verbena extract (50% v/v EtOH) on 6-OHDA-induced degeneration in nematodes Worms + 6-OHDA + +6-OHDA + Worms + 6-OHDA bupropion 100 A. citriodora Untreated (negative μg/ml (positive extract Treatment worms control) control) 600 μg/ml Fluorescence 100.0 ± 2.6 34.3 ± 4.3 68.2 ± 7.7 43.1 ± 5.0 intensity (p < 0.05) (p = 0.07) [% of untreated group]

[0096] The damaging of neurons with 6-OHDA in an animal model is a generally recognized experimental set-up for ADHD (Kostrzewa R M et al.: Pharmacological models of ADHD; J Neural Transm (Vienna) 2008; 115: 287-98). The positive control confirms this in the C. elegans model employed here. The A. citriodora extract according to the invention, like the positive control, has been reported to inhibit the reuptake of NA and DA (see Example 5 above). The A. citriodora extract according to the invention can reach about ⅓ of the protective function of the positive control.

Example 10: Influence on β-Amyloid-Induced Toxicity in C. elegans Model

[0097] In order to test the effect on β-amyloid toxicity, the transgenic C. elegans strain CL4176, which can express human β-amyloid (Aβ 1-42), was employed. Aβ expression can be induced by increasing the temperature. Age-synchronized and treated worms were incubated at 16° C. for 48 hours. After increasing the temperature to 25° C. and incubation for 24 hours, the worms began to get paralyzed because of the toxicity of the Aβ oligomer. The paralysis was evaluated every 2 hours (experimental set-up according to Heiner et al.: Sideritis scardica extracts inhibit the aggregation of α-synuclein and β-amyloid peptides in Caenorhabditis elegans used as a model for neurodegenerative diseases. Planta Medica 81—PW_127, 2015). Substances that counteract this toxic effect of β-amyloid lead to a slower or later paralysis of the animals. Such substances delay the neurodegeneration, inter alia, in Alzheimer's dementia. The median (PT.sub.50), which is the time when exactly 50% of the nematodes were paralyzed, was used as a comparative value.

TABLE-US-00007 Negative +A. citriodora +acteoside single control extract substance in (no treat- 50% EtOH A. citriodora Treatment ment) 600 μg/ml 200 μg/ml PT.sub.50 value 31.0 ± 0.4 32.5 ± 0.9 32.8 ± 0.3 [hours] (p = 0.17) (p < 0.05) Prolongation reference +1.5 hours +1.8 hours of lifespan

TABLE-US-00008 TABLE 6 Influence of lemon verbena extract (50% v/v EtOH) on β-amyloid-induced toxicity +Sideritis scard. extract 50% EtOH Negative control 600 μg/ml Treatment (no treatment) positive control according to Heiner et al. PT.sub.50 value [hours] 33.5 ± 0.5 37.0 ± 0.0 (p < 0.001) Prolongation of lifespan reference +3.5 hours

[0098] Treatment with the lemon verbena leaf extract (50% v/v ethanol) delayed the β-amyloid-induced paralysis by 1.5 hours. The acteoside, which occurs as an individual substance in this extract, positively contributes to the protective effect (+1.8 hours). A plant extract from Sideritis scardica (50% EtOH), which has been reported for its suitability in ADHD (EP 2 229 950 B1), was tested in the same model and used as a positive control and showed a highly significant effect (+3.5 hours).

Example 11: Influence on β-Amyloid-Induced Neuronal Dysfunction (Limited Cognition)

[0099] β-Amyloid species can damage neurons and trigger a dysfunction of neuronally controlled behavior. The transgenic C. elegans strain CL2355 expresses human β-amyloid on a pan-neuronal level. This leads to cognitive disorders, such as reduced chemotactic movement towards an attractant (benzaldehyde). In the experiments, the chemotaxis index, i.e., the proportion of worms moved towards the attractant on the agar plate, was measured. The control strain CL2122 does not express β-amyloid and shows no behavior disorders; its chemotaxis index was correspondingly high. The experiment was performed in accordance with Heiner et al.

TABLE-US-00009 TABLE 7 Influence of lemon verbena extract on neuronal dysfunction Treatment Chemotaxis index CL2355 test strain untreated 0.10 ± 0.04 (negative control) CL2122 control strain 0.50 ± 0.09 (positive control) CL2355 + A. citriodora extract 0.23 ± 0.02 (p < 0.05) (50% EtOH) 600 μg/ml CL2355 + acteoside individual substance in 0.45 ± 0.02 (p < 0.05) A. citriodora 200 μg/ml

[0100] Treatment with a lemon verbena leaf extract (50% v/v ethanol) according to the invention showed a significant chernotaxis index that was increased by a factor of 13. The improvement over the untreated test strain was 46%. This is due to a reduced β-amyloid-induced degeneration of the neurons that are important o this cognitive ability. The acteoside, which occurs as an individual substance in this extract, is demonstrably involved in the neuroprotective effect (reached about 88% of the chemotaxis index of the control strain CL2122).

Example 12: Behavior Examinations in a Morris Water Maze (MWM)

[0101] According to the animal model “Morris water maze”, the increase of the cognitive performance becomes visible especially under an existing stress situation by showing an increase in the learning performance, especially retentiveness. In a round pool filled with muddy water provided with lateral distinctive marks, so-called external cues, test animals, in this case mice, are trained over several days to independently find a hidden platform provided under the water's surface, and to memorize its spatial position. The mice are put into water at an approximate distance from the edge of about 30 cm, whereupon the animals immediately try to reach the saving platform with swimming movements. The advantage of this measuring system, which has been known since the beginning of the 1980's, over conventional simple mazes in animal experiments is the fact that there are no local landmarks, but only global ones, and that the task has a high motivation factor because of the animals' escape behavior. The experiment is mainly directed to the examination of (spatial) learning (recognition and memorization) of the animals under stress conditions, and the measurement of possible influences thereon. The measuring parameters include the time until they find the platform, the distance traveled to there, as well as the relative stay time in the correct quadrant of the pool. These parameters are influenced by the training effect. Thus, the time of meeting and the journey is usually reduced, and the time spent in the quadrant is prolonged. The effect of training can also be influenced by different concentrations of neurotransmitters [Doctoral Thesis, University of Freiburg 2004, Theresa Schweizer: 3,4-Diaminopyridin evozierte Freisetzung von Neurotransmittern aus Hirnschnitten von Ratten: Untersuchungen im Kortex und Hippocampus an alten Ratten, sowie an Ratten mit serotonergen Läsionen hippocampaler Afferenzen und intrahippocampalen Raphé-Transplantaten].

[0102] In this test arrangement, 2 groups of 6 mice were studied, inter alia. The first control group was formed by transgenic animals treated with water (strain AD-B6) that due to their genetic disposition manifested in the interval of 50 days after their birth a strong β-amyloid deposition, or they became ill with Alzheimer's disease. The other two control groups consist of transgenic animals (strain AD-B6) that from their d=50 day of life were treated with a solution of a lemon verbena extract according to Example 2 or Example 3. In each case, the dosage was 400 mg of native extract per kg of body weight.

[0103] At the age of about 95 days a behavioral biological trial began by means of the Morris water maze (95-100 d). The trial comprised one test/learn unit of daily learning early and late for four days. The early unit begins with a run without a platform for 30 seconds, and the time in which the mouse stays in the quadrant in which the platform is usually located (target quadrant) is recorded. The other four runs are carried out with a hidden platform and with 4 variable starting positions.

[0104] The parameter analyzed was the time until reaching the platform (escape latency), graphically evaluated in FIG. 2. The evaluation shows that treatment with the lemon verbena extract according to the invention from 20% v/v EtOH leads to an impressive increase of the memory performance. Thus, the escape latency improved from relative 100% on day 1 to only 24.6% on day 2, or 22.4% on day 3. The slightly more lipophilic lemon verbena extract with 50% v/v EtOH augmented with some delay and reached the same order of magnitude. The escape latency improved from relative 100% on day 1 to 42.0% on day 2, and only 19.1% on day 3. Thus, the animals treated with lemon verbena extracts tended to be quicker already on days 2 and 3, and were significantly more quick on day 4 (p<0.05) in reaching the platform. On day 4, the learning performances of treated and healthy animals reach the same level in this model; however, they are significant different as compared to the transgenic animals with β-amyloid plaques. These results surprisingly show that lemon verbena extract is capable of increasing the cognitive performance

Example 13: EEG Profiles on Healthy Rats

[0105] Electroencephalography (from Greek encephalon=brain, graphein=write), abbreviated EEG, is a procedure of medical diagnosis for the measurement of the total electrical activity of the brain by recording voltage variations. The electroencephalogram offers a standardized study procedure in neurology as a graphic representation of such variations. For the clinical evaluation, a registration is required in at least twelve channels of different combinations of electrodes. The resulting data can be studied by expert specialists to determine striking patterns. A wide-spread mathematical method for analyzing the EEG is Fourier transformation into the frequency domain. Frequently the EEG is divided into frequency bands (so-called EEG bands), wherein the number of bands and the exact division is stated differently in part. The division of the frequency bands and their limits is historically conditioned and do not consistently coincide with boundaries that are considered useful due to more modern studies. Thus, for example, the theta band was divided into theta 1 and theta 2 intervals to consider the different meanings of the partial intervals. In particular, for long duration and sleep EEGs, software algorithms are used for assisted or automatic evaluation that reproduce a pattern recognition.

[0106] However, brain waves can not only be measured, but they can also be influenced. This can be done, inter alia, as neurofeedback, a special form of biofeedback, as a consequence of pharmacologically active substances, such as psychotropic drugs [Dimpfel W et al. (1996) Source Density Analysis of Functional Topographical EEG: Monitoring of Cognitive Drug Action. Eur J Med Res 1: 283-290]. The evaluation is also referred to as an electropharmacogram. In neurofeedback, it is usual to subdivide the EEG bands more finely and interpret them in more detail than in the classical EEG. An increased amplitude within the frequency ranges correlates with certain mental states or activities. Theta-2 waves can correlate, for example, with memory and learning ability, concentration, and/or creativity. Likewise, after extensive calibration, conclusions can be drawn to neurotransmitter-mediated SNC activities, which can be classified into dopaminergic, serotoninergic, cholinergic or noradrenergic subgroups.

[0107] A group of n=7 Fischer-344 rats were implanted in each case 4 semimicroelectrodes into the 4 brain areas “frontal cortex”, “hippocampus”, “striatum”, and “formatio reticularis”. The measurable changes of the potential field were transmitted by radio and evaluated to give an electropharmacogram.

[0108] The animals were treated with a lemon verbena dry extract formulation according to Example 3 with a dosage equivalent of 150 mg/kg of body weight. For this purpose, the individual doses were dissolved in water and administered once. Water served as a control experiment. After a pre-drug observing phase of 45 minutes, the test liquid was administered to the animals orally by gavage, followed by a setting phase of 5 minutes for the animal. Subsequently, the measurement was started for a measurement period of 5 hours. The frequency data were obtained by fast Fourier transformation (FFT) and averaged over periods of 60 minutes. The statistical evaluation was carried out by means of the Wilcoxon-Mann-Whitney U test against the control (water).

[0109] The examination of a lemon verbena 50% v/v EtOH extract according to Example 3 showed a statistically significant change in different frequency bands within one hour after the administration (see FIG. 3, EEG graph). In particular, theta, alpha1, alpha2 and beta1 frequencies were affected, which indicates an influence on the noradrenergic, serotonergic, dopaminergic and glutaminergic systems. This complex kind of activity was previously observed in medicaments having an inhibiting effect on monoaminoxidase. Comparable electropharmacograms are seen for, for example, moclobemide, a monoaminoxidase inhibitor approved as an antidepressive, but also methylphenidate, a dopamine and noradrenaline reuptake inhibitor with additional agonistic activity on the serotonergic receptors 5-HT1A and 5-HT2B, approved for the treatment of ADHD.

[0110] These results show that the lemon verbena extract is suitable for treating ADHD.

[0111] In the serum of the mice, an increased level of the stress hormone cortisol was not found, which is why it can be considered that the usual mental stress of the measurement could be prophylactically prevented or at least decreased by the extract.

[0112] Also, an EEG profile comparable with that of a ginkgo extract (Ph. Eur. grade) could be seen, which shows the properties of lemon verbena extract for improving the cognitive performance. The data found in a rat EEG confirm the in vitro results obtained for neurotransmitter binding studies. To conclude, all preclinical data together give a clear evidence of the use of lemon verbena leaf extracts as CNS-active extracts, preferably in the application fields of improving the cognitive performance, or the attention deficit hyperactivity disorder.