Uses of Lemon Verbena (Aloysia citriodora) Leaf Extract to Increase Neuronal and Cerebral Availability of Neurotransmitters Selected from the Group of Serotonin, Dopamine, and Noradrenaline

Abstract

The object of the present invention is to provide preparations and extracts of lemon verbena (Aloysia citriodora or its synonyms) for the prevention of mental stress, for increasing cognitive ability, and for the treatment of 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 may be used in food, dietary supplements, supplementary balanced diets, or pharmaceutical preparations.

Description

Technology Field

[0001] Botanically, lemon verbena (plant species: Aloysia citriodora PALÅU) is a genus of beebrushes of the family Verbenaceae ( Aloisia It belongs to ). Background Technology

[0002] In the literature on lemon verbena Aloysia tripila ( Aloysia triphylla) (L'Herit.) Kuntze / Ripia Citriodora ( Lippia citriodora) Kunth / Verbena tripilla ( Verbena triphylla) There are various synonyms for L'Herit., which can also be found in the European Pharmacopoeia monograph (Ph. Eur. Chapter 1834).

[0003] scientific name Aloysia citriodora (Previously Ripia Citriodora ) refers to "citriodora" and indicates the specific smell and taste of the leaves, such as a citrus scent. Aloysia tripila (Previously Verbena tripilla For synonyms of ), see " Tripillar " = Represents the arrangement of leaves on a tree branch with 3 leaves.

[0004] The correct international name for Lemon Verbena is as follows:

[0005] German: Zitronenverbene, Zitronenstrauch

[0006] Austria: Luiserkraut

[0007] English: lemon verbena, lemon-scented verbena

[0008] French: verveine citronelle, verveine odorante

[0009] Dutch: Citroenverbena, Citroenstruik

[0010] Portuguese: limonette, buona Luisa

[0011] Spanish: cedron, hierba Luisa

[0012] In colloquial German, names such as "Zitronenkraut" (lemon herb) and "Verbenenkraut" (verbena herb) are often mistakenly used interchangeably. In some publications, primarily in French, all genera of the Verbenaceae family are sometimes included under the name "verbena," but verbena It is also used as a synonym for *jong*, that is, iron herbs, and is sometimes used for lemon verbena. Therefore, *jong* Aloysia citriodora or Verbena officinalis ( Verbena officinalis The clear assignment of the term "verbane" to ) is not always clearly derived, and it is always necessary to clarify it within the context of each individual case.

[0013] Non-brush within the Verbenaceae family ( Aloisia The genus is, for example, "Aztec sweet herb" Lipia Dulcis ( Lippia dulcis ) TREV.) or "Mexican oregano" Ripia Graveolens ( Lippia graveolens Sweet herbs represented by ) HB & K.) Ripia ), and the traditional medicinal plant "common vervain" ( Verbena officinalis About 250 species of verbena ( to which L.) belong Verbena It is different from ).

[0014] The most notable feature botanically is Aloisia While plants of the genus grow as perennial deciduous shrubs up to 6 m in height, Verbena mostly grows as an annual herb reaching about 70 cm in height, whereas Aztec sweet herb grows flat and stays close to the soil, reaching about 20 cm in height.

[0015] Lemon verbena is a plant native to South America and can be found growing wild primarily in the regions of Chile and Uruguay. Due to its lack of frost tolerance in Europe, lemon verbena is cultivated mainly only in the southern regions of France and Spain. The leaves of lemon verbena are generally light green with somewhat serrated margins and are smooth, lanceolate or acute in shape. The length of the leaves varies from 3 to 10 cm, while the width can be 0.5 to 3 cm. The leaves usually have small dots (so-called oil glands) that contain characteristic essential oils and release a lemon-like scent when touched.

[0016] Aloysia citriodora In [the text], the leaves are generally used, whereas in the Aztec Sweet Herb's common verbena, the entire aerial plant part (stems, shoot tips, leaves, and flowers) is used. Due to recurring confusion in current terminology, the European Pharmacopoeia refers to lemon verbena leaf (Ph.Eur. 1834, monograph lemon verbena leaf) or the herb of common verbena (Ph.Eur. 1854, monograph Verbena officinalis It appears in two separate monographs referencing herba. The analytical lead compound derived from the leaves of lemon verbena, described as acteoside according to Ph.Eur. 1834, is found in the literature under the synonym verbascoside, which often makes clear naming more difficult.

[0017] DE 10 2011 078 432 A1 describes the use of essential oils derived from various plants and their components in the treatment of hyperkinetic disorders. In particular, the description of the invention in paragraph

[0014] states " Verbena officinalis"Although it correctly translates 'general verbena', in paragraphs

[0036] ,

[0046] and

[0049] , 'general verbena' Ripia Citriodora It is incorrectly identified with Kunze. In the examples, plant names are no longer used, but the claims are Verbena officinalis It is changed back to. This shows in an exemplary way how inaccurately terms are handled.

[0018] Traditionally, fresh or dried leaves of lemon verbena are used as flavoring additives or as medicinal plants. Described therapeutic applications include antioxidant, galactogogue, analgesic, antibacterial, antipyretic, muscle relaxant, and diuretic properties. The components of the essential oil and the flavonoid group are jointly responsible for a very wide range of active substances. Consistently, the Ph. Eur. monograph specifies a minimum of 3 ml / kg of essential oil and 2.5% acteoside and DC trace amounts (fingerprint) for the presence of flavonoids in lemon verbena leaves (lemon verbena folium) in the quality specifications for dried whole leaves.

[0019] While essential oils are frequently cited as a group of active ingredients, characteristic chromatographic analysis of specific oil components is not precisely explained along with their effects. Therefore, the essential oil components of lemon verbena are used in aromatherapy. In the treatment of nervous restlessness conditions and stress-related fatigue, particularly when consumed orally as tea, they serve as agents used for delayed sleep onset or mild digestive disorders.

[0020] Wannmacher et al. investigated the hypothesized sedative and anxiety-relieving effects of lemon verbena tea preparations, but found no significant effect in a human randomized, double-blind, parallel-group study of 40 healthy subjects (Wannmacher L. et al. - Plants employed in the treatment of anxiety and insomnia: II. Effect of infusions of Aloysia triphylla on experimental anxiety in normal volunteers. Fitoterapia 1990; 61: 449-453). Therefore, simple aqueous liquid extracts with a low proportion of essential oils did not show significant effects on the central nervous system (CNS).

[0021] It is stated that an aqueous extract of lemon verbena named "Planox L" contributes to anti-aging effects. This is preferably based on the antioxidant and anti-inflammatory properties of epithelial cells occurring in the skin and intestinal regions.

[0022] CN 107397011 (A) describes a Chinese herbal tea mixed with nine different plants, which is described as restoring vitality. It is an aqueous extract. The ingredient listed as verbena is regular verbena, not lemon verbena.

[0023] CN 102309622 (B) concerns individual glycosides of Verbena for use in cerebral ischemia. In paragraph

[0007] , this refers to the plant being discussed as common Verbena ( Verbena officinalis It can be seen that )

[0024] Erping Xu et al., Saudi Pharmaceutical Journal 25 (2017) 660-665 describes the testing of different plant components in cerebral ischemia. In particular, preparations of total verbena glycosides are tested. The composition of the mixture of substances is unclear. The glycosides are derived from the English term "verbena," which refers to common verbena rather than lemon verbena.

[0025] Carpinella et al. Phytotherapy Research 24 (2010), 259-263 describe pure ethanol extracts from 73 different plants, which are subsequently separated into aqueous and organic fractions. The applied measurement system, namely "acetylcholinesterase inhibition," is used for screening diseases such as Alzheimer's disease.

[0026] DE 10 2007 052 ​​223 A1 is Verbena officinalis L., that is, the extract of common verbena (see paragraph

[0002] ).

[0027] WO 2005 / 058338 A1 is about extracts of lemon verbena. The depletion of essential oils from extracts obtained in this way is not described.

[0028] In the case of extracts prepared by freeze-drying and via an essential oil-free aqueous route, Ragone et al. also GABA AIt is hypothesized that the sedative and anxiety-relieving effects are mediated through receptor affinity. In open field tests, this was investigated in mice and demonstrated by exhibiting heart-sparing negatively inotropic activity in isolated rat hearts (Ragone MI, Sella M, Pastore A, Consolini AE. Sedative and Cardiovascular Effects of Aloysia citriodora Palau, on Mice and Rats. Latin American Journal of Pharmacy (Lat Am J Pharm) 2010; 29(1): 79-86.). Therefore, CNS activity can also be demonstrated for dry extracts prepared by the aqueous route and without essential oil fractions.

[0029] This was confirmed by an investigation by Veisi et al., who also prepared a dried extract via the aqueous route through heat drying and administered it orally to rats. Therefore, although a high anxiety-relieving effect was not found in the elevated-plus maze from 10 mg of extract / kg body weight (Veisi M, Shahidi S, Komaki A, Sarihi A.: Assessment of aqueous extract of Lemon verbena on anxiety-like behavior in rats. J Pharm Negative Results 2015; 6: 37-9), an effect that promotes anxiety ("anxiety-like behavior") was found.

[0030] Ceuterick et al. investigated the evaluation of the ethnobotanical use of medicinal plants among Colombians in London, including lemon verbena applied as an anti-anxiety, sedative, and antidepressant (Ceuterick M., Vandebroek I., Torry B., Pieroni A. - Cross-cultural adaptation in urban ethnobotany: The Colombian folk pharmacopoeia in London, Journal of Ethnopharmacology 120 (2008) 342-359). Regarding the substance, the use of fresh or dried leaves and teas derived from these pure aqueous extracts was always described. The impact of changing the production method (e.g., extractant) on the range of components of lemon verbena leaf extract regarding properties affecting the CNS has not been described to date. There were also no additional analytical hints regarding the group of components related to the effects of properties affecting the CNS.

[0031] There is no literature demonstrating the effectiveness of an extract prepared with aqueous ethanol derived from lemon verbena, which contains almost no essential oil. The problem to be solved

[0032] The objective of the present invention is to discover additional fields of application for lemon verbena. This objective is to use lemon verbena ( Aloysia citriodora This is achieved with leaf extract. means of solving the problem

[0033] The present invention relates to lemon verbena ( Aloysia citriodora ) Explains the effects of standardized formulations of leaf extracts and the correlation of specific CNS-mediated clinical manifestations affecting neurotransmitters. Therefore, the application fields of the former can be extended to include the range of signs of attention deficit disorder and / or an increase in cognitive ability as a preliminary stage of Alzheimer's disease.

[0034] Attention Deficit Hyperactivity Disorder (ADHD) is a mental disorder that begins in childhood and is characterized by problems with attention and impulsivity, and often also hyperactivity. Up to 10% of all children in Europe show symptoms of ADHD (it occurs much more frequently in boys than in girls). Symptoms can persist into adulthood with varying intensities. In 30 to 70% of adolescents with ADHD, the disorder persists into adulthood (persistence).

[0035] In adulthood, hyperactivity alters personality, resulting in increased inner anxiety. Individuals with ADHD also frequently exhibit various other mental disorders, such as depression, anxiety disorders, and impairments in self-awareness, self-esteem, and social phobia. Current evidence suggests that Attention Deficit Hyperactivity Disorder is a multifactorial disorder with a genetic predisposition that indicates signs of the condition.

[0036] At a neurobiological level, ADHD is often explained as striatofrontal dysfunction. The affected area of ​​the brain—more precisely, the frontal lobe—is where motivation, cognition (information transformation), emotions, and movement behaviors are regulated or their interactions are coordinated. Doctors discuss striatofrontal dysfunction because the so-called striatum (part of the basal ganglia belonging to the cerebrum) is affected by factors other than the frontal lobe.

[0037] Prevalence estimates in Germany from 2009 to 2014 support the increase in the frequency of ADHD diagnoses from 5.0% to 6.1% in patients aged 0 to 17 and from 0.2% to 0.4% in patients aged 18 to 69. It is one of the most widespread chronic diseases among children and adolescents. It occurs more frequently in boys than in girls, and more frequently in children than in adolescents. It was found that up to 13.9% of boys aged approximately 9 years were affected (Bachmann CJ, Philipsen A, Hoffmann F: ADHD in Germany: trends in diagnosis and pharmacotherapy ― a country-wide analysis of health insurance data on attention-deficit / hyperactivity disorder (ADHD) in children, adolescents and adults from 2009-2014. Dtsch Arztebl Int 2017; 114: 141-8. DOI: 10.3238 / arztebl.2017.0141).

[0038] While the prescription of ADHD medications for adults diagnosed with ADHD has increased (the most frequently prescribed drugs are methylphenidate, followed by atomoxetine and risdextramphetamine), the prescription rate for children and adolescents has decreased. There is a great need for alternative medications.

[0039] In the case of ADHD medications, stimulants that affect the brain's dopamine metabolism are primarily used. These include methylphenidate and amphetamine derivatives (DL-amphetamine) that have been used since about the mid-20th century. About two-thirds of affected people respond to these drugs. Additionally, antidepressants that affect dopamine or norepinephrine metabolism may also be used for treatment.

[0040] One of the causes of ADHD is an abnormality in the brain's signal processing. This disorder is based on a deficiency or reduced effects of the neurotransmitters norepinephrine and dopamine. For example, attention is controlled through norepinephrine, while motivation is controlled through dopamine. Therefore, as a result of disruptive signal processing, affected individuals have difficulty focusing on one thing at a time and filtering external stimuli based on their importance (i.e., being easily distracted and overstimulated). Furthermore, the involvement of the neurotransmitter serotonin in ADHD has been established. Since serotonin regulates impulses, the disorder consequently increases impulsivity, lowers frustration tolerance, and prevents affected individuals from adapting to specific situations.

[0041] Methylphenidate, the substance most frequently used today for the treatment of Attention Deficit Hyperactivity Disorder (ADHD), inhibits the reuptake of dopamine and norepinephrine at the presynapse, thereby increasing their concentrations in the synaptic cleft. This increases the signal density of receptors and, in particular, leads to an increase in sympathetic tone. Therefore, substances that selectively inhibit the reuptake of norepinephrine and dopamine (and also slightly serotonin) are promising candidates for ADHD treatment. However, this active substance is also characterized by a wide range of potential side effects. Commonly occurring side effects are known, as listed below: rhinopharyngitis, decreased appetite, moderate weight and size loss (when applied extensively to children), mental imbalance, anxiety, depression, irritability, cardiac arrhythmia, etc.

[0042] Clinical experience suggests that methylphenidate may improve symptoms of behavioral and thought disorders in children with psychosis. There are no treatment recommendations for children under 6 years of age. Side effects appear to be most likely with long-term administration. Chronic inappropriate use can even lead to a loss of methylphenidate's effectiveness (development of tolerance) and induce psychological dependence.

[0043] Cognition (the conversion of information) is a measurable criterion within striatal frontal dysfunction. While "cognition" is frequently used with various meanings, in a comprehensive sense, it refers to thinking. Many human cognitive processes are conscious, but "cognition" and "consciousness" do not have the same meaning. Certain human processes can be unconscious yet cognitive. An example of this is unconscious learning. Human cognitive abilities include, for instance, attention, memory, learning, creativity, planning, orientation, imagination, argumentation, self-observation, will, and belief. Cognitive abilities are investigated in various sciences, such as psychiatry, psychology, philosophy, and neuroscience.

[0044] Therefore, cognitive performance is a complex process that can be quantified by measurable parameters of brain performance. Learning ability includes memorization (the duration and amount of remembered input) and reactivity, logical computational ability (speed and accuracy), or spatial thinking ability (e.g., the stage of orientation under new and changed conditions). Impairment of these cognitive abilities is specifically described by MCI syndrome (Mild Cognitive Impairment). MCI represents a specific condition of age-related decline in cognitive function and ability. In healthy individuals, MCI testing can be considered a preindicator of late-stage dementia.

[0045] Surprisingly, at this clinic, lemon verbena ( Aloysia citriodoraIt was revealed that the extract of the ) leaves significantly inhibits the reabsorption of norepinephrine and dopamine, and inhibits serotonin in synaptosomes of the rat cortex. In addition, In vitro The suitability for ADHD found through the results was based on the use of nematodes. In Vibo This can be confirmed in experiments, mouse EEG recordings, and additionally in mouse behavioral models (Morris water maze).

[0046] It has been newly revealed that the cognitive domain, in particular, is favorably supported by oral administration of a hydroethanol extract of lemon verbena.

[0047] In addition, it has been revealed that consuming regular doses of lemon verbena extract, similar to drinking instant supplements, shows a significant therapeutic effect in ADHD patients.

[0048] Accordingly, the present invention relates to the treatment of ADHD and / or cognitive and / or mild cognitive impairment and / or stress-related fatigue pathology / stress disorder. Aloysia citriodora This concerns the use of leaf extracts of a species (or its synonym).

[0049] Diseases that can be treated or prevented according to the present invention include somatoform disorders (burnout syndrome), acute and post-traumatic stress disorders, chronic fatigue syndrome; neurotic anxiety (mental stress), anxiety and depression as symptoms of neurological disorders, and stress symptoms such as fatigue and exhaustion.

[0050] Preferably, the neurological disorder is not a neurodegenerative disease associated with accumulated proteins / peptides, and in particular is not Alzheimer's disease or Parkinson's disease.

[0051] The present invention also relates to a method for manufacturing a drug for the treatment of ADHD. Aloysia citriodora This relates to the use of an extract derived from the leaves of a species (or its synonym).

[0052] The present invention also relates to a therapeutic agent for improving cognitive ability and / or mild cognitive impairment. Aloysia citriodora This concerns the use of extracts from the leaves of a species (or its synonym).

[0053] The present invention also relates to manufacturing a therapeutic agent for use in nervous anxiety (mental stress). Aloysia citriodora This concerns the use of extracts from the leaves of a species (or its synonym).

[0054] The present invention is described below by means of preferred embodiments and individual examples relating to preferred embodiments. In a preferred use according to the present invention, dried and cut leaves of lemon verbena that meet the quality standards of the Ph. Eur. monograph are used for the aforementioned purposes. In a more preferred embodiment of the present invention, an extract prepared from the dried leaves of lemon verbena Ph. Eur. is used. Alternatively, leaves and extracts derived from lemon verbena leaves may be used together.

[0055] For example, when using lemon verbena leaf extracts, they are alcoholic aqueous extracts. In this context, "alcohol" refers to all lower (C1 to C4) alcohols that are miscible with water.

[0056] Preferred solvents include methanol, ethanol, isopropanol, and mixtures thereof, with ethanol being particularly preferred. The volume ratio of the alcohol-water mixture for obtaining the lemon verbena leaf extract may vary within a wide range. This is preferably 99:1 to 1:99 volume %, more preferably 70:30 to 30:70 volume %, and most preferably 55:45 to 45:55 volume % (based on the ratio of alcohol to water in the total mixture used for extraction). Preferred ratios include 10:90 to 70:30 as volume % and as alcohol to water, or 10:90 to 60:40, or 20:80 to 70:30, or 20:80 to 60:40.

[0057] Preferably, a mixture of ethanol and water is used as an extractant, wherein the mixing ratio is 70:30 to 30:70 volume %, particularly 60:40 to 40:60 volume %, or 55:45 to 45:55 volume % is particularly suitable. In other embodiments, a particularly suitable extractant comprises a mixture of water having a content of 10:90 to 30:90 volume %. Preferred ratios are 10:90 to 70:30, or 10:90 to 60:40, or 20:80 to 70:30, or 20:80 to 60:40, respectively, as alcohol and water in volume %. All values ​​of volume % are based on volume at 21 °C.

[0058] However, other mixtures, such as a mixture of water and an organic solvent like a ketone or organic acid, may also be used as extractants. A characteristic of the selected organic solvent is that it is removed from the concentrated extract by distillation, sublimation, or freezing after the extraction process.

[0059] In a further preferred embodiment of the present invention, the extract prepared by the exemplary extractant mentioned above is not used as is but is further purified as a primary extract. This treatment step may be as follows:

[0060] - Enzymatic treatment of aqueous extracts in the presence of cellular components;

[0061] - Removal of essential oil fractions and volatile organic components by distillation, preferably azeotropic distillation;

[0062] - Removal of essential oil fractions and lipophilic components, such as plant resins and chlorophyll, by liquid-liquid purification using aliphatic hydrocarbons;

[0063] - Concentration of polar valuable components by liquid-liquid extraction using n-butanol or ethyl acetate;

[0064] - Concentration of polar valuable components by liquid-solid extraction from a solid phase, such as organic adsorbent polymers.

[0065] The performance of the purification step mentioned and the recovery of the extract fraction obtained from the primary extract mentioned can be carried out in any manner known to a person skilled in the art, and therefore no further explanation is required here.

[0066] Additionally, in another preferred embodiment according to the present invention, extracts from lemon verbena leaves and / or fractions prepared from such extracts may be used alone or in combination with one another. Similarly, according to the present invention, it is preferable to use extracts from lemon verbena leaves and / or fractions prepared from such extracts alone or in combination with chemically synthesized substances, for example, non-selective monoamine reuptake inhibitors (NSMRIs), selective serotonin reuptake inhibitors (SSRIs), selective noradrenaline reuptake inhibitors (NARIs), serotonin-noradrenaline reuptake inhibitors (SNRIs), dopamine reuptake inhibitors (DRIs), selective noradrenaline-dopamine reuptake inhibitors (NDRIs), or MAO inhibitors.

[0067] The use of the present invention according to the specific embodiments described above may be in the form of a pharmaceutical formulation. As such, it may take the form of a conventional formulation known to those skilled in the art, comprising a lemon verbena extract of a specific concentration in a form that enables dose administration in a conventional pharmaceutical formulation. In a manner known to those skilled in the art, the formulation may depend on the route of administration (oral, intravenous, intramuscular, nasal) and may be solid, semi-solid, liquid, spray, gas, or other forms of administration acceptable for the desired route. Additionally, the use according to the present invention may also be as a food supplement in combination with one of the aforementioned formulations.

[0068] The following preferred embodiments describe the present invention. Brief explanation of the drawing

[0069] Figure 1 shows a thin-layer fingerprint comparison of flavonoid substances (yellow) and CQA compounds (light blue) using the extract fraction of lemon verbena and the primary 50% v / v EtOH extract as examples. The same natural fraction of the extract was applied. Fig. 2a is In Vibo This represents the Morris underwater maze behavior test. Figure 2b shows the test setup for the Morris underwater maze behavior test. Figure 2c shows the effect of lemon verbena extract (20% v / v and 50% v / v EtOH) on memory ability in a mouse cognitive test model (n = 6). Fig. 3a is In Vibo Remote stereo EEG measurements indicated; when using the lemon verbena extract (50% v / v EtOH) according to the present invention, n = 7 rats; 150 mg / kg body weight and rapid CNS activity within 65 minutes. Figure 3b shows the control group of rats administered water at a rate of 1 ml / kg body weight. In ViboIt shows remote stereo EEG measurements. FIG. 3c shows the lemon verbena extract (50% v / v EtOH) according to the present invention when using In Vibo Remote stereo EEG measurements are shown for healthy rats (n = 8) compared with Ph. Eur. grade synthetic active substance and Ginkgo biloba extract in batch UB2010-98. Specific details for implementing the invention

[0070] Preparation and Characterization of Lemon Verbena Leaf Extract:

[0071] Example 1: Extraction with water

[0072] 1500 g of lemon verbena leaves (folia) Aloysia citriodora The mixture was mixed twice with 15 liters of osmotic water at 80 °C and extracted for 8 hours with occasional stirring (transfer immersion). The mixture was left overnight to cool. The eluent was separated from the extracted starting material and filtered cleanly through a cellulose filter. The filtrate was evaporated in a plate evaporator to form a viscous spissum extract. After adding water to remove all volatile components, the evaporation process was repeated twice. 190 g of maltodextrin, a drying additive, was added to 622 g of spissum extract evaporated to 71.4% solids. This 70% natural extract preparation was spray-dried to form a beige powder (spray head temperature: 185 °C, spray tower outlet temperature: 105 °C). The resulting extract preparation is a beige powder characterized by a fraction of < 0.01% essential oil, a flavonoid content of 0.06%, and 1.8% verbascoside.

[0073] Example 2: Extraction from water with 20 volume % ethanol

[0074] 1700 g of lemon verbena leaves (Folia Aloysia citriodoraThe mixture was mixed with 10 liters of 20% (v / v) ethanol twice at 50 °C and percolated for 8 hours (transfer extraction). The mixture was separated from the extracted starting material and filtered cleanly through a 40 μm cellulose filter. The filtrate was separated from its ethanol fraction and evaporated in a Sambay evaporator to form a homogeneous spius extract. Volatile components were removed from the extract by repeatedly adding water and evaporating it again under reduced pressure. 182 g of dry additives was added to 611 g of spius extract, which had evaporated to a solid content of 69.5%. This 70% natural extract formulation contained 28% maltodextrin and 2% Aerosil (dry silica) and was dried in a vacuum drying oven at 45 °C. Subsequently, the extract was 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 <0.01% essential oil fraction, 0.06% flavonoid content, and 4.3% berbascoside.

[0075] Example 3: Extraction from water with 50 volume % ethanol

[0076] 1700 g of lemon verbena leaves (folia Aloysia citriodoraThe mixture was mixed with 10 liters of 50% (v / v) ethanol twice at 50 °C and percolated for 8 hours (transfer extraction). The mixture was separated from the extracted starting material and filtered cleanly through a 40 μm cellulose filter. The filtrate was pre-evaporated in a plate evaporator and finally evaporated in a rotary evaporator under vacuum at 45 °C to form a solvent-free spisom extract. The evaporation process was repeated twice by adding water to remove all volatile components. 206 g of dry additive was added to 750 g of the spisom extract evaporated to a solid content of 64.3%. This 70% natural extract formulation contained 28% maltodextrin and 2% Aerosil (dry silica) and was dried in a vacuum drying oven at 45 °C. Subsequently, the extract was ground in a sieve grinder with a 0.315 mm sieve to form a uniform brown powder. The resulting extract formulation is characterized by < 0.01% essential oil fraction, 0.51% flavonoid content, and 11.6% berbascoside.

[0077] Example 4: Extraction with 96 volume % ethanol

[0078] 1500 g of lemon verbena leaves (folia Aloysia citriodoraThe mixture was mixed twice with 15 liters of 96% (v / v) ethanol at room temperature and extracted for 8 hours with occasional stirring (transfer hot maceration). The mixture was separated from the dry extract and filtered cleanly through a 40 μm cellulose filter. The clear green filtrate was evaporated in a plate evaporator to form the spius extract. The evaporation process was repeated twice by adding water to remove all volatile components. 54 g of dry additives was added to 190 g of spius extract evaporated to a solid content of 65.8%. This 70% natural extract formulation contained 20% maltodextrin and 10% aerosil. The increased silica content is necessary to bind the increased chlorophyll fraction with this lipophilic extract and to facilitate drying. Finally, the material was dried in a vacuum drying oven at 45 °C and ground in a ball grinder to form a powder. The resulting extract preparation is a green-brown powder characterized by an essential oil fraction of < 0.1%, a flavonoid content of 1.70%, and 19.6% berbascoside.

[0079] Characterization of extracts

[0080]

[0081] Table 1: Characterization of extraction properties as a function of extraction solvent

[0082] The ratio of drug to extract represents the concentration factor; arithmetically, it is the reciprocal of the yield of the naturally extracted material.

[0083] All comparable mixtures were produced from one and identical batches of lemon verbena leaves. This batch corresponds to the Ph. Eur. Grade (essential oil content: 0.5%, acteoside content: 5.6%). All formulation variants resulted in essential oil-free extract formulations with different content of different solution properties, appearances, and polar components, such as flavonoids or berbascoside (phenylethanoid). Flavonoid measurements were performed as group measurements by UV spectroscopy, similar to the Ph. Eur. method, using birch leaves containing hyperoside as the reference substance. Verbascoside, a synonym for acteoside, was measured similarly to the acteoside HPLC method for lemon verbena leaves, similar to Ph. Eur. 1834.

[0084] Example 5: Measurement of properties affecting the neurotransmitters norepinephrine, dopamine, and serotonin by lemon verbena leaf extract

[0085] Freshly isolated synaptosomes from rats were used according to Perovic and Muller [Perovic S, Muller WE, 1995, Arzneimittelforschung 45; 1145-1148] for dopamine reuptake, serotonin reuptake, and norepinephrine reuptake tests. 3 H]-dopamine, [ 3 H]-Noradrenaline and [ 3[H]-serotonin acted as the respective ligands. The test substances were incubated with synaptosomes and corresponding ligands in the dark at 37°C for 15 minutes. Then, the samples were transferred to GF / C filter plates, washed twice with cold water, and dried; afterward, filter-bound radioactivity was measured using a microtitration plate counter (Microbeta, Wallac, Finland). To measure 100% reuptake, the experiment was performed without the test substance. Measurements for incubation with corresponding inhibitors (GBR12909 for dopamine reuptake inhibition, protriptyline for noradrenaline reuptake inhibition, and imipramine hydrochloride for serotonin reuptake inhibition) were considered as 0% reuptake.

[0086] For the first screening of the suitability of extracts for ADHD, Sommer et al. recommend mean reuptake inhibition of dopamine or noradrenaline in the < 50 μg / ml range for a combination drug with about 20 μg / ml as a potent example (Sommer et al.: Ein pflanzliches Arzneimittel im Vergleich zu Methylphenidat: Ein alternativer Weg in der zukunftigen Behandlung von ADHS?, GPT poster contribution to the GPT Phytokongress in September 2017 in Munster, Germany, Zeitschrift für Phytotherapie Issue S 01 · Volume 38 · DOI: 10.1055 / s-007-34868). Markowitz et al. found a significantly weak effect on serotonin and confirmed the effects on norepinephrine and dopamine (Moskowitz et al.: A Comprehensive In VitroScreening of d-, l-, and dl-threo-Methylphenidate, Journal of child and adolescent Psychopharmacology 16, 2006, 687-698).

[0087]

[0088] Table 2: Inhibition of neurotransmitter reuptake by lemon verbena leaf extract compared to methylphenidate

[0089] Surprisingly, excellent potential for inhibiting the reuptake of three neurotransmitters was found in all four tested lemon verbena extracts. The highest potential was found for norepinephrine, and all four extracts demonstrated very potent reuptake inhibition of < 25 μg / ml. Additionally, all four extracts were able to inhibit dopamine reuptake in a dose-dependent manner. The three hydroethanol extracts showed excellent IC50 values ​​of up to 30 μg / ml; while the aqueous extracts still showed excellent effects, they lagged behind the hydroethanol extracts by at least 2.6 times. A similar pattern was observed regarding the effects on serotonin. Here too, the aqueous extracts exhibited the lowest activity. The hydroethanol extracts showed excellent (Extract 4), very excellent (Extract 2), and moderate (Extract 3) activity, demonstrating a clear dependence on the extractant. However, generally speaking, the extracts exhibited much higher activity in inhibiting serotonin reuptake compared to methylphenidate. Thus, the ratio of serotonin activity to norepinephrine was 86-fold for methylphenidate, whereas for Extract 2 or Extract 4, it was approximately 4.1-fold. Therefore, surprisingly, fundamentally similar activity profiles regarding the effects on norepinephrine and dopamine were found for the lemon verbena leaf extracts, with an additionally improved effect on serotonin. Considering the three most important neurotransmitters tested, the extracts are highly suitable candidates for the treatment of ADHD.

[0090] Furthermore, when the mentioned results are taken in relation to the concentration factor (natural DER value) and the pharmacological properties of the extracts, a desirable range is obtained for intermediate polarity extracts such as Extract No. 2 or 3. Here, pharmacological properties are significantly enhanced compared to pure water as an extractant, while the concentration is nearly the same and the pharmacological properties are similar. Compared to Extract No. 4, a highly lipophilic extractant with 96% v / v EtOH, the degree of concentration is three times higher in Extract No. 4 than in Extract No. 2, and the DER is 12:1. ICs obtained for the three neurotransmitters 50 Dividing the value by an additional concentration factor of 3, the IC for an arithmetically similar total concentration in dried leaves 50 A value is obtained, where natural DER is 4:1.

[0091]

[0092] This numerical comparison shows that the lipophilic extract with pure ethanol (Extract 4) contains effect-co-determining components of the starting material at the same level as the medium-polarity-aqueous-ethanol extract (Extract 2) when based on the same extract yield (here DER = 4:1). Considering a relative gain error of 5–10%, there are virtually identical results for all three neurotransmitters.

[0093] This is not an advantage, even when considering the economic efficiency of the higher extraction cost (requiring higher material usage: 12 times vs. 4 times). Furthermore, compared to extracts No. 2 and 3, extract No. 4 exhibits worse pharmacological properties in terms of appearance (green-brown), handling (hygroscopic dry extract), and solubility (almost insoluble in aqueous media). This is due to the migration of cuticular wax and chlorophyll fractions from the leaves, which are favored by the 96% v / v EtOH extract. These problems can be avoided by using up to 50% or 60% (v / v) ethanol.

[0094] Additional refining

[0095] The primary extract obtained with 50% v / v ethanol was processed to perform further separation into strongly lipophilic, nonpolar, and polar hydrophilic components in terms of production technology. Thus, a total of six subfractions were obtained using known separation techniques such as liquid-liquid partitioning, liquid-solid phase separation, and ethanol reprecipitation. These were investigated for recovery (= concentration factor), typical components, and fingerprints by thin-layer chromatography (see Fig. 1).

[0096] Example 6: Liquid-liquid extraction:

[0097] The primary extract of 50% v / v ethanol, prepared similarly to Example 3, was redissolved in deionized water to 10% dry matter and stirred continuously. Thus, tri-addition was performed in increments of one-third of the volume of the primary solution of n-butanol, followed by vigorous stirring. Subsequently, phase separation was performed in a separation funnel. The three combined butanol phases were evaporated in a rotary evaporator under vacuum until no solvent remained. Subsequently, this extract of 70% natural / 30% dry additive (28% maltodextrin and 2% silica) was dried in a vacuum drying oven at 45°C and then ground into a homogeneous powder. Similarly, the remaining aqueous phase was processed into a 70% natural extract powder and dried.

[0098] Example 7: Reprecipitation in ethanol:

[0099] A primary extract from 50% v / v ethanol prepared similarly to Example 3 was partially added to 96% v / v ethanol until a total of 10% by weight of dry material was present in a dissolved form. After stirring for 60 minutes, the solution was left in a cool place at 6°C for 24 hours. The hydrophilic material precipitated.

[0100] The upper ethanol phase was separated by decanting, and the precipitate was absorbed into water. The mixture was evaporated in a rotary evaporator under vacuum until the solvent was gone. Subsequently, this extract of 70% natural / 30% dry additive (28% maltodextrin and 2% silica) was dried in a vacuum drying oven at 45°C and then ground into a uniform powder. The ethanolic supernatant was also evaporated in a rotary evaporator under vacuum until the solvent was gone and dried into a 70% natural extract powder.

[0101] Example 8: Liquid-Solid Distribution:

[0102] The primary extract in 50% v / v ethanol, prepared similarly to Example 3, was redissolved in deionized water to 10% dry matter and stirred for 30 minutes. The solution was left overnight in a cool place for 12 hours. A small fraction of the extract precipitated as an insoluble solid. This was filtered through a folded filter and discarded. The clear eluent was placed on a glass column filled with an XAD-7HP type adsorbent. The fill weight of the dissolved natural extract was 25 wt% of the dry weight of the adsorbent. The adsorbent was highly selective, allowing it to bind to polar medium-sized substances such as flavonoids and phenols, while allowing smaller molecules such as sugars, salts, and amino acids to pass through. The filtration effect of the adsorbent kept the material inside the adsorbent. These were washed by eluting with twice the volume (bed volumes) of 96% v / v ethanol. The obtained ethanol eluent was evaporated in a rotary evaporator under vacuum until the solvent was removed. Subsequently, this extract phase of 70% natural / 30% dry additive (28% maltodextrin and 2% silica) was dried in a vacuum drying oven at 45°C and then ground into a uniform powder. In addition, the previously collected aqueous phase, which had passed through an adsorbent, was processed and dried into a dry extract powder having 70% natural fraction and 30% additive.

[0103]

[0104] Table 3: Extract characteristics of 6 lemon verbena extract fractions

[0105]

[0106] Table 4: Inhibition of neurotransmitter reuptake by lemon verbena leaf extract fractions compared to 50% v / v EtOH primary extract

[0107] Furthermore, considering the analytical data related to the components, fractionation demonstrated that concentration from polar lipophilic substances toward intermediate polarity can enhance the active effect. As the flavonoid, caffeoylquinic acid (CQA compound), and berbascoside groups were concentrated, the inhibition of reuptake for all three tested neurotransmitters increased.

[0108] This is particularly evident in extracts No. 9 and 10 resulting from adsorbent resin separation. The adsorbent selectively separated the target substances from the aqueous phase (Extract No. 9), resulting in a clear decrease in activity against serotonin and dopamine compared to the primary extract. The fact that norepinephrine was affected by nearly the same amount also demonstrates that the entire extract, rather than individual marker substances / groups of substances, determines the activity. Implicationally, the ethanol extract phase from the adsorbent (Extract No. 10) was able to achieve a concentration 3.2 times higher in terms of production technology (12.9 for the fraction vs. 4.0 for the primary extract). The overall effect on neurotransmitters was improved precisely to approximately this magnitude.

[0109] Taking extracts No. 7 and No. 8 (re-precipitated with ethanol) as examples, fractionation showed approximately equivalent pharmacological activity results even when the relative quantitative distribution was 50% each and there may be no difference in the degree of concentration. Here, too, it was found that even though the distribution of some analytical marker substances (flavonoids, berbascoside) differed, they contributed to the effect but were by no means the only active substances. This all depends on the method of preparing the extract. Furthermore, compared to the primary extract, this example further supports the effect of lemon verbena extract on all three neurotransmitters tested herein.

[0110] These positive In vitro When looking at the data, this In ViboQuestions are raised regarding the extent to which this can be proven even in this context. A suitable model for this is the nematode. C. Elegance . Caenorhapditis Elegance ( Caenorhabditis elegans The parasite is a nematode primarily explored as a model organism in developmental biology and genetics. An important reason for this is that, despite having nearly 1,000 somatic cells, it contains over 20,000 genes (compared to nearly 40,000 in humans), most of which perform functions similar to those in mammals. Nearly half of all proteins encoded in the parasite's genome include a wide variety of known human disease genes. sapient It has homologues. Therefore, it is a representative model organism regarding metabolism, target gene expression, and neuronal behavior. The complete nervous system of a nematode consists of approximately 300 neurons and includes two independent parts: a larger somatic nervous system and a smaller pharyngeal nervous system. Nematodes use acetylcholine, glutamate, GABA (γ-aminobutyric acid), and biogenic amines such as serotonin and dopamine as neurotransmitters.

[0111] C. Elegance It inhabits soil in temperate climates and feeds on bacteria. The animal reaches a length of about 1 mm and has a transparent cuticle layer (cuticula). C. Elegance They have a short lifespan of about 15 to 20 days (depending on the temperature and amount of food). On agar plates Esherikia Kolai ( Escherichia coli ) They can be cultured in a reproducible manner using bacteria as food. Depending on the problem, transgenic worms are selectively used in which the conversion product having a defined substance can be directly detected through fluorescence measurement or behavioral tests.

[0112] Example 9: Protection of dopaminergic neurons from 6-OHDA-induced degeneration:

[0113] 6-hydroxydopamine (6-OHDA) damages dopaminergic neurons used in other animal models. Gene transplantation C. Elegance Strain BZ555 expresses green fluorescent protein in dopaminergic neurons. Therefore, neurodegeneration induced by 6-OHDA or its reduction can be quantified by fluorescence microscopy. Parasites were exposed to 50 mM 6-OHDA, which reduces the fluorescence intensity of the neurons due to neuronal degeneration. Bupropione, a selective noradrenaline and dopamine reuptake inhibitor (NDRI), served as a positive control. Bupropione prevents the neurotoxin 6-OHDA from entering and damaging neurons by inhibiting noradrenaline (NA) or dopamine (DA) transporters (reuptake). Treatment with 600 µg / ml of lemon verbena extract from 50% ethanol according to Example 3 resulted in approximately 20% higher fluorescence intensity (protective function) compared to the negative control.

[0114]

[0115] Table 5: Effects of Lemon Verbena Extract (50% v / v EtOH) on 6-OHDA-induced regression in nematodes

[0116] Neuronal damage caused by 6-OHDA in animal models is a generally accepted experimental setup for ADHD (Kostrzewa RM et al.: Pharmacological models of ADHD; J Neural Transm (Vienna) 2008; 115: 287-98). The positive control used here is C. Elegance This is verified in the model. According to the present invention A. Citriodora The extract was reported to inhibit the reabsorption of NA and DA, similar to the positive control (see Example 5 above). According to the present invention A. Citriodora The extract was able to reach about one-third of the protective function of the positive control.

[0117] Example 10: C. Elegance Effects on β-amyloid-induced toxicity in models:

[0118] Gene transplants capable of expressing human β-amyloid (Aβ 1-42) to test the effects on β-amyloid toxicity C. Elegance Strain CL4176 was used. Aβ expression can be induced by increasing the temperature. Age-synchronized and treated parasites were incubated at 16 °C for 48 hours. After raising the temperature to 25 °C and incubating for 24 hours, parasites began to become paralyzed due to the toxicity of Aβ oligomers. Paralysis was assessed every 2 hours (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. Experimental setup according to Planta Medica 81 - PW_127, 2015). Substances acting counter to these toxic effects of β-amyloid induced paralysis in animals more slowly or later. These substances delay neurodegeneration, particularly in Alzheimer's dementia. The median (PT), which is the time when exactly 50% of nematodes were paralyzed. 50 ) was used as the comparison value.

[0119]

[0120]

[0121] Table 6: Effect of Lemon Verbena Extract (50% v / v EtOH) on β-amyloid-induced toxicity

[0122] Treatment with lemon verbena leaf extract (50% v / v ethanol) delayed β-amyloid-induced paralysis by 1.5 hours. Acteosides, which occur as individual substances in this extract, contribute positively to the protective effect (+1.8 hours). Reported to be suitable for ADHD (EP 2 229 950 B1). Sideritis Skadica ( Sideritis scardica The plant extract (50% EtOH) of ) was tested in the same model and used as a positive control, showing a highly significant effect (+ 3.5 hours).

[0123] Example 11: β - Effects on amyloid-induced neurological dysfunction (limited cognition):

[0124] β-amyloid tumors can damage neurons and cause dysfunction in neuronal control behaviors. Gene transplantation C. Elegance Strain CL2355 expresses human β-amyloid at the pan-neuronal level. This induces cognitive impairments, such as reduced chemotactic movement toward an attractant (benzaldehyde). In the experiment, the chemotactic index, i.e., the proportion of parasites that moved from the agar plate toward the attractant, was measured. The control strain CL2122 did not express β-amyloid and did not exhibit behavioral impairments; its chemotactic index was correspondingly high. The experiment was performed according to Heiner et al.

[0125]

[0126] Table 7: Effects of Lemon Verbena Extract on Neurological Dysfunction

[0127] Treatment with the lemon verbena leaf extract (50% v / v ethanol) according to the present invention showed a significant 2.3-fold increase in the chemotactic index. The improvement for the untreated test strain was 46%. This is due to the degeneration of neurons important for cognitive ability caused by reduced β-amyloid. Acteosides occurring as individual substances in this extract are clearly involved in the neuroprotective effect (reaching approximately 88% of the chemotactic index of the control group CL2122).

[0128] Example 12: Behavioral Test in Morris Underwater Maze (MWM) :

[0129] According to the animal model "Morris Underwater Maze," the increase in cognitive ability is visualized by demonstrating an increase in learning capacity, particularly retention, especially under existing stressful situations. In a circular pool filled with muddy water featuring lateral unique markings known as so-called external cues, test animals (in this case, mice) were trained over several days to independently discover a hidden platform provided below the surface and to remember its location. When a mouse is placed in the water at an approximate distance of about 30 cm from the edge, the animal immediately attempts to reach the rescue platform by swimming. The advantage of this measurement system, known since the early 1980s, is that compared to conventional simple mazes in animal experiments, it features only overall landmarks without local ones, and the task has a high motivational factor due to the animal's escape behavior. The experiment primarily concerns testing the animals' (spatial) learning (recognition and memorization) under stressful conditions and measuring the possible impacts thereon. Measurement parameters include the time taken to discover the platform, the distance traveled to reach it, and the relative time spent in the correct quadrant of the pool. These parameters are influenced by the effects of training. Therefore, generally, the time of encounter and journey decreases, and the time spent in the quadrant increases. The effect of training may also be influenced by the concentration of neurotransmitters [Doctoral Thesis, University of Freiburg 2004, Theresa Schweizer: 3,4-Diaminopyridin evozierte Freisetzung von Neurotransmittern aus Hirnschnitten von Ratten: Unter-suchungen im Kortex und Hippocampus an alten Ratten, sowie an Ratten mit serotonergen Lasionen hippocampaler Afferenzen und intrahippocampalen Raphe-Transplantaten].

[0130] In this test batch, two groups consisting of six mice were specifically studied. The first control group was formed by water-treated genetically modified animals (strain AD-B6) that, due to their genetic substrate, developed potent β-amyloid deposition or Alzheimer's disease at 50-day intervals after birth. The other two control groups consisted of genetically modified animals (strain AD-B6) treated with a lemon verbena extract solution according to Example 2 or Example 3 starting from d = 50 days after birth. In each case, the dosage was 400 mg of the natural extract per kg of body weight.

[0131] Behavioral biological tests were initiated using the Morris Water Maze (95–100 d) at approximately 95 days of age. The tests consisted of one test / learning unit performed daily during the initial and later stages for four days. The initial unit began with a 30-second run without a platform, and the time the mouse spent in the quadrant where the platform was typically located (target quadrant) was recorded. The other four runs were performed with a hidden platform and four variable starting positions.

[0132] The analyzed parameter is the time to reach the platform (escape latency), which was evaluated graphically in Figure 2. The evaluation shows that treatment with the lemon verbena extract according to the present invention derived from 20% v / v EtOH leads to an impressive increase in memory ability. Thus, escape latency improved from 100% relative on day 1 to 24.6% on day 2, or 22.4% on day 3. A somewhat more lipophilic lemon verbena extract containing 50% v / v EtOH showed a slight increase in latency and reached a magnitude of the same order. Escape latency improved from 100% relative on day 1 to 42.0% on day 2, and 19.1% on day 3. Thus, animals treated with lemon verbena extract already tended to be faster on days 2 and 3, and were much faster to reach the platform on day 4 (p < 0.05). On day 4, the learning ability of the treated and healthy animals reached the same level in this model; However, they differ significantly when compared to genetically modified animals with β-amyloid plaques. This result surprisingly demonstrates that lemon verbena extract can increase cognitive ability.

[0133] Example 13: EEG profile of a healthy rat

[0134] Electroencephalography (EEG) (derived from the Greek words encephalon = brain, graphein = write) is a medical diagnostic method that measures the brain's total electrical activity by recording voltage changes. The electroencephalogram provides a standardized research procedure in neurology as a graphical representation of these changes. For clinical evaluation, registration is required across more than 12 channels with different electrode combinations. The resulting data can be studied by experts to determine noticeable patterns. A widely used mathematical method for analyzing EEG is the Fourier transform into the frequency domain. Often, EEG is divided into frequency bands (so-called EEG bands), although the number of bands and the precise division vary. The division of frequency bands and their boundaries has been historically regulated and does not consistently align with the boundaries considered useful by more modern research. Therefore, for example, to consider different meanings of sub-intervals, the theta band was divided into theta1 and theta2 intervals. In particular, for organ and sleep EEG, software algorithms are used for assisted or automated evaluations that reproduce pattern recognition.

[0135] However, brainwaves can not only be measured but also be influenced. This can occur as neurofeedback, a special form of biofeedback, particularly as a result 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]. This evaluation is also known as an electropharmacogram. In neurofeedback, it is common to subdivide EEG bands more finely and interpret them in greater detail than in classical EEG. Increased amplitude within a frequency range correlates with specific mental states or activities. Theta-2 wavelengths, for example, may correlate with memory and learning ability, concentration, and / or creativity. Likewise, after extensive correction, conclusions can be drawn regarding neurotransmitter-mediated SNC activity that can be classified into dopaminergic, serotonergic, cholinergic, or noradrenergic subgroups.

[0136] In each case, four semimicroelectrodes were implanted in a group of n = 7 Fischer-344 rats in four brain regions: the "frontal cortex," "hippocampus," "striatum," and "formatio reticularis." Measurable changes in the potential field were wirelessly transmitted and evaluated to provide electropharmacograms.

[0137] Animals were treated with the lemon verbena dry extract preparation according to Example 3 at a dose equivalent of 150 mg / kg body weight. For this purpose, individual doses were dissolved in water and administered as a single dose. Water was used as a control. After a 45-minute pre-drug observation phase, the test solution was orally administered to the animals via gastrointestinal feeding, followed by a 5-minute setup phase. Subsequently, measurements were initiated for a 5-hour measurement period. Frequency data were obtained by Fast Fourier Transform (FFT) and averaged over 60 minutes. Statistical evaluation was performed using the Wilcoxon-Mann-Whitney U test against the control (water).

[0138] Tests of the lemon verbena 50% v / v EtOH extract according to Example 3 showed statistically significant changes in different frequency bands within 1 hour of administration (see Fig. 3, EEG graph). In particular, theta, alpha1, alpha2, and beta1 frequencies were affected, indicating that it affects the noradrenergic, serotonergic, dopaminergic, and glutaminergic systems. This complex type of activity has previously been observed in agents with inhibitory effects on monoamine oxidases. For example, similar electropharmacokinetics were shown for moclobemide, a monoamine oxidase inhibitor approved as an antidepressant, as well as for methylphenidate, a dopamine and noradrenergic reuptake inhibitor approved for the treatment of ADHD due to its additional efficacious activity against serotonin receptors 5-HT1A and 5-HT2B.

[0139] These results show that lemon verbena extract is suitable for treating ADHD.

[0140] Since no increased levels of the stress hormone cortisol were detected in the serum of mice, it can be considered that the usual mental stress of the measurement can be preventively prevented or at least reduced by the extract.

[0141] In addition, an EEG profile similar to that of ginkgo extract (Ph. Eur. grade) can be observed, demonstrating the properties of lemon verbena extract for improving cognitive abilities.

[0142] Data found in rat EEGs was obtained from neurotransmitter binding studies In vitro The results were verified. In conclusion, all preclinical data provide clear evidence that lemon verbena leaf extract is preferably used as a CNS active extract in the field of cognitive improvement or application for attention deficit hyperactivity disorder.

Claims

Claim 1 Lemon Verbena ( Aloysia citriodora ( Aloysia citriodora A pharmaceutical composition for use in treating attention deficit hyperactivity disorder (ADHD) or improving cognitive performance, comprising an extract of a leaf, wherein the extract is an alcoholic aqueous extract having an alcohol concentration of 20-50% v / v, and the extract has an essential oil content of less than 0.05% by weight based on an extract without additives. Claim 2 A pharmaceutical composition according to claim 1, wherein the extract is secondarily purified by removing lipophilic components by: - ​​liquid / liquid treatment using a solvent incompatible with water; or - liquid / solid treatment using adsorber resins / adsorbents that concentrate polar components. Claim 3 A pharmaceutical composition according to claim 1, characterized in that an extract fraction in liquid or dry form is used. Claim 4 A pharmaceutical composition according to any one of claims 1 to 3, wherein the pharmaceutical composition is administered orally. Claim 5 In claim 4, the pharmaceutical composition is a beverage formulation prepared from an instant formulation. Claim 6 A pharmaceutical composition according to claim 4, wherein the pharmaceutical composition is a tablet, capsule, lozenge, chewing preparation, wafer, or fused tablet. Claim 7 A pharmaceutical composition according to any one of claims 1 to 3, wherein the extract has a verbascoside content of 2 weight% or more based on the extract without additives. Claim 8 A pharmaceutical composition according to any one of claims 1 to 3, wherein the extract has a flavonoid content of 0.05 weight% or more based on the extract without additives. Claim 9 A pharmaceutical composition according to any one of claims 1 to 3, wherein the extract has an essential oil content of less than 0.01 weight% based on the extract without additives. Claim 10 delete Claim 11 delete Claim 12 delete Claim 13 delete Claim 14 delete Claim 15 delete Claim 16 delete

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