They were harvested prior to flowering. The M. officinalis samples were prepared for two separate analyses. In the first analysis, a crude ethanolic extract of the whole herb was assessed, in separate assays, for receptor binding to human cortex nicotinic and muscarinic receptors. In the second analysis, a moderately polar fraction containing terpenoids and phenolic materials was isolated for muscarinic analysis, and a basic fraction ionizable basic materials was isolated for nicotinic analysis.

The fractionation served two purposes. Firstly, it was intended to confirm that any displacement properties of the crude extract were receptor specific, as opposed to being a result of adherence by incidental plant components hindering the access of the radiolabels.

Secondly, it served to highlight inhibitory or synergistic properties of the crude extract by demonstrating whether the activity in the fractions was less or greater than that of the whole extract.

The ethanolic solutions were then decanted from the extracted leaf residues, filtered through Whatman No. The methanolic melissa extracts were fractionated using differences in pH and hydrophobicity. Each extract was separated into a moderately polar fraction terpenoids and phenolic materials for muscarinic analysis, and a basic fraction ionizable basic materials for nicotinic analysis.

Preparation of brain membranes: Human cortex from the occipital lobe obtained from the Newcastle General Hospital Brain Bank was homogenized and prepared as described by Wake et al Nicotinic and muscarinic displacement assays: The displacement of radiolabeled [ 3 H]-nicotine and [ 3 H]-scopolamine from the tissue homogenates by both the crude ethanolic extracts and methanolic fractions prepared from the M.

officinalis samples was undertaken using the methods described in detail by Wake et al Human AChE inhibition: Ethanolic extracts of the dried leaf samples and post-mortem samples of normal human brain tissue were prepared using the methodology described in Perry et al AChE activity was measured in homogenates using the spectrophotometric method of Ellman et al following the methodology of Perry et al A total of 14 female and six male undergraduate volunteers mean age Prior to participation, each volunteer signed an informed consent form and completed a medical health questionnaire.

All participants reported that they were in good health, and were taking no illicit social drugs. Owing to possible confounding effects on CNS cholinergic status, smokers were excluded from the study.

The CDR computerized assessment battery has been used in hundreds of European and North American drug trials, and has been shown to be sensitive to acute cognitive improvements eg Moss et al, ; Scholey et al, as well as impairments with a wide variety of substances eg Ebert et al, ; O'Neill et al, A tailored version of the CDR battery was used.

This has previously been found to be sensitive to modulation of cognitive function as a consequence of acute ingestion of M. officinalis Kennedy et al, b , Ginkgo biloba Kennedy et al, , a , and Panax ginseng Kennedy et al, a , a , and acute and chronic administration of a G.

ginseng combination Kennedy et al, b , a ; Wesnes et al, , In the case of the current study, the additional RVIP task was included in the battery. The running order of the tasks is illustrated in Figure 2. The selection of computer-controlled tasks from the system was administered with randomly ordered parallel forms of the tests being presented at each testing session.

Effects of M. The table presents means with standard errors in italics of baseline scores and change from baseline scores for each dose of M.

Word presentation : A total of 15 words, matched for frequency and concreteness, were presented in random order on the monitor for the participant to remember.

The task was scored as the number of words correct and the resulting score was converted into a percentage. In all, 50 stimuli were presented with an interstimulus interval that varied randomly between 1 and 3. Reaction times were recorded in ms. Digit vigilance task : A target digit was randomly selected and constantly displayed to the right of the monitor screen.

There were 50 trials, of which the stimulus word was chosen randomly with equal probability, with a randomly varying interstimulus interval of between 1 and 3. Spatial working memory : A pictorial representation of a house was presented on the screen with four of its nine windows lit.

The participant was instructed to memorize the position of the illuminated windows. In 36 subsequent presentations of the house, one of the windows was illuminated and the participant decided whether or not this matched one of the lighted windows in the original presentation.

Numeric working memory : Five digits were presented sequentially for the participant to hold in memory. This was repeated two further times with different stimuli and probe digits. The task was scored for number of words correct and the resulting score was converted into a percentage.

Delayed word recognition : The original words plus 15 distracter words were presented one at a time in a randomized order. Delayed picture recognition : The original pictures plus 20 distracter pictures were presented one at a time in a randomized order.

The original factor analysis of data from the battery is described in detail in Wesnes et al The contribution of each individual task outcome to the outcome factors is included in Figure 1. The factors are described below. officinalis on the individual task outcome measures from the CDR battery.

Mean baseline and change from baseline scores with standard errors are presented. Tasks are displayed in order of completion with a diagrammatic representation of which cognitive factor they contribute to on the left.

Rapid visual information processing task RVIP : This task has been widely used to study the cognitive effects of psychotropic drugs, and has been shown to be sensitive to cholinergic modulation eg Wesnes and Warburton, ; Wesnes et al, The participant monitors a continuous series of digits for targets of three consecutive odd or three consecutive even digits.

The task is scored for percentage of target strings correctly detected, average reaction time for correct detections, and number of false alarms. This task was not included in the original factor analysis Wesnes et al, and is therefore analyzed separately here.

Treatments: On each study day, participants received eight capsules that were of identical appearance on each occasion. officinalis leaf. To maintain the double blind, a disinterested third party was responsible for preparing treatments and the code remained unbroken until the initial statistical analysis had been completed.

All treatments were identical in appearance and scent. Procedure: Each participant was required to attend a total of five study days that were conducted 7 days apart, to ensure a sufficient washout between conditions.

Testing took place, commencing at the same time on each day, in a suite of laboratories with participants visually isolated from each other.

On arrival at their first session on the first day, participants were randomly allocated to a treatment regime using a Latin square design that counterbalanced the order of treatments across the four active days of the study.

The first day was identical to the following four, except that no treatment active or placebo was offered, to allow familiarization with the test battery and procedure.

Data from the four sessions of this practice day were not included in any analysis. Each active study day comprised four identical testing sessions. The first was a predose testing session that established baseline performance for that day, and was immediately followed by the day's treatment on days 2—5.

Each testing session comprised completion of the Bond—Lader visual analog scales, and the CDR test battery. Prior to carrying out planned comparisons, an analysis of variance ANOVA General Linear Model , with terms fitted to the model for dose, visit, dose × visit, and subject Kirk, , was carried out to identify the main effects and interaction effects on each measure.

To ensure the overall Type I error protection level, only those planned comparisons associated with measures that generated a significant main effect or interaction effect, or a trend towards the same, on the initial ANOVA are reported.

Furthermore, all testing was two-tailed, comparisons were strictly planned prior to the study, were restricted to the number of conditions minus one at each time point, and only probabilities associated with these preplanned comparisons were calculated.

IC 50 values for the displacement of radio-labeled [ 3 H]-nicotine and [ 3 H]-scopolamine from human occipital cortex tissue are shown in Table 1. The relative activity is ranked in order of effectiveness for both nicotine and scopolamine displacement, by both the whole ethanolic extract and the active fractions.

In the case of the fractions, in order to make a valid comparison between samples, the rank reflects both displacement and the comparative yield of the fraction data not shown from the leaf for each sample. The primary indicator of relevant activity is displacement by the whole ethanolic melissa extract, which reflects the combined effects of coligands, synergists, and binding inhibitors present in the dried leaf.

However, in order to validate this activity, displacement by the basic fraction or moderately polar fraction must also be substantial for any given sample. This indicates that the demonstrated displacement is due to a direct influence on the receptor-binding site rather than an indirect property of the extract eg interference with binding.

All of the assessed samples demonstrated substantial levels of displacement of radioligand from muscarinic receptors. However, in the case of nicotinic receptors, samples 5, 7, and 8 were excluded from further consideration on the basis of an interference pattern on the binding curve data not shown of either the ethanolic extract or basic fraction a sigmoidal dose—response curve is indicative of genuine receptor interaction.

Of the remaining samples 1, 2, and 3, all exhibited IC 50 values suggesting relatively high displacement of both nicotine and scopolamine. Reference to their respective binding curves suggested that sample 1 had the most favorable profile, with sigmoidal dose—response curves for both fractions.

This sample was taken forward into the behavioral experiment. AChE inhibition: None of the extracts from the eight samples of dried M. officinalis exhibited any human AChE inhibitory properties, with zero percent inhibition of the enzyme at a final assay concentration of 0.

officinalis for each primary outcome cognitive factor scores, RVIP scores, and Mood scale scores were subjected to a one-way, repeated-measures ANOVA.

There were no significant differences on any measure. Individual task outcome measures: Task outcomes in chronological order , a diagrammatic representation of which factor they contribute to, and performance data on the individual task outcome measures are presented in Figure 1. Results on the RVIP are described separately below.

Cognitive factor outcome measures: The mean raw baseline scores and change from baseline scores for each condition across each session are presented in the tables and graphs of Figure 2.

Working memory factor: There were no significant differences on the initial ANOVA for the working memory factor. Planned comparisons revealed that all three doses were associated with slowing across the timed memory tasks. Planned comparisons showed decrements for all three doses. Speed of attention factor: There was no effect on this factor, or the component single task outcomes.

Quality of attention factor: There was no effect on this factor, or the component single task outcomes. officinalis led to increased ratings of calmness, in comparison to placebo.

The effects of M. officinalis on mood measures are presented in the table and graphs of Figure 3. officinalis on self-rated mood as measured using Bond—Lader visual analogue scales.

The table presents raw scores and change from baseline scores means with standard errors in italics. The results of the current study confirm both that M. officinalis has cholinergic receptor-binding properties, and that the ingestion of single doses can modulate both the mood and cognitive performance of healthy young volunteers in a dose- and time-dependent manner.

Modulation of cognitive performance was reflected both in improved memory performance, and in decrements on the speed of the timed memory tasks and both accuracy and speed of the RVIP task.

While performance following all the three doses was subject to modulation, the general pattern was of more beneficial effects as the dose increased. This pattern of results shows some intriguing similarities, and differences, to those obtained with the commercial preparation administered in the only previous human study of the cognitive and mood effects of M.

officinalis Kennedy et al, b. The most striking similarity is that the decrements seen within the CDR battery in both studies are largely confined to the same tasks, that is, the timed memory tasks, specifically the spatial memory and delayed word-recognition tasks with the addition in this case of delayed picture recognition.

The single proviso to this is that in the previous study, decrements were seen in terms of accuracy, while in the current study, such decrements were manifested as a slowing of performance.

Similarly, the dose showing overwhelmingly positive mood effects in the first study led, as here, to increased calmness, a finding that is wholly consistent with contemporary usage of the herb.

The most striking difference then is that the dose-related pattern of results with regard to both the negative cognitive and positive mood effects are in the opposite direction in the case of each study. In the first study, the lowest dose led to the least impaired performance, but improved calmness.

However, decrements on the three timed memory tasks increased with dose, and the highest dose was associated with a negative effect on mood, and specifically reduced alertness. In the current study, the performance decrements on the timed memory tasks and RVIP were largely restricted to the lower doses, with improvements in mood increasing with dose.

These tasks draw on different cognitive, and presumably neural, substrates eg successful completion of the RVIP requires working memory processes and also draws heavily on executive function.

Nevertheless, the most parsimonious explanation for these differences is that they possibly reflect the modulation of shared neural mechanisms. This would suggest that the decreasing decrements with dose on the timed memory tasks following administration of the concentrated manufactured extract, and the increased decrements following the dried leaf, reflect the descending and ascending arms, respectively, of a U-shaped dose—response curve.

Similarly, increasing calmness with dose in the previous study and decreasing calmness in the current study may represent the ascending and descending arms of an inverted U-response curve, respectively.

This possibility also suggests that the highest dose in the current study, utilizing dried leaf and therefore providing lower overall dosages of the relevant constituent, and only approaching the herbalist's recommended dose Bisset and Wichtl, , might have only just reached the therapeutic window in the M.

officinalis dose—response curves. By the same token, the lowest dose in the first study, using the more concentrated manufactured extract, may have bordered on or exceeded the beneficial dose range.

An explanation that would satisfy Occam's razor is that the tasks involved are simply the most cognitively demanding tasks within the battery. Reference to the reaction times for the relevant tasks confirms this, as do the similar decrements that were seen on the nonmemory, but equally demanding, RVIP.

If it is accepted that the decrements in performance are not memory specific and, given the lack of nicotinic receptor binding in the first study Kennedy et al, b , are probably not attributable to direct interaction with nicotinic receptors, then the improved memory performance can be viewed as being attributable to a separate mechanism.

Naturally, given the rationale of the study, it is tempting to suggest that the improved secondary memory performance in the absence of working memory effects for the highest dose is directly attributable to the demonstrated cholinergic receptor-binding properties of the dried leaf used in the present study.

Such a pattern of results is broadly consistent with demonstrations of cholinergic modulation of long-term memory Drachman and Leavitt, ; Peterson, , but not working memory Rusted and Warburton, Given that the treatments in both this and the former study possessed measurable muscarinic receptor-binding properties, it is possible that the decrements on the cognitively demanding timed tasks are attributable to interaction with these receptors.

However, it is equally likely that this specific effect is mediated through another mechanism, and, of course, interaction with other neurotransmitter systems cannot be ruled out. One speculative possibility is that the decrements could be related to modulation of noradrenergically mediated vigilance, which has been shown to preferentially disrupt tasks that require a more difficult sensory discrimination.

The possibility that M. officinalis interacts with a number of neurotransmitter systems deserves further investigation. Indeed, the wider question of how the manufacturing process defines the active properties of herbal extracts, and thereby the behavioral consequences of their administration, is one that has ramifications for all herbal supplements.

The clear demonstration of differential patterns of both in vitro properties and behavioral consequences following the manufactured extract Kennedy et al, b and the dried leaf employed in the current study certainly suggests that this matter deserves further research attention.

This study shows, for the first time, a profile of acute cognitive effects in an extract of M. officinalis with demonstrable cholinergic activity, while the chosen extract had relatively high binding levels compared with other extracts both here Table 1 and elsewhere Perry et al, ; Wake et al, It is clearly of great interest to identify such active agents.

On the broader question of a possible medicinal role for M. officinalis , it is notable that the results evinced here support the suggestion that M. officinalis may eventually have a role to play in the treatment of dementia Perry et al, , What is particularly interesting about melissa's potential as a treatment is that the pharmacological arsenal for Alzheimer's disease currently lacks a well-tolerated cholinergic receptor agonist.

It is conceivable that the cholinergic effects of M. officinalis may be of value to those individuals with a loss of nicotinic receptors. officinalis also has other properties that might recommend it as a candidate treatment.

The first is that advanced Alzheimer's disease commonly involves severe agitation and mood disturbance. In line with the results of both this and the former study Kennedy et al, b , and both traditional and contemporary usage of M.

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The funders played no role in this study. NICM Health Research Institute, Western Sydney University, Locked Bag , Penrith, NSW, , Australia. Adele E. Cave, Dennis H. Chang, Gerald W. School of Medicine, Western Sydney University, Penrith, NSW, , Australia.

Translational Health Research Institute THRI , Western Sydney University, Locked Bag , Penrith, NSW, , Australia. You can also search for this author in PubMed Google Scholar. AEC and GZS contributed to the conceptualisation and design of the review.

AEC took the lead in drafting the review paper. GZS provided guidance throughout. All authors provided critical feedback and reviewed the final manuscript.

Correspondence to Genevieve Z. As a medical research institute, NICM Health Research Institute receives research grants and donations from foundations, universities, government agencies, individuals, and industry. Sponsors and donors provide untied funding for work to advance the vision and mission of the institute.

The project that is the subject of this article was not undertaken as part of a contractual relationship with any organisation other than the funding declared below. It should also be noted that NICM conducts clinical trials relevant to this topic area, for which further details can be provided on request.

AEC has received PhD scholarship support from FIT-BioCeuticals Pty Ltd. We are more concerned if it begins to limit their ability to function normally or if there is a change over time.

Jessica Kalender-Rich , a geriatric medicine physician at The University of Kansas Health System who was not involved in the study, told Medical News Today. In the Australian study, researchers examined the safety and efficacy of a herbal supplement called SaiLuoTong SLT.

It contains extracts including panax ginseng , ginkgo biloba and crocus sativus L. To test the effectiveness of the supplement, the researchers enlisted 78 participants who were 60 years and older and had mild cognitive impairment. The participants were either given mg of SLT every day or a placebo.

The supplements were provided by a pharmaceutical company. The researchers reported that after 12 weeks, those who were given SLT had a statistically significant improvement in their logical memory delayed recall scores compared with those who were given the placebo.

SLT was also found to improve performance in executive function. Victor Henderson , a professor of epidemiology, population health, and neurology and neurological sciences at Stanford University who was not involved in the study, said the results are intriguing, but more research is needed.

But the actual difference in the trials is very, very small. If SLT is found to be effective in a larger sample group, one potential benefit is that the supplement can be given via tablet rather than an IV infusion.

While treatment options for mild cognitive impairment are limited, Henderson is optimistic that with more research, effective therapies will be found.

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However, as with many other formulations used in traditional medicine, scientific evidence on the specific effects of this herb and on its mechanisms of action is lacking. Previously, however, several studies have reported that the ingestion of a specific green oat preparation in healthy subjects has acute positive effects on cognitive functions and neurophysiological parameters.

Positive effects have been observed in measures of speed of performance global , executive function, working and episodic memory, attention, and quantitative electroencephalography qEEG Berry et al.

In all these studies, a specific oat extract Neuravena ® was used. Neuravena ® is green oat herb extract containing a range of potentially bioactive constituents. In bio-assay guided approaches, it has been shown to have a significant inhibitory effect on the monoamine oxidase B MAO-B and phosphodiesterase 4 PDE-4 enzymes Moccetti et al.

Based on this finding, it has been proposed that increased dopaminergic availability and cerebral vasodilation underlie its reported acute positive effects Moccetti et al. Chronic intake of the extract has also shown to improve vasodilator function in peripheral and cerebral arteries Wong et al.

Dopamine plays a cardinal role in movement, cognition, and motivation. Proper functioning of the structures conveyed along basal ganglia-thalamo-cortical dopaminergic pathways is crucial for the optimal performance of several cognitive processes ascribed to frontal-executive functions.

These include action and conflict monitoring, inhibitory control, attention, target detection, and processing speed Goldman-Rakic, ; Nieoullon, ; Remy and Samson, ; Grahn et al. Event-related brain potentials ERPs derived from EEG recordings enable the study of neurophysiological activity and behavioral parameters in relation to these cognitive processes Luck, The P3a and P3b components obtained during auditory oddball paradigms selectively reflect the automatic orientation of attention to novel stimuli frontal P3a , target detection, and contextual update of information parietal P3b Sutton et al.

Dopaminergic activity has been strongly associated with both P3a and P3b. Specifically, it appears to play a cardinal role in the frontal-central P3a and related frontal functions such as working memory and focal attention, but it also plays a partial role in the amplitude of P3b Courchesne et al.

Error-related negativity ERN is a negative deflection that occurs after error responses, and it is part of the action monitoring system Holroyd and Coles, ; Yeung et al.

It has been the subject of intensive research and has been interpreted as a neural correlate of error detection, originated due to the phasic dopaminergic changes of the neurons projecting from the mesencephalon to the anterior cingulate cortex ACC Gehring and Knight, ; Luu and Tucker, Similarly, in conflict priming, it has been suggested that the frontal-central N2 component reflects cognitive control Kopp et al.

The morphology of these ERPs has been found to be sensitive to changes caused by several conditions affecting the central nervous system CNS , and also to changes induced by pharmacological agents Aron et al.

Considering the previously reported findings from studies addressing the effects of Neuravena ® , and taking into account the proposed mechanisms of action of this compound, we aimed to examine the relationship between indices of attention, performance-monitoring and inhibitory control abilities, such as the P3a, P3b, ERN, and N2 amplitudes and cognitive performance, as measured by neuropsychological tests in healthy individuals.

This study had a single-session, randomized, double-blind, placebo-controlled design with two parallel study arms: Active intervention or placebo. Assessments were performed at baseline and 60 min after administering either the active intervention or placebo.

The total running time of the experimental session was the same for all participants and consisted of a 60 min session of neurophysiological acquisition and task performance baseline , a 60 min resting period at the beginning of which the active intervention or placebo was administered, and a 60 min session of neurophysiological acquisition and task performance after active intervention or placebo.

The experimental session was conducted in the facilities of the Laboratory of Cognitive Neurophysiology and Human Neuropsychopharmacology at the Biomedical Research Institute Sant Pau, in an isolated room specifically prepared for conducting neurophysiological studies.

As per protocol, before the experimental session was conducted, all participants were interviewed regarding adherence to the instructions provided in relation to the evening meal and breakfast as well as the quality of sleep and rest during the previous night.

To explore the acute effects of the studied compound, the experimental session was conducted on a single day, in the morning. Participants were requested to have their standard evening meal on the previous day but to avoid alcohol.

In order to get a relative homogeneity on evening meal, participants were provided by a set of recommendations the amounts to be consumed and the types of foods that were recommended or should preferably be avoided.

At breakfast, calorie intake was restricted to kcal, and participants were required to take their normal amount of coffee to prevent potential caffeine withdrawal effects. Similar to what was done with evening meal, a list with possible breakfasts to consume and avoid was provided to participants.

After the baseline assessment, during the resting period, participants received a kcal snack followed by the intervention.

Participants were pre-selected from a database of healthy volunteers. Inclusion criteria were males or females aged 20 to 50 years and able to freely decide to participate in the study by signing the proper informed consent.

From the pre-selected volunteers, twenty participants were included in the study and randomly allocated to receive the placebo or the active compound. The study included two groups of ten participants matched for main sociodemographic variables age, gender, years of education, and BMI, summarized in Table 1.

Both the placebo and encapsulated active intervention were provided by Frutarom Switzerland Ltd. The active intervention consisted of an extract Neuravena ® manufactured from the dried above-ground parts of a selected variety of Avena sativa L.

Characteristics of the extract are drug-to-extract- ratio DER 3. The dosage form consisted of two hard-gelatin capsules, each containing mg of extract or maltodextrin as placebo for a total of mg.

The reason for choosing mg in the current study relies on a previous study Kennedy et al. We used a modified version of the Eriksen flanker task Lopez-Gongora et al.

Participants were required to respond to the orientation of a central green arrow target with two additional green arrows on either side. To do so, they were instructed to signal with the right hand after a right-pointed target arrow and with the left hand after a left-directed target arrow.

The four surrounding arrows either primed the target response compatible trials, or or primed the opposite direction incompatible trials, or. In these trials the central green arrow changed to red for instance: after a delay of ms and participants had to inhibit response in these trials.

Duration of the stimuli was ms. A random Stimulus-Onset Asynchrony SOA between ms and ms was used. The experiment consisted of 3 blocks of 4 min, each with stimuli. A s rest period was allowed between blocks. Subjects were required to respond to the stimuli as fast and as accurate as possible and to inhibit their responses whenever a stop trial appeared.

The percentage of total correct responses, and the reaction times RT to correct responses, and to error responses were recorded. Same measures were separately obtained for the compatible and incompatible trials. A standard auditory oddball task with a set of frequent, infrequent, and novel stimuli was used.

Frequent stimuli Hz, 60 ms duration occurred with a probability of 0. Over a period of 15 min, participants were requested to respond as fast as possible to infrequent target sounds with the right index finger, and to ignore all other sounds. Percentage of correct responses to target and percentage or responses to non-target novel stimuli were recorded.

Vertical eye movements were recorded using a bipolar montage with two electrodes linked together and placed below each eye referenced to a third electrode placed centrally above the eyes. Horizontal eye movements were monitored using two electrodes placed on the external canthi of each eye.

Electrode impedances were kept below 5 kOhm. The electrophysiological signals were filtered with a bandpass of 0. To maximize the information available for the subsequent event-related potential analysis ERPs , raw EEG signals were subjected to an ocular artifact minimization algorithm.

This algorithm is based on an eigenvalue decomposition of time-delayed covariance matrices. After identifying the source signals associated with eye movements, we obtained corrected EEG signals from the remaining components. The algorithm was implemented using Brain Vision Analyzer Brain Products GmbH; Germany.

In the auditory oddball task, the continuous EEG recording was segmented in epochs of ms starting ms before the stimulus presentation and until ms post-stimulus. Epochs were baseline-corrected, subtracting the mean amplitude in the ms before the presentation of the stimulus.

A two-step artifact rejection procedure was then used. First epochs were rejected if the signal in any of the 19 channels showed amplitude values greater than ± μV.

Subsequently, additional epochs were excluded if amplitude values were greater than ± 40 μV in Fz, Cz or Pz channels. After these preprocessing steps, three types of trials were averaged separately: epochs containing frequent stimuli, epochs containing infrequent stimuli, and epochs containing novel stimuli.

These averages were obtained for each study participant and the ERP components were identified and quantified. The P3b was identified as the most positive deflection in the ERP between and ms post-stimulus in the infrequent trials. The P3a was identified as the most positive deflection in the ERP between and ms post-stimulus in the novel trials.

The mean amplitude was calculated in the time-window defining each ERP and was introduced into the statistical analysis.

In the stop task, ERPs were obtained following response and stimuli-locked average procedure. For the response-locked ERPs, the continuous EEG was segmented in epochs of ms, starting ms before the commission of stop errors and before the correct responses until ms thereafter. The 0-time point thus corresponded to a button press when the participant should withhold the response, and to the emission of a correct response.

Baseline correction was performed subtracting the mean amplitude in the ms before button press. For the stimulus-locked ERPs, we performed the same procedure as described for the acquisition and averaging of P3a or P3b, but on these occasions, we created two types of epochs: epochs containing trials compatible with correct responses and epochs containing trials incompatible with correct responses.

Following preprocessing, the epochs of the response-locked condition were averaged in order to obtain the ERN wave following commission errors. The ERN was identified as the negative deflection in the ERP appearing between 0 and ms following a stop error. Quantification methods were used at Fz and Cz, because the ERN shows a frontocentral distribution.

Accordingly, the average mean voltage was calculated between 0 and ms following stop errors and correct responses. The N2 was identified as the negative deflection in the ERP appearing between and ms following the presentation of compatible and incompatible trials with correct responses.

The mean amplitude was calculated at the defined time-window. As with the ERN, given the frontocentral distribution of the N2, it was quantified at Fz and Cz.

The obtained average mean amplitudes for each subject were introduced into the statistical analysis described below. For depicting purposes, the signals obtained for each participant were averaged together providing a single grand average.

Sociodemographic and baseline characteristics were subjected to independent t -test comparisons between the two groups of interest for continuous variables and χ2 for categorical variables.

Intervention effect on cognitive performance variable was analyzed using repeated-measures analyses of variance ANOVA. Within each intervention arm, the effect was tested using paired t -test analysis. ERP effects were quantified for the three midline electrodes Fz, Cz, and Pz , and the resulting data were subjected to repeated-measures ANOVAs with the Greenhouse-Geisser correction applied when necessary.

Post hoc comparisons were performed using paired t -test comparisons. The statistical analysis was conducted using SPSS v25 software package. Twenty healthy adults participated in this study 9 females and 11 males. Sociodemographic and baseline characteristics of the participants are shown in Table 1.

Participants had a mean age of The two groups were matched for all the sociodemographic and baseline characteristics. Table 2. Behavioral performance in the Stop-signal task and in the Auditory oddball task at baseline and endpoint.

Post hoc paired t -test comparisons using the difference score between pre- and post-intervention administration showed that these effects were driven by significant decreases in several measures of RT in the active intervention group Table 2. Bivariate correlation analysis showed significant negative effects after intervention in the placebo group data not shown.

Figure 1. Stimulus-locked and response-locked ERN grand average ERPs for the whole sample in the stop-signal task. A Stimulus-locked grand average ERP at Fz electrode for compatible grey line and incompatible black line trials. The N2 is identified as the negative deflection in the ERP appearing between and ms grey area.

The topographical map shows the increased frontocentral negativity for incompatible trials. B Response-locked grand average ERPs at Fz electrode for correct grey line and error black line responses.

The ERN is identified as the negative deflection in the ERP appearing between 0 and ms grey area. The topographical map shows the frontal-central ERN. C ERPs associated with frequent, infrequent and novel stimuli in auditory oddball task. Grand average stimulus-locked ERPs at Fz and Pz electrodes for the whole sample.

The P3a was identified as the most positive deflection in the ERP between and ms grey area post-stimulus in the novel trials. The topographical map shows the P3a and P3b. No significant differences were found at baseline between groups in the mean amplitude of N2 and ERN.

Figure 2. Stimulus-locked grand average ERPs for the N2 in the two groups. B The topographical map shows the pre-post effect on the mean amplitude of the N2 for incompatible condition.

C Mean percentage of N2 change in mV for incompatible condition between study groups. Figure 3. A ERPs comparison in the placebo and in the active intervention group.

B The topographical map shows the pre-post effect on the mean amplitude of the ERN. C Mean percentage of ERN change in mV between study groups. No differences were found between groups at baseline neither post-intervention in the percentage of correct responses to target and to non-target novel stimuli.

No significant differences were found between groups at baseline on the mean amplitude of the P3a and P3b components. Figure 4. B Topographical maps showing the pre-post intervention effects on the P3a and P3b. C Mean percentage of P3a and P3b change in mV between study groups.

In the present study we explored the acute effects of ingestion of mg of a green-oat herb extract Neuravena ® on a set of behavioral and neurophysiological parameters.

While previous studies have shown a potential modulatory effect of Neuravena ® on neurophysiological activity as measured by EEG in the fronto-temporal brain region, and successive improvements in several measures of cognitive function Berry et al.

We also looked at the association between these neurophysiological measures and several behavioral variables. Our results show that the two groups were strictly matched for the main variables of interest and that before the intervention there were no differences in any of the studied variables.

However, in the active-intervention group, we found a set of post-intervention differences with respect to baseline and with respect to the placebo group in several neurophysiological and related behavioral parameters.

Specifically, after active-intervention administration, we found significant reductions in several ERPs, and a significantly faster performance in several task components. Importantly, this faster performance was not associated with a worsening in accuracy. In contrast, in the placebo group, we found worsening effects both in RT and in accuracy.

We interpret the reduction in the mean amplitude of several ERPs as signatures of an acute effect of optimization of neural resources and cognitive performance in participants receiving the active intervention. These effects likely influence the maintenance of optimal performance in the task, as obvious signatures of tiredness were found in the placebo group.

In previous studies focusing on neurological disorders, compensatory mechanisms were observed during performance in the same tasks as those we used in the present study Lopez-Gongora et al. Specifically, in ERP studies, the engagement of efficient compensatory mechanisms has been seen as a significant increase in ERP amplitude in association with the normalization of related behavior Lopez-Gongora et al.

This suggests that in these cases, participants use additional neural resources to perform tasks with no apparent difficulties. Similarly, throughout neurodevelopment, a progressive reduction in the amplitude of several ERPs is found in association with better performance in related tasks Abdul Rahman et al.

This progressive reduction may reflect progressive specialization along neurodevelopment of the neural resources needed to overcome task performance Davis et al. Taken together, it is reasonable to assume in the present study that the reductions found in the ERPs in the group receiving the active intervention and the related positive behavioral consequences of these effects reflect better optimization of the neural resources required to perform the tasks, and better capacity to maintain optimal levels of performance, avoiding the tiredness effects found in the placebo group.

Taking into account the functional meaning of the studied ERPs and the related behavioral parameters, our results are consistent with previous data suggesting that Neuravena ® induces positive acute effects in processing speed and measures of attention and executive function Berry et al.

More specifically, our data supports significant effects of Neuravena ® in terms of improving efficiency in action monitoring, cognitive control over interference and attention. Here, we demonstrate that in participants receiving a single dose of mg of Neuravena ® , a reduction in the amplitude of the ERN is found with no deleterious effects on performance.

Similarly, in the present study, participants in the active-intervention arm showed a reduction in the N2 component that was associated with faster performance both in compatible and incompatible trials. Specifically, incompatible trials are usually associated with greater RT than compatible trials.

This greater RT is assumed to reflect the higher processing demands of the conflict monitoring system when participants are faced with incompatible trials. Thus, the significant reduction in RT in the active-intervention arm suggests increased efficiency of the conflict monitoring system to solve the processing demands driven by incompatibility.

Conversely, increased latency of RT was found in the placebo group, affecting specific trials and thus suggesting a loss of performance efficiency along the experimental session.

During the auditory oddball task, both P3a and P3b were lower in the active-intervention group after intervention administration. The P3b is one of the most frequently studied ERPs in the history of cognitive neurophysiology.

Although its specific functional meaning is only partially understood, it is clear that it is associated with processing speed and integration of information. Participants in the active-intervention group showed a significant decrease in the P3b component, with no deleterious effects on performance.

Again, this finding supports the idea that more efficient neural resources are deployed by those using mg of Neuravena ® , promoting better performance. Equivalent findings were found for P3a component.

The frontal P3a is specifically associated with automatic orientation of attention to novelty. Here, participants in the active-intervention group showed a significant reduction in the P3a component in association with a better performance in terms of accuracy.

Again, more efficient neural resources and performance appear to be enhanced after the administration of mg of Neuravena ®. The main strengths of the present study are the inclusion of a carefully matched sample of healthy individuals in each study arm and the use of several behavioral and neurophysiological metrics to assess the effects pre and post intervention.

Treatment with PF, ALWPs, and LPS did not reduce LPS-induced IL-1β mRNA levels compared with treatment with PF and LPS Figures 5C,D.

These results suggest that ALWPs regulate FAK signaling to modulate LPS-stimulated proinflammatory responses. NF-κB plays an important role in neuroinflammation Lawrence, ; Cao et al.

NF-κB in microglia is activated by viruses and bacterial toxins such as LPS. Therefore, we examined whether ALWPs can regulate NF-κB subcellular localization.

We observed that pretreatment with ALWPs followed by LPS did not alter p-IκBα, IκBα, or NF-κB levels in the cytosol compared with LPS treatment Figures 6A—D. In the nuclear fraction, LPS increased NF-κB levels compared with control treatment Figures 6E,F.

Pretreatment with ALWPs followed by LPS resulted in decreased LPS-induced nuclear NF-κB levels compared with treatment with LPS alone in BV2 microglial cells Figures 6E,F. FIGURE 6. ALWPs decreased LPS-induced nuclear NF-κB Ser levels. Western blotting was conducted on the cytosolic fraction using antibodies against p-IκBα, IκBα, NF-κB, and β-actin as a cytosolic marker.

E Western blotting was performed on the nuclear fraction using antibodies against NF-κB and PCNA as a nuclear marker. Cells were fixed and immunostained with anti-CD11b and anti-p-NF-κB Ser antibodies 40× confocal images.

Cells were fixed and immunostained with anti-CD11b and anti-p-NF-κB Ser antibodies. Scale bar 20 μm 40× confocal images. Immunocytochemistry was then performed with anti-CD11b and anti-p-NF-κB antibodies. LPS alone significantly increased nuclear p-NF-κB levels, whereas ALWPs significantly decreased LPS-induced nuclear p-NF-κB levels in BV2 microglial cells Figures 6G,H.

These data suggest that ALWPs modulate LPS-induced NF-κB nuclear translocation in BV2 microglial cells. We then examined whether ALWPs modulate FAK signaling to alter LPS-induced nuclear p-NF-κB levels. Immunocytochemistry was then conducted with anti-CD11b and anti-p-NF-κB antibodies. Treatment with PF, ALWPs, and LPS further decreased LPS-stimulated nuclear p-NF-κB levels compared with treatment with PF and LPS or ALWPs and LPS Figures 6I,J.

These data suggest that ALWPs alter FAK signaling to affect nuclear p-NF-κB signaling to modulate the neuroinflammatory response. Several studies have shown that microglial cell migration is associated with the neuroinflammatory response Choi et al.

We found that LPS alone significantly increased BV2 microglial cell migration compared with control treatment Figures 7A,B. In addition, pretreatment with ALWPs followed by LPS treatment significantly suppressed BV2 microglial cell migration compared with LPS treatment Figures 7A,B.

These data suggest that ALWPs can inhibit LPS-mediated BV2 microglial cell migration. FIGURE 7. ALWPs inhibited LPS-induced BV2 microglial cell migration. Images of the wound gap were acquired at 0 h i.

Scale bar μm 4× confocal images. C A working model of the regulation of the LPS-induced neuroinflammatory response by ALWPs. Several studies have shown that microglial activation is a cause of AD and other neurological disorders Wee Yong, ; Hirsch et al. In addition, enhanced neuroinflammation can lead to memory impairment and eventually results in neurodegenerative diseases Fang et al.

Thus, we investigated whether ALWPs can regulate cognitive function in vivo. In addition, training sessions of the novel object recognition test NOR were performed 1 h after LPS injection, followed by the NOR test 24 h later.

The LPS-injected wild-type mice exhibited significantly reduced short- and long-term memory compared with PBS-injected wild-type mice Figures 8A,B. Pretreatment with ALWPs and injection with LPS rescued short- and long-term memory compared with LPS injection alone Figures 8A,B , suggesting that ALWPs can affect learning and memory in LPS-injected wild-type mice.

FIGURE 8. Y-maze tests and training sessions of the novel object recognition test NOR were performed 1 h after LPS injection. Next, we examined whether ALWPs can regulate microglial activation in vivo. Three hours later, immunohistochemistry was conducted with an anti-Iba-1 antibody, a marker for activated microglia.

The LPS-injected wild-type mice exhibited significantly increased microglial activation in the hippocampus Figures 8C—E and cortex Figures 8F,G compared with PBS-injected wild-type mice. Four chemical standards corresponding to components of ALWPs were used to confirm the ALWP formula by UHPLC as described in the Materials and Methods: 5-hydroxymethylfuraldehyde 5-HMF , morroniside, loganin and paeoniflorin.

glutinosa , morroniside is present in L. chinense , loganin is present in C. Officinalis in, and paeoniflorin is present in Moutan Cortex Radicis. We observed that the retention times of 5-HMF, morroniside, loganin and paeoniflorin were approximately 4.

These results indicate that ALWPs contain the corresponding herbal ingredients. To verify each herbal component of ALWPs by identifying the principal markers and to determine the content of the principal markers, UHPLC was performed.

Using these equations, the quantities of 5-HMF, morroniside, loganin, and paeoniflorin in ALWPs were calculated Table 1. TABLE 1. The contents of 5-HMF, morroniside, loganin, and paeoniflorin in ALWPs.

Many studies have shown that neuroinflammation is tightly associated with neurodegenerative diseases, including AD. However, very little research has focused on elucidating the molecular mechanisms underlying neuroinflammation. In this study, we examined the novel effects of ALWPs on cognitive performance and neuroinflammatory responses.

Specifically, we found that ALWPs suppressed LPS-induced IL-1β levels in BV2 microglial cells but not in primary astrocytes.

In addition, ALWPs regulated the nuclear localization of the transcription factor NF-κB, thereby alleviating neuroinflammatory responses. Proinflammatory cytokines and neuroinflammation have been linked to neurodegenerative disease Downen et al. IL-1 exists in two isoforms, IL-1a and IL-1β, and both are highly involved in neurodegenerative diseases Shaftel et al.

A recent study showed that chronic activation of LPS or IFN-γ regulates proinflammatory cytokines, including IL-1β, leading to neuronal dysfunction and neuronal death Papageorgiou et al. Another study found that activated microglial cells release IL-1β, leading to dopaminergic neuronal death in a PD animal model Chung et al.

Other studies have demonstrated that the natural compound resveratrol inhibits LPS-induced IL-1β expression in a murine microglial cell line N9 cells and in primary microglial cells but not in primary astrocytes Lu et al. Interestingly, we observed that ALWPs affected LPS-stimulated IL-1β levels in BV2 microglial cells Figure 2 , whereas no effect on LPS-induced changes in proinflammatory cytokine levels was observed in primary astrocytes Supplementary Figures 1, 2.

Our findings indicate that ALWPs specifically modulate LPS-induced proinflammatory cytokine IL-1β levels and have different effects depending on cell type. We speculate that ALWPs reduce IL-1β levels to regulate neuroinflammation as well as cognitive function.

However, we have not fully addressed why ALWPs only regulate IL-1β levels in microglial cells. The effects of ALWPs on LPS-induced IL-1β levels will be investigated at the molecular level in future studies.

Lipopolysaccharide binds to TLR4 on the cell surface of microglial cells, which then release proinflammatory cytokines Gaikwad et al.

Therefore, TLR4 inhibitors or antagonists are candidates as therapeutic agents targeting neuroinflammation-related diseases.

In the present study, ALWPs altered TLR4 signaling to modulate LPS-induced IL-1β levels Figure 4. It is possible that ALWPs interfere with the association between LPS and TLR4 or between LPS and unknown receptors that interact with LPS because we observed that ALWPs decreased LPS-induced IL-1β levels in the absence of a TLR4 inhibitor.

In addition, we observed that ALWPs significantly decreased cell-surface levels of TLR4 Figure 4. Thus, ALWPs inhibit the interaction between LPS and TLR4 on the cell surface, thereby affecting neuroinflammatory responses.

However, it is not clear whether ALWPs affect the interaction between LPS and unknown receptors to alter neuroinflammation. A future study will explore whether ALWPs can modulate the interaction between LPS and TLR4 or unknown receptors to regulate the LPS-induced neuroinflammatory response.

MAPK signaling is a key factor for regulating proinflammatory cytokines in microglial cells. The expression of IL-1β is regulated by the MAPK signaling pathway in neuroglial cells, including microglia, astrocytes, and a microglial cell line BV2 Kaminska et al.

Unexpectedly, we found that ALWPs did not alter the LPS-induced MAPK signaling pathway Supplementary Figure 3.

Thus, we examined another potential target, the FAK signaling pathway, which is a downstream signaling pathway activated by LPS Wong et al. LPS treatment increases autophosphorylation of FAK Tyr in murine macrophages and human synoviocytes. In addition, a study has shown that LPS-induced IL-6 levels are associated with FAK phosphorylation Tyr Zeisel et al.

Thus, we examined the effects of ALWPs on FAK phosphorylation and observed that ALWPs decreased p-FAK Tyr in LPS-stimulated BV2 microglial cells Figure 5. In addition, treatment with a FAK inhibitor, ALWPs, and LPS did not reduce LPS-induced IL-1β levels compared with treatment with a FAK inhibitor and LPS Figure 5.

These data suggest that ALWPs suppress FAK phosphorylation Tyr to regulate LPS-induced IL-1β levels. NF-κB is a key transcription factor regulating inflammatory cytokines Shaftel et al.

Several studies have shown that LPS increases the phosphorylation of NF-κB-p65 in vivo and in vitro Stylianou et al.

In addition, NF-κB binds to the IL-1β promoter region Hiscott et al. Here, we observed that ALWPs downregulated p-NF-κB and translocation in the nucleus in LPS-stimulated BV2 microglial cells Figure 6.

In addition, ALWPs but not the individual components significantly decreased LPS-stimulated proinflammatory cytokine IL-1β levels Figure 3 , suggesting that ALWPs play an important role in regulating NF-κB and IL-1β levels and providing a new mechanism of the anti-inflammatory response.

Microglial cell migration is associated with neuroinflammation. Specifically, microglial migration and the related phagocytic activity can be activated by G-coupled receptors, which include chemokines Guida et al. Karlstetter et al. found that curcumin, one of the major components of turmeric, has anti-inflammatory effects by inhibiting microglial cell migration Choi et al.

Therefore, we examined the effects of ALWPs on microglial cell migration and found that ALWPs significantly inhibited LPS-induced BV2 microglial cell migration Figure 7. Based on the literature and our findings, we speculate that ALWPs modulate BV2 microglial cell migration by altering LPS-mediated IL-1β levels.

Thus, we will further investigate how ALWPs regulate BV2 microglial cell migration. In this study, we examined the anti-inflammatory effects of ALWPs. ALWPs contain ten different herbs, and some of the components of ALWPs are known to be involved in inflammation.

For instance, 5-HMF, one of the components of R. glutinosa , a component of ALWPs, inhibits reactive oxygen species ROS and NF-κB activity in TNF-α-induced vascular endothelial cells Kim et al.

Corni fructus , a component of ALWPs, has anti-inflammatory effects by suppressing COX-2 and iNOS levels through the downregulation of NF-κB binding activity in macrophages Sung et al. Morroniside is a major component of C. officinalis , a component of ALWPs, and reduces proinflammatory cytokine IL-6 and IL-1β levels in a rat model of acute myocardial infarction Yu et al.

In addition, type 2 diabetic rats orally administered morroniside, a component of ALWPs, exhibit downregulated NF-κB activity in hepatic tissue Park et al.

Loganin is one of the components of C. officinalis , a component of ALWPs, and has been shown to suppress ApoCIII-induced proinflammatory cytokines and NF-κB phosphorylation in mouse adipocytes Li et al.

Thus, each component of ALWPs has antioxidant and anti-inflammatory effects in vascular endothelial cells, hepatic tissue, or adipose cells. However, whether the ten individual components of ALWPs have anti-inflammatory effects in LPS-induced BV2 microglial cells and in the brain is not clear.

To test this, we examined the effects of each component of ALWPs on proinflammatory cytokine IL-1β levels and found that ALWPs but not the individual components decreased LPS-mediated IL-1β mRNA levels in BV2 microglial cells Figure 3. Although each component of ALWPs is known to have a positive effect at the cellular level, we suggest that the combination of these ten ingredients in ALWPs has synergistic effects to reduce neuroinflammation.

Memory loss is one of the clinical symptoms of neurodegenerative diseases and is most commonly associated with AD. Memory deficits have been shown to be closely related to neuroinflammation.

Specifically, activated microglia release proinflammatory cytokines that exacerbate memory loss in various diseases Perry and Holmes, ; Lelios and Greter, ; Wendeln et al. In particular, several studies have demonstrated critical roles for IL-1β and TNF-α in the formation of learning and memory.

For instance, increased levels of IL-1β are correlated with cognitive deficits in sepsis-associated encephalopathy and a repeated social defeat model Imamura et al. Other studies reported that intrahippocampal injection or chronic overexpression of IL-1β in the hippocampus region leads to impairments of spatial memory Gonzalez et al.

Wang et al. reported that the proinflammatory cytokine IL-1β induces memory deficits by modulating the expression of GABA A receptors through the P38 signaling pathway Wang et al.

Similarly, overexpression of TNF-α in neurons or glial cells impairs synaptic plasticity and learning and memory Fiore et al. In addition, chronic LPS administration impairs learning and memory via TNF-α Belarbi et al. These studies indicate that inflammation can affect cognitive function. Accordingly, several studies have evaluated the potential of traditional herbal medicines to inhibit neuroinflammation and ameliorate memory loss in inflammation-related diseases.

For example, chrysophanol a component extracted from Rhubarb , a traditional herbal medicine, attenuates memory deficits and neuronal death by inhibiting inflammation in diabetic mice Chu et al.

In addition, Gu et al. Another group reported that loganin, a component of ALWPs, decreases Aβ-induced inflammatory responses and ameliorates memory deficits induced by scopolamine Hwang et al.

However, there are few studies on the therapeutic effects and related mechanisms of each of the components of ALWPs in inflammation-related cognitive deficits. Based on the literature, we hypothesized that ALWPs affect cognitive performance by inhibiting neuroinflammation.

To test this hypothesis, we examined the effects of ALWPs on cognitive function in LPS-injected wild-type mice. Interestingly, we found that ALWPs had anti-inflammatory effects Figures 2 , 3 and attenuated LPS-induced short- and long-term memory deficits in wild-type mice Figure 8.

Future studies will examine whether ALWPs regulate cognitive function through IL-1β as well as whether the individual components of ALWPs are sufficient to enhance learning and memory. In addition, treatment with ALWPs followed by LPS alters the subcellular localization of the transcriptional factor NF-κB.

Moreover, ALWPs promote short- and long-term memory and inhibit microglial activation. Taken together, these data indicate that ALWPs might be a potential therapeutic anti-neuroinflammatory drug for neuroinflammation-related diseases, including AD. Y-MW, JWK, and H-SH conceived and designed the study.

J-YL, JN, WL, BJ, HN, H-JC, H-JK, YN, and YS acquired the data. J-YL, HN, JN, BJ, YN, and JK prepared the figures. YS, YJ, and Y-MW prepared the tables. J-YL, JN, JK, YN, YJ, Y-MW, JWK, YN, and H-SH wrote the manuscript. Confocal microscopy Nikon, TI-RCP data were acquired at the Advanced Neural Imaging Center at the Korea Brain Research Institute KBRI.

This work was supported by the KBRI basic research program through the Korea Brain Research Institute funded by the Ministry of Science, ICT and Future Planning Grant No.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. We thank So Yeon Koo, Dr. Young-Pyo Choi, and Bae-Jin Kim for editing, valuable comments, and assistance with the behavior studies in our manuscript.

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