Jean-Arthur Micoulaud-Franchi

Iliana Kotwas

Laura Lanteaume

Christelle Berthet

Mireille Bastien

Jean Vion-Dury

Aileen Mcgonigal

Fabrice Bartolomei

Christelleberthet

Skin conductance biofeedback training in adults with drug-resistant temporal lobe epilepsy and stress-triggered seizures: A proof-of-concept study

à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction

Antiepileptic drugs (AEDs) are the main treatment in the management of epilepsy. However, more than 30% of patients with epilepsy have inadequate control of seizures despite optimal AED treatment [START_REF] Kwan | Early identification of refractory epilepsy[END_REF]. Thus, throughout the past decade, research into nonpharmacological treatments [START_REF] Sheth | Nonpharmacological treatment options for epilepsy[END_REF] and, in particular, psychological managements of epilepsy (i.e., cognitive behavioral therapy, relaxation therapy, biofeedback, cognitive rehabilitation, and educational intervention) have gained greater attention [START_REF] Schmid-Schonbein | Improvement of seizure control by psychological methods in patients with intractable epilepsies[END_REF][START_REF] Boylan | Peri-ictal behavioral and cognitive changes[END_REF][START_REF] Engelberts | The effectiveness of psychological interventions for patients with relatively well-controlled epilepsy[END_REF][START_REF] Ramaratnam | Psychological treatments for epilepsy[END_REF]. Indeed, it was found that the majority of patients could identify factors that trigger their seizures (i.e., a factor whose presence is associated with an increased probability of seizures over a relatively brief, defined time period) [START_REF] Boylan | Peri-ictal behavioral and cognitive changes[END_REF][START_REF] Spector | Seizure precipitants and perceived self-control of seizures in adults with poorly-controlled epilepsy[END_REF][START_REF] Spatt | Subjective perception of seizure precipitants: results of a questionnaire study[END_REF][START_REF] Sperling | Self-perception of seizure precipitants and their relation to anxiety level, depression, and health locus of control in epilepsy[END_REF] and that some report the ability to terminate, prevent, or reduce their seizures [START_REF] Boylan | Peri-ictal behavioral and cognitive changes[END_REF][START_REF] Hether | Spontaneous strategies for psychological control of seizures[END_REF]. It is, thus, possible to train patients with drug-resistant epilepsy to identify habitual triggers of their seizures and to develop counter measures using cognitive and behavioral methods that may interrupt seizure onset and neutralize triggering factors [START_REF] Schmid-Schonbein | Improvement of seizure control by psychological methods in patients with intractable epilepsies[END_REF][START_REF] Boylan | Peri-ictal behavioral and cognitive changes[END_REF].Researchinthisfield may enhance the possibility of management of drug-resistant epilepsy, in particular when surgical treatment is contraindicated, which is the case in 30% of focal drug-resistant epilepsy [START_REF] Engel | Surgery for seizures[END_REF], or when patients prefer nonsurgical management options.

Among psychological treatments in epilepsy [START_REF] Sheth | Nonpharmacological treatment options for epilepsy[END_REF], biofeedback presents two advantages. Firstly, by steady feedback, its use can restore perceived control in the participants [START_REF] Epstein | Biofeedback, self-control, and self-management[END_REF], which is known to be a significant variable in the psychological impact of epilepsy [START_REF] Spector | High and low perceived self-control of epileptic seizures[END_REF]. Indeed, pervasive loss of control has been associated with a negative effect on seizure control [START_REF] Velissaris | The psychological impact of a newly diagnosed seizure: losing and restoring perceived control[END_REF] and with a low quality of life [START_REF] Jacoby | Exploring loss and replacement of loss for understanding the impacts of epilepsy onset: a qualitative investigation[END_REF]. Secondly, biofeedback is more than a psychological treatment because it enables the participant, by online feedback to a covert physiological activity, to actively learn self-directed strategies to obtain and control a physiological response. Thus, biofeedback can be considered as a psychophysiological treatment that enables targeting, in a noninvasive and drugfree manner, specific physiological activity related to a disorder [START_REF] Yucha | Evidence-based practice in biofeedback and neurofeedback[END_REF].

A number of biofeedback approaches have been suggested for the management of epilepsy [START_REF] Sheth | Nonpharmacological treatment options for epilepsy[END_REF][START_REF] Micoulaud-Franchi | Biofeedback and drug-resistant epilepsy: back to an earlier treatment?[END_REF][START_REF] Nagai | Biofeedback and epilepsy[END_REF][START_REF] Rockstroh | Cortical self-regulation in patients with epilepsies[END_REF]. Among these approaches, biofeedback using skin conductance presents two advantages. Firstly, skin conductance is an easily recorded physiological activity compared with brain electrical activity. Secondly, skin conductance changes are related to peripheral sympathetic nervous activity changes that can be considered as a relevant physiological target for the nonpharmacological management of epilepsy. An increase in skin conductance (i.e., an increase in the electrical conductance measured in microsiemens) is related to an increase of arousal level [START_REF] Grapperon | The measurement of electrodermal activity[END_REF]. Moreover, it has been shown that an increase in arousal level might be associated with a decrease in cortical excitability, thus with a decrease in the probability of seizures [START_REF] Nagai | Changes in cortical potential associated with modulation of peripheral sympathetic activity in patients with epilepsy[END_REF].Biofeedback methods that teach the patient how to increase skin conductance have shown a significant reduction in seizure frequency [START_REF] Nagai | Clinical efficacy of galvanic skin response biofeedback training in reducing seizures in adult epilepsy: a preliminary randomized controlled study[END_REF], which was maintained up to 3 years after the termination of biofeedback treatment [START_REF] Nagai | Long-term effects of electrodermal biofeedback training on seizure control in patients with drug-resistant epilepsy: two case reports[END_REF].

However, limitations of biofeedback efficacy studies have been that the type of epilepsy has often not been taken into account and that analysis of efficacy has not been based on a homogenous sample of patients with similar seizure types [START_REF] Micoulaud-Franchi | Biofeedback and drug-resistant epilepsy: back to an earlier treatment?[END_REF][START_REF] Nagai | Clinical efficacy of galvanic skin response biofeedback training in reducing seizures in adult epilepsy: a preliminary randomized controlled study[END_REF]. Temporal lobe epilepsy (TLE) is the most common drug-resistant epilepsy. Moreover, TLE is characterized by abnormal electrical changes starting from one region or several regions of the temporal lobe including the amygdala [START_REF] Bartolomei | Neural networks involving the medial temporal structures in temporal lobe epilepsy[END_REF],w h i c hi s known to be involved in the regulation of arousal and anxiety level and in the response of emotional negative stimuli [START_REF] Morris | Parallel neural responses in amygdala subregions and sensory cortex during implicit fear conditioning[END_REF]. Thus, it would be interesting to investigate the efficacy of biofeedback in TLE. More specifically, patients with TLE who could identify stress factors that trigger their seizures have been described [START_REF] Lanteaume | How do cognition, emotion, and epileptogenesis meet? A study of emotional cognitive bias in temporal lobe epilepsy[END_REF]. Stress is a complex phenomenon but can be defined in terms of its biological, environmental, and psychological aspects, whereby external demands exceed adaptive capacity [START_REF] Novakova | The role of stress as a trigger for epileptic seizures: a narrative review of evidence from human and animal studies[END_REF].Thirtytofifty percent of patients with TLE are reported to perceive stress as a factor whose presence is associated with an increased probability of seizures [START_REF] Spector | Seizure precipitants and perceived self-control of seizures in adults with poorly-controlled epilepsy[END_REF][START_REF] Spatt | Subjective perception of seizure precipitants: results of a questionnaire study[END_REF][START_REF] Sperling | Self-perception of seizure precipitants and their relation to anxiety level, depression, and health locus of control in epilepsy[END_REF]. These patients disclosed specific attentional biases towards negative emotional stimuli compared with patients without a stress factor trigger for seizures. Moreover, the degree of attentional biases correlated with abnormal metabolism in the amygdala [START_REF] Lanteaume | Cognitive and metabolic correlates of emotional vulnerability in patients with temporal lobe epilepsy[END_REF].

To the best of our knowledge, no previous studies have investigated nonpharmacological treatment in TLE with stress as a trigger for seizures. Thus, the aim of the present study was to be a proof of concept to demonstrate feasibility of skin conductance biofeedback in TLE with stress-triggered seizures. We aimed to replicate the methodology used in the study of Nagai et al. and to verify that it is well tolerated in this specific type of epilepsy. For that purpose we (i) included patients with TLE with seizures triggered by stress [START_REF] Lanteaume | How do cognition, emotion, and epileptogenesis meet? A study of emotional cognitive bias in temporal lobe epilepsy[END_REF], (ii) treated these patients with skin conductance biofeedback using a method similar to that used previously [START_REF] Nagai | Clinical efficacy of galvanic skin response biofeedback training in reducing seizures in adult epilepsy: a preliminary randomized controlled study[END_REF], and (iii) evaluated the effect of biofeedback on seizure frequency, on psychometric evaluation of stress and affectivity, and on cognitive evaluation of attentional biases towards negative emotional stimuli. As skin conductance biofeedback increases levels of peripheral sympathetic arousal, which can be associated with stress response, anxiety, and exposure to negative emotional stimuli [START_REF] Lang | Looking at pictures: affective, facial, visceral, and behavioral reactions[END_REF][START_REF] Öhman | Orienting and attention: preferred preattentive processing of potentially phobic stimuli[END_REF][START_REF] Dawson | Concurrent measurement of awareness and electrodermal classical conditioning[END_REF][START_REF] Gross | Antecedent-and response-focused emotion regulation: divergent consequences for experience, expression, and physiology[END_REF],in this proof-of-concept study, we sought to verify that patients with TLE and stress-triggered seizures are not worsened in terms of stress, anxiety, and negative emotional response to this nonpharmacological treatment. We aimed to check the following: i) whether patients with TLE with stress-triggered seizures, treated with biofeedback, did not worsen on psychometric evaluation of stress and affectivity and on cognitive evaluation of attentional biases towards negative emotional stimuli and ii) whether patients showed reduction in seizure frequency. In cases of a positive result concerning seizure frequency, we expected a correlation between changes in seizure frequency and changes in skin conductance over the period of biofeedback treatment.

Methods and materials

Participants

The study duration was 17 months (January 2013-May 2014). Patients were selected if they presented with both drug-resistant TLE and stress-triggered seizures. Five patients were selected from an existing database established in our center, having already been identified and previously included in the studies by Lanteaume et al. [START_REF] Lang | Looking at pictures: affective, facial, visceral, and behavioral reactions[END_REF].An additional 6 patients were prospectively recruited over the study period. Thus, 11 patients with TLE with stress-triggered seizures were recruited in the Epilepsy Unit of the Clinical Neurophysiology Department of the Marseille University Hospital (France). According to the previous study of Nagai et al., a sample size of more than 10 would have sufficient power to find an effect. Inclusion criteria were as follows: TLE with stress-triggered seizures, drug-resistant epilepsy, age between 18 and 60 years, duration of epilepsy more than 3 years, frequency of seizures more than 1 per month, stable medication one month before the study and during the study duration, and sufficient cognitive ability to keep a seizure diary. The diagnosis of TLE was documented clinically and confirmed with video-EEG investigations. All patients underwent brain magnetic resonance imaging (MRI) and fluorodeoxyglucosepositron emission tomography (FDG-PET).

The presence of a stress trigger for seizures was assessed with the impact of seizure precipitant scale described previously [START_REF] Lanteaume | How do cognition, emotion, and epileptogenesis meet? A study of emotional cognitive bias in temporal lobe epilepsy[END_REF]. A face-toface interview was carried out in which patients were questioned about several clinical triggers. The importance of stress triggers was evaluated on a balanced 10-point Likert scale ranging from 0 ("never") to 10 ("always"). Patients with TLE had to have a score of more than 5 to be included in the present study.

Exclusion criteria were as follows: reduced capacity to consent, poor antiepileptic drug compliance, mental disorders, severe cognitive impairment, and comorbid chronic medical conditions (other than epilepsy), e.g., severe diabetes, cerebrovascular disease, or chronic obstructive bronchopneumonia. Patients were screened for any current or lifetime history of a DSM-IV axis I disorder based on the Mini-International Neuropsychiatric Interview (MINI) [START_REF] Sheehan | The Mini-International Neuropsychiatric Interview (M.I.N.I.): the development and validation of a structured diagnostic psychiatric interview for DSM-IV and ICD-10[END_REF].

Gender, age, laterality, age at onset of epilepsy, number and type of AEDs, and number of seizures were collected as data (Table 1).

After receiving a detailed description of the study, participants gave their informed written consent. This study was conducted in accordance with the Declaration of Helsinki and French Good Clinical Practices.

2.2. Procedure 2.2.1. Clinical evaluation 2.2.1.1. Seizure frequency evaluation. Patients were asked to keep a seizure diary for 3 months before (baseline seizure frequencies) and 3 months after the biofeedback treatment. Average seizure frequency (per month) was calculated for each of these periods.

Psychometric evaluation of stress and affectivity.

Patients completed the State-Trait Anxiety Inventory (SAI) in order to evaluate the state of anxiety [START_REF] Spielberger | Psychometric properties of the STAI: a reply to Ramanaiah, Franzen, and Schill[END_REF], the Positive and Negative Affect Schedule (PANAS) in order to evaluate the negative affectivity (NA) and the positive affectivity (PA) [START_REF] Watson | Development and validation of brief measures of positive and negative affect: the PANAS scales[END_REF], and the Beck Depression Inventory (21 items, BDI-21) in order to evaluate depressive mood [START_REF] Beck | An inventory for measuring depression[END_REF]. These evaluations were completed the week before and the week after the biofeedback treatment.

Cognitive evaluation of attentional biases.

The cognitive paradigm we used to study attentional biases towards negative emotional stimuli was the probe detection task. This task measures the modulation of spatial attention by negative and neutral stimuli and allows the assessment of attentional vigilance towards negative stimuli and attentional disengagement from negative stimuli. As previously described [START_REF] Lanteaume | How do cognition, emotion, and epileptogenesis meet? A study of emotional cognitive bias in temporal lobe epilepsy[END_REF][START_REF] Lanteaume | Cognitive and metabolic correlates of emotional vulnerability in patients with temporal lobe epilepsy[END_REF], each experimental trial consisted of three successive steps: i) display of a black fixation cross (500 ms), ii) display of a word pair (500 ms): "negative-neutral" or "neutral-neutral", and iii) a dot display (until the response was given). Participants were asked to give the location of the dot by pressing the corresponding button as quickly and as accurately as possible. In each "negative-neutral" pair, the combination of the location of the words and the location of the dot led to congruent conditions (i.e., dot replaces negative word) and to incongruent conditions (i.e., dot replaces neutral word). For each "neutral-neutral" pair, all conditions were considered as control conditions. Manipulation of congruent, incongruent, and control pairs allows measurements of 3 indices related to attentional biases. The most important is a score of congruency (a positive score indicates a modulated attention by the negative stimuli). A modulated attention by the negative stimuli could be related to two kinds of attentional biases: i) a facilitated effect of negative stimuli presented congruently to the dot (measured by a positive score of vigilance) and ii) a difficulty in disengaging attention from the negative stimuli presented incongruently to the dot (measured by a positive score of disengagement). These scores are calculated by the differences of reaction time between the congruent, the incongruent, and the control conditions as described previously [START_REF] Lanteaume | How do cognition, emotion, and epileptogenesis meet? A study of emotional cognitive bias in temporal lobe epilepsy[END_REF][START_REF] Lanteaume | Cognitive and metabolic correlates of emotional vulnerability in patients with temporal lobe epilepsy[END_REF].T h e cognitive evaluation was conducted the week before and the week after the biofeedback treatment.

Skin conductance biofeedback

2.2.2.1. Material. Measurement of skin conductance and implementation of biofeedback sessions were undertaken using a biofeedback system (Biograph, Thought Technology Ltd., Montreal, Quebec, Canada) that had been modified for experimental use in the present study.

Dry nickel-plated electrodes were placed on the palmar surface of the participant's index and middle finger of the left hand. A computergenerated graphic was presented visually on a computer monitor. Two simultaneous feedbacks were presented to the patient. The first feedback was presented to the patients in the form of a graphical thermometer, inspired by previous MRI feedback studies [START_REF] Micoulaud-Franchi | Towards a new approach of neurophysiology in clinical psychiatry: functional magnetic resonance imaging neurofeedback applied to emotional dysfunctions[END_REF], which displayed a green bar that increased when skin conductance activity increased and a red bar that decreased when skin conductance activity decreased. For the feedback, the bar was continually updated with new skin conductance information. The second feedback was presented to the patients in the form of a female facial continuum from sad to happy. The facial images were created from a set of black and white photographs [START_REF] Pollak | Early experience is associated with the development of categorical representations for facial expressions of emotion[END_REF]. The two prototype endpoint photographs (100% sad and 100% happy) were morphed to create a linear continuum of nine facial images. Each intermediate image was transformed by a 10% increment according to a morphing technique described previously [START_REF] Pollak | Early experience is associated with the development of categorical representations for facial expressions of emotion[END_REF].The feedback facial display moved towards the happy face when skin conductance activity increased and towards the sad face when skin conductance activity decreased. For the feedback, the facial display was continually updated at intervals of 1.5 s. We chose this kind of feedback because we posited that emotional feedback would be more adapted to patients with TLE and stress-triggered seizures than a neutral feedback used by Nagai et al. [START_REF] Nagai | Clinical efficacy of galvanic skin response biofeedback training in reducing seizures in adult epilepsy: a preliminary randomized controlled study[END_REF]. The aim of the teaching was that patients learn to increase their arousal level while associating this with positive emotion.

Biofeedback session.

Patients attended a total of 12 sessions (1 session/week) in a quiet room kept as constant as possible at 23 °C to avoid variation of skin conductance in relation to ambient temperature [START_REF] Grapperon | The measurement of electrodermal activity[END_REF][START_REF] Boucsein | Electrodermal activity[END_REF]. Each session lasted 1 h. During the first session, information about biofeedback was given. Patients were instructed that the use of arousal and alertness cognitive activity combined with the feedback could enable them to control the thermometer bar and the face. However, no specific strategies were provided. Before beginning the following session, a short face-to-face interview was carried out in which patients were questioned about evolution since the last session in terms of seizure frequency, intensity of stress triggers, perceived self-control, motivation, and learning of self-strategies to obtain and control the skin conductance.

A baseline block was conducted during 2 min before the biofeedback. Patients were instructed to relax in order to bring skin conductance back to the baseline level. It, thus, enabled calibration of the baseline and the range of the thermometer. During baseline blocks, a landscape screen was presented to the patients without any feedback. Each training session lasted 30 min, during which the patients received biofeedback and were instructed to enhance skin conductance activity. At the end of each biofeedback session, patients were actively asked to report any discomfort during the feedback, in particular concerning feelings of stress. The training also explicitly emphasized the goal of transfer of the skills learned during the session into everyday life. The patient was encouraged to train at home as if he or she were sitting in front of the monitor at home, and a downscaled picture of the feedback computer screen was given as a memory aid handout to the patients. Moreover, patients were also encouraged to practice self-regulation skills in seizure-prone situations.

Statistical analyses

Descriptive statistics of the obtained data included frequencies and percentages of categorical variables together with means and standard b Percentage of seizure frequency changes 3 months before and 3 months after the biofeedback treatment, as also shown in Fig. 1.

deviations of continuous variables. Data analyses were performed using SPSS software (Version 18 for Mac, PASW Statistics). For all the tests, the accepted significance level was 5%.

Comparison statistics

Clinical evaluations (seizure frequency, SAI, PA, NA, BDI-21, score of congruency, vigilance, and disengagement) were compared before and after the 12 sessions of the biofeedback treatment using the Wilcoxon signed-rank test.

Relation between skin conductance changes and seizure frequency changes

For each patient, change in seizure frequencies was calculated as the percentage of variation compared with baseline seizure frequencies.

Change in skin conductance during a session was calculated by subtracting the mean skin conductance level for the first 2 min and for the last 2 min and by calculating the percentage of variation compared with the first 2 min. Percentage change in skin conductance was averaged for each patient over the 12 biofeedback sessions.

The progress in the training over the course of the 12 biofeedback sessions was calculated by subtracting the average percentage change in the first 3 sessions and the last 3 sessions and by calculating the percentage of variation compared with the first 3 sessions according to the methodology used in the study of Nagai et al. [START_REF] Nagai | Clinical efficacy of galvanic skin response biofeedback training in reducing seizures in adult epilepsy: a preliminary randomized controlled study[END_REF].Thepercentage change in the first 3 sessions and that in the last 3 sessions were compared using the Wilcoxon signed-rank test.

Spearman's rank correlation was used to analyze the relationship between change in seizure frequency and change in skin conductance over the biofeedback treatment and progress in the training during the 12 biofeedback sessions.

Relation between psychometric changes and seizure frequency changes

For each patient, changes in psychometric evaluation (SAI, PA, NA, and BDI-21) were calculated as the percentage of variation compared with baseline. Spearman's rank correlation was used to analyze the relationship between change in psychometric evaluation and change in seizure frequency.

Results

Sample characteristics

The mean age was 36.72 years (SD = 7.28, range: years old); 9 women and 2 men. The mean age at onset of epilepsy was 17.54 (SD = 7.69). One patient was on no medication by choice. All of the other patients were treated by AEDs. The mean impact of seizure precipitant scale was 6.91 (SD = 1.45), indicating a high prevalence of stress-triggered seizures [START_REF] Lanteaume | How do cognition, emotion, and epileptogenesis meet? A study of emotional cognitive bias in temporal lobe epilepsy[END_REF]. None of the patients included was taking antidepressant medication.

Seizure frequency, psychometrics, and cognitive changes

Clinical evaluations are detailed in Table 2. There was a significant difference before and after the biofeedback treatment concerning the seizure frequency with a mean reduction of -48.61% (SD = 27.79), with 5 (45%) patients having more than 50% seizure reduction. Table 1 and Fig. 1 show individual patient's percentage seizure frequency change after the biofeedback treatment.

Concerning the psychometric and cognitive changes, there were significant differences before and after the biofeedback treatment for the NA, with a mean reduction of -20.21% (SD = 19.23), and the BDI, with a mean reduction of -37.65% (SD = 36.99). There was a trend for the SAI, with a mean reduction of -9.71% (SD = 19.54). Note that all the Z scores of the Wilcoxon signed-rank test were negative.

None of the patients reported any stress discomfort, nor did any seizure occur during the biofeedback session. None of the patients dropped out of the study, suggesting that the treatment was well tolerated and good observance of the sessions.

Relation between skin conductance changes and seizure frequency changes

The mean change in skin conductance activity over the biofeedback treatment was 30.15% (SD = 14.92). A significant correlation was found between seizure frequency change and skin conductance activity change over the biofeedback treatment (r(11) = .62, P = .042) (Fig. 2).

The progress in the training through the course of the 12 biofeedback sessions showed a trend towards significance: the mean change in the first 3 sessions was 45.72% (SD = 30.85), and the mean change in the last 3 sessions was 55.44% (SD = 33.35, P = .091). The progress in training was 13.61% (SD = 61.44). No significant correlation was found between progress in training and seizure frequency change (r(11) = -.24, P = .47).

Relation between psychometric changes and seizure frequency changes

There was no significant correlation between seizure frequency change and SAI change (r(11) = -.19, P = .57), PA change (r(11) = -.11, P = .76), NA change (r(11) = -.024, P = .95), or BDI change (r(11) = .17, P = .62).

Discussion

While there is interest in the use of nonpharmacological treatments in epilepsy [START_REF] Sheth | Nonpharmacological treatment options for epilepsy[END_REF], there is currently a lack of robust data to support the use of psychological therapies [START_REF] Boylan | Peri-ictal behavioral and cognitive changes[END_REF][START_REF] Engelberts | The effectiveness of psychological interventions for patients with relatively well-controlled epilepsy[END_REF][START_REF] Ramaratnam | Psychological treatments for epilepsy[END_REF]. To the best of our knowledge, this is the first study to investigate psychological management in drugresistant TLE with stress-triggered seizures. Patients treated with biofeedback did not worsen on subjective psychometric evaluation of stress and affectivity or on objective cognitive evaluation of attentional biases towards negative emotional stimuli. In fact, significant improvement was found for negative affect and depressed mood symptoms, with a nonsignificant trend to improvement for the anxiety state. Moreover, the face-to-face interview carried out in each biofeedback session revealed no stress discomfort and a high level of motivation of each patient. No patient dropout occurred as in the biofeedback group of Nagai et al. [START_REF] Nagai | Clinical efficacy of galvanic skin response biofeedback training in reducing seizures in adult epilepsy: a preliminary randomized controlled study[END_REF]. Thus, despite the fact that increases in skin conductance could be related to increases in stress level, this effect was not shown in our study. These results indicate that the skin conductance biofeedback used in the present study, which teaches an increased level of peripheral sympathetic arousal, was a well-tolerated nonpharmacological treatment. Two explanations can be emphasized in line with the biofeedback protocol specifically designed in the present study. The first is the use of an emotional facial continuum as feedback that allowed patients to associate an increase of arousal with positive emotion. The second is the use of a graphical thermometer that allowed a continuous feedback. Indeed, there are differences between the presentation of discrete and continuous stimulionskinconductance [START_REF] Dawson | The electrodermal system[END_REF]. Discrete stimuli induce phasic increase in skin conductance. The most useful measure is the skin conductance response (SCR) following stimulus onset [START_REF] Dawson | The electrodermal system[END_REF]. The increase of this phasic arousal is known to be related to stress stimuli such as shock [START_REF] Dawson | Concurrent measurement of awareness and electrodermal classical conditioning[END_REF],fear stimuli [START_REF] Öhman | Orienting and attention: preferred preattentive processing of potentially phobic stimuli[END_REF], or negative emotional stimuli [START_REF] Lang | Looking at pictures: affective, facial, visceral, and behavioral reactions[END_REF][START_REF] Gross | Antecedent-and response-focused emotion regulation: divergent consequences for experience, expression, and physiology[END_REF]. Continuous stimuli modulate increases or decreases in skin conductance. The most useful measure is the skin conductance level (SCL), which was the measure performed in our biofeedback protocol [START_REF] Dawson | The electrodermal system[END_REF]. Skin conductance level can be considered to reflect the tonic arousal level. The increase of tonic arousal level is known to be related to tasks requiring an effortful allocation of attentional resources [START_REF] Jennings | Bodily changes during attending[END_REF]. The skin conductance biofeedback protocol in the present study has been developed to reinforce essentially SCL, as in the previous study of Nagai et al. [START_REF] Nagai | Clinical efficacy of galvanic skin response biofeedback training in reducing seizures in adult epilepsy: a preliminary randomized controlled study[END_REF]. Thus, the strategies learned during the treatment to obtain a control of the physiological response are thought to be more related to attentional cognitive strategies than to emotional strategies. This explanation is supported by the following: i) patients' reporting in the present study that they tried to focus their attention on something in their environment or in their mind, ii) a study showing that cognitive strategies were more likely used in response to seizure-prone situations than behavioral and emotional strategies [START_REF] Hether | Spontaneous strategies for psychological control of seizures[END_REF], and iii) the relation between SCL and slow cortical potentials (SCP) [START_REF] Nagai | Changes in cortical potential associated with modulation of peripheral sympathetic activity in patients with epilepsy[END_REF]. Indeed, an increase in SCL is associated with a reduction of SCP that caused decreased cortical excitability [START_REF] Nagai | Changes in cortical potential associated with modulation of peripheral sympathetic activity in patients with epilepsy[END_REF]. Thus, SCP neurofeedback has been used in drug-resistant epilepsy, according to the same argument as skin conductance biofeedback, and has shown efficacy [START_REF] Kotchoubey | Modification of slow cortical potentials in patients with refractory epilepsy: a controlled outcome study[END_REF].T h e modulation of SCP has been associated with attentional cognitive modulation [START_REF] Nagai | Changes in cortical potential associated with modulation of peripheral sympathetic activity in patients with epilepsy[END_REF][START_REF] Mayerk | Onesizefits all? Slow cortical potentials neurofeedback: a review[END_REF][START_REF] Elbert | Biofeedback of slow cortical potentials[END_REF]. Moreover, Nagai et al. [START_REF] Nagai | Changes in cortical potential associated with modulation of peripheral sympathetic activity in patients with epilepsy[END_REF] found that skin conductance biofeedback and SCP neurofeedback were probably more mediated by the attentional regulation of the sensory input to the cortex via the thalamus than the regulation of brain structures involved in emotions and stress (such as amygdala and limbic temporal regions) [START_REF] Nagaiy | Brainactivity relating to the contingent negative variation: an fMRI investigation[END_REF]. These anatomofunctional explanations remain speculative and need further investigation. However, it could explain the efficacy, without side effects, of skin conductance biofeedback that increased the arousal level in patients with TLE shown in the present study despite stress vulnerability.

Concerning the efficacy of the treatment, given the lack of wellcontrolled conditions and a randomized group, the present result can only be considered as preliminary. However, we replicated the results of Nagai et al. [START_REF] Nagai | Clinical efficacy of galvanic skin response biofeedback training in reducing seizures in adult epilepsy: a preliminary randomized controlled study[END_REF]. Firstly, we found a very close mean percentage change in seizure frequency in our sample (-48.61%) to that observed in Nagai et al's biofeedback sample (-49.26%) and a similar rate of response (45% versus 60%). Secondly, we found a correlation between changes in seizure frequency and changes in skin conductance over the biofeedback treatment that suggests a causal relationship between the efficacy of skin conductance biofeedback activity and a reduction in seizure frequency.

Some limitations in the current study have to be considered. Firstly, using the methodology of Nagai et al. [START_REF] Nagai | Clinical efficacy of galvanic skin response biofeedback training in reducing seizures in adult epilepsy: a preliminary randomized controlled study[END_REF]., we found only a trend in the improvement in the degree to which the patients changed their skin conductance level in the first 3 sessions compared with the last 3 sessions. Thus, the progress in training of biofeedback over the 12 sessions was smaller in our study (13.61%) than in the study of Nagai et al. (51.98%). Moreover, in contrast to Nagai et al., we did not find a significant correlation between this progress and reduction in seizure frequency. However, we found that those patients who had the greatest increase in skin conductance during the first 3 sessions maintained their performance across the session, and that they benefited the most from the treatment. One explanation could be that the main difference in our study was to perform the biofeedback session only once per week, contrary to 3 per week in Nagai et al. [START_REF] Nagai | Clinical efficacy of galvanic skin response biofeedback training in reducing seizures in adult epilepsy: a preliminary randomized controlled study[END_REF], which could modify the training to increase the skin conductance level. Secondly, biofeedback could be efficacious apart from specificeffectsbecause of learned self-regulation of a physiological activity. The information given to the patients about the neurophysiological background of epilepsy, the proposition to treat them with a psychophysiological and a electrophysiological method, as well as the therapeutic relationship established once a week during 12 weeks all likely contributed to the positive effects of the training, since positive expectations and the experience of self-efficacy are important nonspecific variables in biofeedback [START_REF] Gevensleben | Neurofeedback in children with ADHD: validation and challenges[END_REF] and the clinical course of epilepsy [START_REF] Velissaris | The psychological impact of a newly diagnosed seizure: losing and restoring perceived control[END_REF][START_REF] Jacoby | Exploring loss and replacement of loss for understanding the impacts of epilepsy onset: a qualitative investigation[END_REF]. Moreover, the improvements in anxiety, negative affect, and depressed mood symptoms in our study could be an intermediate variable to explain the effect of biofeedback in seizure frequency. Indeed, it was found that anxiety [START_REF] Beyenburg | Anxiety in patients with epilepsy: systematic review and suggestions for clinical management[END_REF] and depression [START_REF] Crail-Melendez | Cognitivebehavioral therapy for depression in patients with temporal lobe epilepsy: a pilot study[END_REF] are important components to manage with psychological interventions in order to improve seizure frequency. However, the lack of any relationship in our study between seizure reduction and reduced anxiety and depression scores is not in favor of this hypothesis. Thirdly, 81% of the patients were women. This marked female predominance is in contrast to the study by Nagai et al. that included around 50% women. This predominance of female subjects could potentially influence the results because of the known effect of the menstrual cycle on resting skin conductance. This parameter could be controlled in further studies. Lastly, the use of paper-based seizure diaries to assess seizure frequency could be inaccurate [START_REF] Elger | Seizure prediction and documentationtwo important problems[END_REF][START_REF] Cook | Prediction of seizure likelihood with a long-term, implanted seizure advisory system in patients with drug-resistant epilepsy: a first-in-man study[END_REF].Theactof self-reporting seizures may itself influence the observation by making the subject attend more vigilantly to seizures after biofeedback treatment. However, seizure diaries currently remain a widely accepted means of estimating seizure frequency in the context of trials to validate antiseizure drugs or devices. Since some studies have documented patient preference and increased compliance for electronic versus paper diaries [START_REF] Fisher | Seizure diaries for clinical research and practice: limitations and future prospects[END_REF], future biofeedback studies could use electronic diaries. Moreover, the advantages of electronic diaries include the possibility of linkage to a biosensor [START_REF] Fisher | Seizure diaries for clinical research and practice: limitations and future prospects[END_REF], which would enable recording of the physiological parameters targeted during biofeedback sessions in ecological, "real life" conditions [START_REF] Poh | A wearable sensor for unobtrusive, long-term assessment of electrodermal activity[END_REF].

In conclusion, both subjective and objective evaluations of stress and affectivity showed that skin conductance biofeedback is a well-tolerated nonpharmacological treatment in patients with drug-resistant TLE and seizures triggered by stress. Moreover, this treatment could be effective and, thus, could potentially be used in the nonpharmacological management of this type of epilepsy. Thus, although methodological limitations necessitate cautious interpretation of results, the present study encourages further well-controlled studies in this field of nonpharmacological treatment for epilepsy.

  b A positive score indicates a modulated attention by the negative stimuli. c A positive score indicates a facilitated effect of negative stimuli presented congruently to the dot. d A positive score indicates a difficulty in disengaging attention from the negative stimuli presented incongruently to the dot.

Fig. 1 .

 1 Fig. 1. Percentage of seizure frequency changes 3 months before and 3 months after the biofeedback treatment. In x axis, result for each patient.

Fig. 2 .

 2 Fig. 2. Relation between seizure frequency changes 3 months before and 3 months after the biofeedback treatment and skin conductance change over the 12 sessions.

Table 1

 1 Patient demographic and clinical characteristics. CBZ: carbamazepine, LCM: lacosamide, LEV: levetiracetam, LMT: lamotrigine, OXC: oxcarbazepine, PGB: pregabalin, TPM: topiramate, VPA: valproic acid.

		Gender	Age Laterality	Age at onset	Number and type of	Total number of seizures during	Total number of seizures during	Percentage of seizure
				of the TLE	of epilepsy	antiepileptic drugs a	3-month period before biofeedback	3-month period after biofeedback	frequency changes b
	P1	Women 47	Left	6	CBZ, PGB	3	2	-33.33
	P2	Men	24	Left	19	CBZ, TPM	21	9	-57.14
	P3	Women 45	Left	27	LMT, LEV	30	6	-80
	P4	Men	35	Left	19	CBZ, VPA	30	16	-46.67
	P5	Women 33	Right	7	OXC, TPM, LCM	89	3	-96.63
	P6	Women 39	Left	15	LMT, LEV	8	6	-25.00
	P7	Women 27	Right	17	CBZ, LCM	15	8	-46.67
	P8	Women 33	Right	22	LMT	16	5	-68.75
	P9	Women 44	Left	14	-	48	20	-58.33
	P10 Women 37	Left	32	LMT, LEV	3	3	0
	P11 Women 40	Bilateral	15	CBZ	9	7	-22.22

a

Table 2

 2 Change in seizure frequency per month, psychometric evaluation of stress and affectivity, and cognitive evaluation of attentional biases the week before and the week after the biofeedback treatment (12 sessions).

		Pretreatment		Posttreatment		Z	P value a
		Mean	SD	Mean	SD
	Seizure frequency (per month)	6.17	6.31	1.93	1.39	-2.8	.005
	Psychometric evaluation of stress and affectivity
	State anxiety inventory	48.91	12.17	43.09	11.97	-1.88	.059
	Positive affect schedule	28.30	5.77	33.09	8.90	-1.28	.201
	Negative Affect Schedule	26.40	11.69	21.73	7.55	-2.45	.014
	Beck Depression Inventory	17.82	11.92	9.82	6.89	-2.60	.009
	Cognitive evaluation of attentional biases
	Score of congruency b	2.39	13.86	.69	22.46	-.44	.657
	Score of vigilance c	1.82	14.42	-5.40	19.59	-.71	.477
	Score of disengagement d	.58	19.93	6.09	21.69	-.18	.859

a Wilcoxon signed-rank test.

Fundings

This study was supported by the Fondation Française pour la Recherche contre l'Epilpesie (FFRE) ("Appels à projets 2012").

Conflict of interest statement

The authors report no conflicts of interest.


Nesrine Sadaoui
email: sadaouinesrine@gmail.com

Nicole Bec

Véronique Barragan-Montero

Nabil Kadri

Frédéric Cuisinier

Christian Larroque

Karim Arab

Bachra Khettal

Frederic Cuisinier

The essential oil of Algerian Ammodaucus leucotrichus Coss. & Dur. and its effect on the cholinesterase and monoamine oxidase activities

de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction

The Alzheimer's disease (AD) is a pathology that affects > 46.8 million people, the most common neurodegenerative disease for an estimated worldwide cost of US$818 billion ( [51].).

On the other hand, the Parkinson disease (PD) is the second age related neurodegenerative disease with an estimation of seven to 10 million people living with it. In spite of their description in the early 20th century and recent advancements in the understanding of possible mechanisms relating to their etiology, these neurological disorders remain orphan of effective treatments.

Monoamine oxidases (MAOs) and cholinesterase (ChE) are enzymatic systems that are known to play a pivotal role in the cause of these neurologic disorders. MAO-A and B are ubiquitineous mitochondrial enzymes present in the outer mitochondrial membrane in various tissues such as nerve terminals, gut and liver. They catalyze the oxidative deamination of neurotransmitters, physiologic or food ingested amines. MAO dependent oxidative stress is recognized as a pejorative factor in Abbreviations: AD, Alzheimer's disease; AChE, Acetylcholinesterase; ATCh, Acetylthiocholine; BuchE, Butyrylcholinesterase; ChE, Cholinesterase; DMSO, Diméthylsulfoxyde; DTNB, Dithiobisnitrobenzoate; EO, Essential oil; MAOs, Monoamine oxidase; PD, Parkinson disease; US-FDA, US Food and Drug Administration T 1 the occurrence of neurological disorders such as depression, Parkinson (AD) or Alzheimer (AD) diseases ( [START_REF] Finberg | Inhibitors of MAO-A and MAO-B in Psychiatry and Neurology[END_REF]., [START_REF] Deftereos | From depression to neurodegeneration and heart failure: re-examining the potential of MAO inhibitors[END_REF]). There are two isoforms of MAOs differing by their substrate inhibitor specificity. The MAO-A which metabolizes aliphatic amines (serotonin, noradrenalin), and is selectively inhibited by clorgyline, while the MAO-B oxidizes preferably benzyl or phenyl amines and is selectively inhibited by selegiline. However, despite their potentiality, the clinical use of these inhibitors has been limited by the side effects associated with their prescription [START_REF] Finberg | Inhibitors of MAO-A and MAO-B in Psychiatry and Neurology[END_REF].

One of the factors known in the etiology of AD is the availability of acetylcholine into the neuronal synaptic compartment that is essential for maintaining the cognitive functions and the most consistent biochemical evidenced change in AD [START_REF] Francis | The interplay of neurotransmitters in Alzheimer's disease[END_REF]. In this way, acetylcholinesterase (AChE) inhibitors are widely used for the symptomatic treatment of this pathology and other dementias such as myasthenia gravis. Butyrylcholinestase is the other form of cholinesterase enzyme widely expressed in humans beings, differing by its substrate specificity but both are able to catalyze the degradation of acetylcholine neuromediator [START_REF] Darvesh | Neurobiology of butyrylcholinesterase[END_REF]. It was evidenced that in the AD brain, the AChE activity is maintained or repressed while the BuChE activity tends to increase [START_REF] Mushtaq | Status of acetylcholinesterase and butyrylcholinesterase in Alzheimer's disease and type 2 diabetes mellitus[END_REF]. This last finding raises the interest in the discovery of the molecules that inhibit AChE and BuChE as well as selective BuChE inhibitors.

Since a decade, a new emerging strategy aims at bringing comfort to people suffering from neurological diseases by using of multitarget directed ligands that would be able to target and inhibit both monoamine oxidases and cholinesterase's enzymes ( [START_REF] Gao | Tertiary amine derivatives of chlorochalcone as acetylcholinesterase (AChE) and buthylcholinesterase (BuChE) inhibitors: the influence of chlorine, alkyl amine side chain and α, β-unsaturated ketone group[END_REF][START_REF] Knez | Dual inhibitors of cholinesterases and monoamine oxidases for Alzheimer's disease[END_REF][START_REF] Ramsay | Assessment of enzyme inhibition: a review with examples from the development of monoamine oxidase and cholinesterase inhibitory drugs[END_REF][START_REF] Tripathi | Design, synthesis, and pharmacological evaluation of 2-amino-5-nitrothiazole derived semicarbazones as dual inhibitors of monoamine oxidase and cholinesterase: effect of the size of aryl binding site[END_REF][START_REF] Wang | Dual functional cholinesterase and MAO inhibitors for the treatment of Alzheimer's disease: synthesis, pharmacological analysis and molecular modeling of homoisoflavonoid derivatives[END_REF]). Plants were used for centuries for the treatment of human and animal illnesses and are the source of most common drugs. Numerous articles report about the MAOs [START_REF] Armijos | Phytochemical and ethnomedicinal study of Huperzia species used in the traditional medicine of Saraguros in Southern Ecuador; AChE and MAO inhibitory activity[END_REF][START_REF] Sengupta | Antiparkinsonian effects of aqueous methanolic extract of Hyoscyamus niger seeds result from its monoamine oxidase inhibitory and hydroxyl radical scavenging potency[END_REF][START_REF] Singh | In vitro evaluation of Bacopa monniera extract and individual constituents on human recombinant monoamine oxidase enzymes[END_REF] or ChEs [START_REF] Calva | Acorenone B: AChE and BChE Inhibitor as a major compound of the essential oil distilled from the ecuadorian species niphogeton dissecta (Benth.)[END_REF][START_REF] Jiang | Traditional Chinese medicinal herbs as potential AChE inhibitors for anti-Alzheimer's disease: A review[END_REF][START_REF] Lin | Anti-acetylcholinesterase activities of traditional Chinese medicine for treating Alzheimer's disease[END_REF] inhibitory capability of crude or purified plant extracts. Moreover, some herbal complementary preparations mainly described in the Ayurvedic or traditional Chinese medicines have been used for centuries as a treatment for neurological disorders and have demonstrated their potential both on models [START_REF] Hu | Neuroprotective effects of Kukoamine A on neurotoxin-induced Parkinson's model through apoptosis inhibition and autophagy enhancement[END_REF][START_REF] Jansen | Effects of five Ayurvedic herbs on locomotor behaviour in a Drosophila melanogaster Parkinson's disease model[END_REF][START_REF] Xu | Epigallocatechin gallate has a neurorescue effect in a mouse model of parkinson disease[END_REF] and clinical studies [START_REF] Cilia | em > Mucuna pruriens < /em > in Parkinson disease[END_REF][START_REF] Katzenschlager | Mucuna pruriens in Parkinson's disease: a double blind clinical and pharmacological study[END_REF][START_REF] Lees | Advice for those trying to find a cure for the shaking palsy[END_REF][START_REF] May | Comparisons between traditional medicines and pharmacotherapies for Alzheimer disease: A systematic review and meta-analysis of cognitive outcomes[END_REF][START_REF] Nagashayana | Association of L-DOPA with recovery following Ayurveda medication in Parkinson's disease[END_REF][START_REF] Gandhi | Management of Parkinson's disease in ayurveda: medicinal plants and adjuvant measures[END_REF][START_REF] Shi | Adding Chinese herbal medicine to conventional therapy brings cognitive benefits to patients with Alzheimer's disease: a retrospective analysis[END_REF][START_REF] Wei | Chinese medicine for idiopathic Parkinson's disease: A meta-analysis of randomized controlled trials[END_REF]. Previous study published a dual inhibitory effect on MAOs and AChE of Huperzia extract [START_REF] Armijos | Phytochemical and ethnomedicinal study of Huperzia species used in the traditional medicine of Saraguros in Southern Ecuador; AChE and MAO inhibitory activity[END_REF], but to our best knowledge, there are few articles reporting on a such dual inhibitory effect of natural preparations.

It has been demonstrated that plants belonging to the Lamiaceae, Polygalaceae, and Apiaceae families are capable of exerting effects on the central nervous system [START_REF] Adams | Plants traditionally used in age related brain disorders-a survey of ethnobotanical literature[END_REF]. Ammodaucus leucotrichus C.& D., medicinal plant, belonging to the Apiaceae family,is an endemic plant and comprises one species in Algeria [START_REF] Quezel | Nouvelle flore de l'Algérie et des régions désertiques méridionales[END_REF]. The local name of this plant is"Akaman" (Tamahaq) and "Oumdraiga" [START_REF] Maiza | Biological activities of plants collected in the Algerian Sahara[END_REF]. Its geographical distribution is in the maritime sands in the Saharan and sub-Saharan countries of North Africa; Morocco, Algeria and Tunisia, extending to Egypt and tropical Africa [START_REF] Tejera | Un nuevo taxón del género Ammodaucus Cosson & Durieu (Apiaceae) en el Archipiélago Canario[END_REF].

A. leucotrichus C.& D. is used by the Algerian Saharan population, with different preparations to treat stomach diseases, fever, vomits, allergies, and is also emmenagogue, abortive and aphrodisiac [START_REF] Maiza | Biological activities of plants collected in the Algerian Sahara[END_REF]. In Morocco, it is used in the treatment of cardiac diseases [START_REF] Jouad | Ethnobotanical survey of medicinal plants used for the treatment of diabetes, cardiac and renal diseases in the North centre region of Morocco (Fez-Boulemane)[END_REF]. Several studies have shown that the A. leucotrichus plant is very rich in essential oil [START_REF] El-Haci | Antimicrobial activity of Ammodaucus leucotrichus fruit oil from Algerian Sahara[END_REF][START_REF] Gherraf | Chemical constituents and antimicrobial activity of essential oils of Ammodaucus leucotricus[END_REF].

In this paper we describe the preparation, chemical characterization, Monoamine oxidases and cholinesterases inhibitory potentials of an essential oil prepared from the whole plant Ammodaucus leucotrichus C.& D. collected in the southern Algeria.

Materials and methods

Chemicals and reagents

All reagents were purchased from Sigma Aldrich.

Plant material and sample preparation

The A. leucotrichus C.& D. aerial parts harvest was performed in April 2014 in Ghardaia, Algeria. The plant was identified by Dr. A.M.Chahma (Faculty of Sciences, University of Ouargla, Algeria). The samples were washed with a tap water then distilled water to remove any adhering soil and dust. A. leucotrichus parts were dried for 5 days at room temperature in a ventilated darkroom to protect the active compounds from light oxidation.

Essential oil extraction

The Clevenger-type apparatus was used for the hydrodistillation according to the method recommended by the European Pharmacopoeia [36]. The aerial parts of A. leucotrichus were subjected to hydrodistillation for four hours and the essential oil was recovered directly without any solvent and stored at 4 °C. The essential oil had a blue color, and the yield was 1.3% (volume/dry-weight).

GC-MS of essential oil

The analysis are carried out on a Focus GC (Thermo, country for all material and method), equipped with a BR5-MS (5% phenyl methyl siloxane) column 30 m × 0.25 mm i.d., film thickness 0.25uM (Bruker).

The spectrometer coupled to the chromatographic chain was a DSQII (Thermo) equipped with an EI source at 70 eV. The mass spectrum was recorded between 40 and 500 Da (m/z equivalent unit). The injections were performed in split mode 1/100.

The temperature of the column was programmed from 70 °C to 300 °C with 10 °C/min steps. The injector temperature was programmed at 200 °C. Therefore, the Helium gas was used as a carrier at a linear velocity 1.2 mL/min. The injected volume was 1 μL.

Components identification

The compounds identification was achieved by comparison of their recorded mass spectra with those of a computer library (NIST2008 v2.0/Xcalibur data system) provided by the instrument software.

Enzymatic activities

For all enzymatic assays, stock samples were dissolved in DMSO at 100 mg/mL. ChE and MAO activities were tested in the presence of 1 to 1000 μg/mL of each sample solution in a constant DMSO concentration. All measurements were made in triplicate and performed at 30 °C. When possible, the IC 50 values were calculated using the GNUPLOT package online (www.ic50.tk, www.gnuplot.info).

Determination of cholinesterase inhibition assay

The cholinesterase (ChE) activities were assayed following a colorimetric protocol adapted from adapted from Ellman et al. [START_REF] Ellman | A new and rapid colorimetric determination of acetylcholinesterase activity[END_REF]. ChEs efficiently catalyze the hydrolysis of acetylthiocholine (ATCh), the sulfur analog of the natural substrate of these enzymes. Upon hydrolysis, this substrate analog produces acetate ion and thiocholine. Thiocholine, in the presence of the highly reactive dithiobisnitrobenzoate (DTNB) ion, generates a yellow color, which can be quantitatively monitored by spectrophotometric absorption at 412 nm. A typical 200 μL inhibition assay volume contained phosphate buffered saline solution (pH 7.4), DTNB (1.5 mM), test sample in DMSO (1% v/v final). Both acetylcholinesterase from Electrophorus electricus (Type V-S, lyophilized powder, 744 U/mg solid, 1272 U/mg protein) and butyrylcholinesterase from equine serum (lyophilized powder, ≥900 units/ mg protein) were dissolved in PBS pH 7.4 and used at 25 mU/mL for the assay. After 10 min of pre-incubation, the substrate acetylthiocholine iodide (1.5 mM) was added to start the reaction. During 1 h of incubation, 96-Well microtiter multiplates were read on a PherastarFS (BMG Labtech) detection system at a wavelength of 405 nm. Donepezil was used as reference ChE inhibitor. In this assay, we did not exclude the possibility of false-positive inhibition results, previously described for high concentration (> 100 μg/mL) of amine or aldehyde compounds [START_REF] Rhee | Screening for acetylcholinesterase inhibitors from amaryllidaceae using silica gel thin-layer chromatography in combination with bioactivity staining[END_REF][START_REF] Rhee | Qualitative determination of false-positive effects in the acetylcholinesterase assay using thin layer chromatography[END_REF]. Nevertheless, the lack of inhibition observed for the essential oil versus the AChE strongly minimized this possibility.

Determination of monoamine oxidase inhibition assay

MAO source was prepared by homogenizing rat liver in 2 volumes of cold 50 mM phosphate buffer pH 7.4, containing 0.125 M sucrose. The mitochondrial fraction containing the bulk of MAO activity was obtained after two centrifugations: firstly at 4000g for 5 min to remove cellular debris and secondly at 17000 g for 15 min to spin down mitochondria.

The monoamine oxidase (MAO-A + B) activity was measured in one step, monitoring the conversion of kynuramine to 4-hydroxyquinoline by recording spectral absorbance changes according to Weissbach and colleagues (1960). A typical 200 μL inhibition assay volume contained reaction buffer (PBS, 0.05% NP40), test sample in DMSO (1.5% v/v), mitochondria suspension containing MAOs activities (2.5%). After 10 min of pre-incubation, the substrate kynuramine (0.45 mM) was added to initiate the reaction. The 96-Well microtiter multiplates were incubated and read for at least 1 h on a PherastarFS (BMG Labtech) detection system. In order to increase the sensitivity of the method, enzymatic activities were evaluated by taking into account the sum of the amplitude of absorbance decrease at 357 nm (disappearance of kynuramine) and the amplitude of absorbance increase at 316 nm (appearance of 4-hydroxyquinoline). Clorgyline and deprenyl were used as a reference to MAO-A and B inhibitors respectively. In order to achieve specific measurements of the MAO-A and MAO-B activities, we have combined the use of both selective inhibitors with a non-selective substrate (kynuramine) in order to distinguish between MAO subtypes. Clorgyline and deprenyl concentrations were chosen to cause minimal non-selective inhibition of MAO-B and MAO-A, respectively (usually 1000 nM and 500 nM final).

For all enzymatic activities studied, the percentage of residual activity (% Residual activity) is given as follows:

= %Residual activity 100 (A /A ) sample control

Where A control is the absorbance of the control reaction containing all reagents except the tested sample, and A sample is the absorbance of the tested compounds.

Results and discussion

The essential oil obtained by hydrodistillation from the aerial parts of A. leucotrichus was blue-coloured and the yield of the extraction was about 1.3% (volume/dry weight).

GC/MS results showed that the essential oil contains essentially monoterpenes identified as perillaldehyde (58.3%), limonene (23.33%) and α-pinene (5.74%). Accordingly, β-pinene (2.26%), perillalcohol (1.86%), 3-carene (1.86%),3-thujanone (0.7%), camphene (0.59%), myrcene (0.51%), α-copaen-11-ol (0.34%), globulol (0.34%), α-cardinol (0.22%), caryophyllene (0.16%) and cumin aldehyde (0.15%) were also identified but in less noticeable amounts (Table 1). Our results are in good agreement with the previous studies that have reported perillaldehyde and limonene as principal components of plants essential oil [START_REF] El-Haci | Antimicrobial activity of Ammodaucus leucotrichus fruit oil from Algerian Sahara[END_REF][START_REF] Louail | Antimicrobial and antioxidant activity of essential oil of Ammodaucus leucotrichus Coss. & Dur. seeds[END_REF][START_REF] Manssouri | Antifungal activity of essential oil from the fruits of Ammodaucus leucotrichus Coss. & Dur., in liquid and vapour phase against postharvest phytopathogenic fungi in apples[END_REF][START_REF] Velasco-Negueruela | Analysis by gas chromatography-mass spectrometry of the volatiles from the fruits of Ammodaucus leucotrichus subsp. leucotrichus and subsp. nanocarpus grown in North Africa and the Canary Islands, respectively[END_REF]. The quantitative difference could be attributed to the harvest time, the origin of the plant, climatic, seasonal factors and period of the vegetative cycle.

Cholinesterase inhibition

The low level of acetylcholine in the brain is one of the causing factors of Alzheimer's disease, a progressive neuro-degenerative disorder associated with memory impairment and cognitive deficit.

Currently, a therapeutic approach is followed according to the cholinergic hypothesis to treat Alzheimer's disease. This approach is based on the inhibition of cholinesterase enzymes (ChE) which catalyze acetylcholine hydrolysis to increase its level in the brain and thus improve the cholinergic functions in AD patients [START_REF] Howes | Plants used in Chinese and Indian traditional medicine for improvement of memory and cognitive function[END_REF]. As shown in Table 2, the anticholinesterase activity of the A. leucotrichus and the individual main compounds of the essential oil were compared to Donepezil, used as a standard drug against Alzheimer's disease.

Essential oil and perillaldehyde have not exhibited an activity against the acetylcholinesterase. In contrast, limonene induced high acetylcholinesterase inhibitory activity with an IC 50 of about 51.6 μg/ mL. Essential oil showed also an interesting activity against BuChE (IC 50 = 95.21 μg/mL). This oil appeared to be effective compared to inhibition results of Donepezil, a compound approved by the US Food and Drug Administration (US-FDA) for the treatment of moderate to severe Alzheimer's disease [START_REF] Sugimoto | Donepezil Hydrochloride (E2020) and Other Acetylcholinesterase Inhibitors[END_REF]. The most promising BuChE activity was observed with perillaldehyde (IC 50 = 42.77 μg/mL), followed by limonene (IC 50 = 66.7 μg/mL). Similar results were obtained in E. globulus showing satisfactory inhibitory activity against AChE with IC 50 of 0, 1298 mg/ml [START_REF] Aazza | Antioxidant and antiacetylcholinesterase activities of some commercial essential oils and their major compounds[END_REF]. This study is also focused on pure limonene showing a moderate activity against AChE and the IC 50 value was about 0.5863 mg/mL. On the other hand, for the C.limon plant, despite the strong presence of limonene in its essential oil, the AChE inhibitory activity was lower with IC 50 about 0.8499 mg/mL.

In another study, the ethanol extract from the fruits of Pimpinella anisoides (Apiaceae) has an interesting activity against both enzyme (AChE an BuchE) and the limonene, one of the major compounds has the lowest activity with the IC 50 of 225.9 and 456.2 μg/mL for AChE an BuchE respectively [START_REF] Menichini | Acetylcholinesterase and butyrylcholinesterase inhibition of ethanolic extract and monoterpenes from Pimpinella anisoides V Brig. (Apiaceae)[END_REF]. Our results are better with a low value of IC 50 for AchE and BuchE (51.6 and 66.7 μg/mL respectively). It should be noted that limonene has been reported as inactive by some authors [START_REF] Dohi | Acetylcholinesterase inhibitory activity and chemical composition of commercial essential oils[END_REF].

For anti-butyrylcholinesterase activity, all samples have a high inhibitory activity and the lowest value of IC 50 was obtained with perillaldehyde followed by limonene and essential oil. Anti-butyrylcholinesterase of the essential oil can be attributed to the strong presence of perillaldehyde and limonene (53.3% and 23.33% respectively). To our knowledge, no previous study reported butyrylcholinesterase inhibitory activity of essential oil of A. leucotrichus and perillaldehyde. [START_REF] Chemsa | Chemical constituents of essential oil of endemic Rhanterium suaveolens Desf. growing in Algerian Sahara with antibiofilm, antioxidant and anticholinesterase activities[END_REF] showed that the essential oil of Rhanterium suaveolens Desf rich in perillaldehyde is inactive against the two enzymes (AchE and BuchE) while the methanolic extract has a moderate inhibitory activity against AChE and a good butyrylcholinesterase inhibitory. Although, both major essential oil compounds have exhibited an interesting inhibitory activity and the essential oil has a lower activity. This can be explained by the antagonistic effect among the components of the oil of A. leucotrichus [START_REF] Savelev | Synergistic and antagonistic interactions of anticholinesterase terpenoids in Salvia lavandula efolia essential oil[END_REF].

MAO inhibition

The MAOs inhibitory effect of the A. leucotrichus essential oil, perillaldehyde, and limonene were evaluated and the results are represented in Table 2.

In our study, EO and perillaldehyde were able to inhibit MAO-A in a dose-dependent manner (1-1000 μg/mL) with IC 50 values of 112.5 and 159.1 μg/mL respectively. In comparison to clorgyline as an irreversible MAO-A inhibitor, the essential oil is much less active. With respect to the effect on MAO-B, the EO and perillaldehyde exhibited a high inhibitory activity with IC 50 values of 40.5 and 98.9 μg/mL respectively. On the other hand, for MAO-A + B, both samples cited above showed good inhibitory effect presented with the IC 50 values of 53.4 and 100.4 μg/mL, respectively, and no anti-MAOs activity was observed with limonene. From the obtained values of IC 50 , it could be predicted that the EO and perillaldehyde are better inhibitors for MAO-B than MAO-A.

The highest MAOs inhibition was observed by the essential oil. The MAO-A inhibitors inhibit the catabolism of serotonin, norepinephrine and dopamine which lead to increase the levels of these neurotransmitters in the brain. They are effective in the treatment of depressions in elderly patients [START_REF] Youdim | Monoamine oxidase: Isoforms and inhibitors in Parkinson's disease and depressive illness[END_REF][START_REF] Zisook | A clinical overview of monoamine oxidase inhibitors[END_REF].

The essential oil and perillaldehyde have an affinity for MAO-B. This enzyme is present with a high level in people affected by Parkinson's disease due to the malfunction in the dopaminergic system [START_REF] Youdim | Monoamine oxidase: Isoforms and inhibitors in Parkinson's disease and depressive illness[END_REF]. MAO-B is responsible for the catabolism of neurotransmitters amines; it metabolizes a dopamine in the basal ganglia. For this, the inhibition of this enzyme in Parkinson's patients conserve dopamine in the brain, which leads to relieve symptoms of the Parkinson's disease [START_REF] Collins | Multiple forms of human brain mitochondrial monoamine oxidase[END_REF][START_REF] Youdim | Human brain monoamine oxidase: multiple forms and selective inhibitors[END_REF].

The inhibition of MAOs by the essential oils is not well documented. Some studies have shown that the Myristicin which is an essential oil compound of nutmeg was able to inhibit rodent brain MAO activity [START_REF] Ashwood | Fine particles that adsorb lipopolysaccharide via bridging calcium cations may mimic bacterial pathogenicity towards cells[END_REF]. Eugenol, another constituent of EO has been proven as an inhibitor of MAO-A and B [START_REF] Tao | Eugenol and its structural analogs inhibit monoamine oxidase A and exhibit antidepressant-like activity[END_REF].

To this fact, it is very important to point out that this study is the first report on the inhibition of cholinesterase and monoamine oxidase properties of the essential oil of A. leucotrichus and its major compounds. According to the BuchE and MAOs results of the EO of A. leucotrichus, it seems that this plant may have promising applications in the treatment of the Alzheimer's disease and neurodegenerative disorders.

Conclusion

The results presented in this study are the first information on the anticholinesterase and anti-monoamine oxidase activities of A. leucotrichus C. & D. essential oil, collected from Algeria. The essential oil, as well as its main compounds, perillaldehyde and limonene have exhibited a global cholinesterase and monoamine oxidase inhibitory activity. A selectivity towards BuChE and the MAO isoform B is evidenced. A consequence of this dual inhibition capability is that consumption of A. leucotrichus may protect people against the Alzheimer's disease and neurodegenerative disorders without any side effect as observed from the traditional practice. Yet, further studies, particularly in vivo tests, are needed to understand A. leucotrichus C. & D. essential oil activity in biological systems. a IC50 values determined under identical conditions for Niphogeton dissecta essential oil and its major constituant. [START_REF] Calva | Acorenone B: AChE and BChE Inhibitor as a major compound of the essential oil distilled from the ecuadorian species niphogeton dissecta (Benth.)[END_REF].

Table 1

 1 Chemical composition of the A.leucotrichus aerial parts oil.

	Number	Components	Retention time	Percentage
	1	α-pinene	4.44	5.74
	2	β-pinene	5.02	2.26
	3	Camphene	4.62	0.59
	4	Myrcene	5.18	0.51
	5	3-Carene	5.53	1.69
	6	D-Limonene	5.86	23.33
	7	3-Thujanone	6.78	0.7
	8	Cumin aldehyde	8.71	0.15
	9	Perillaldehyde	9.34	58.3
	10	Perillalcohol	9.62	1.86
	11	Acetic acid, (2	10.87	1.68
		isopropenylcyclopentylidene)-, methyl
		ester
	12	Caryophyllene	11.48	0.16
	13	α-copaen-11-ol	12.83	0.34
	14	Isolongifolan-8-ol	13.12	0.22
	15	Globulol	13.49	0.34
	16	α-cardinol	14.13	0.27
		Total		97.25

Table 2

 2 IC50 values for the inhibition of acetylcholinesterase (AChE), butyrylcholinesterase (BuChE) and the two Monoamine-oxidases isoforms (MAO-A and MAO-B) activities exhibited by Ammodaucus leucotrichus Coss. & Dur essential oil, reference chemical compounds.

	IC50	AChE	BuChE	MAO
				MAO-A	MAO-B	MAO-A + B
	Essential oil, ug/mL	˃1000	95.2 ± 15.0	112.5 ± 21.3	40.5 ± 3.8	53.4 ± 4.1
	Limonene, ug/mL	51.6 ± 0.7	66.7 ± 12.6	˃1000	˃1000	˃1000
	Perillaldehyde, ug/mL	˃1000	42.7 ± 4.8	159.1 ± 25.9	98.9 ± 7.2	100.4 ± 9.2
	Donepezil, nM	95.6 ± 12.0	8600 ± 570	NA	NA	NA
	a Acorenone B μg/mL	40.8	10.9	NA	NA	NA
	a Niphogeton dissecta EO μg/mL	˃1000	11.5	NA	NA	NA
	Clorgyline, nM	NA	NA	7.1 ± 1.7	NA	NA
	Deprenyl, nM	NA	NA	NA	59.1 ± 2.3	NA
	NA = not applicable.

Acknowledgement This work initiated in the Laboratory of plant biotechnologies & ethnobotany, University of Bejaia, Algeria, was carried out in the Laboratory of Bioengineering and Nanosciences, University of Montpellier, France and in the Institut de Recherche en Cancérologie de Montpellier, INSERM, Montpellier, France. It was financed by the Algerian Ministry of Higher Education and Scientific Research as part of the PNE grant program to finalize a doctoral thesis. Nesrine Sadaoui acknowledges Elodie Middendorp for technical assistance.

Conflict of interest

We wish to confirm that there are no known conflicts of interest associated with this publication and there has been no significant financial support for this work that could have influenced its outcome.