Double-blind, Randomized, Placebo-Controlled study to evaluate the Efficacy of an early treatment with Herbal Supplement based in the Prevention of Post-Traumatic Stress Disorder in emergency department (PHYTeS Study)

Introduction: The prevention from Post-traumatic stress disorder (PTSD) is therefore of major public health interest and one of the concerns of any emergency physician. The purpose of our study was to evaluate the efficacy and safety of an herbal supplement to prevent the occurrence of PTSD in high-risk patients. Methods: It is a randomized, double-blind, prospective, interventional study including patients exposed to a potentially traumatic event that meets DSM-V Criterion A and has a Peri-traumatic Distress Inventory score or the Questionnary for traumatic dissociation experiments (PDEQ) and/or L.Crocq score higher than the thresholds between day 1 and day 3. Two hundred patients were included randomly assigned into two groups: Aleozen group and placebo group. Patients included in aleozen group received Aleozen(R) for 10 days while patients in placebo group received Placebo. A CAPS-5 assessment was performed for all patients at different moments. The main objective was to assess the efficacy of Aleozen after 90 days of an exposition to traumatic events according to PTSD. Secondary objectives were to evaluate the safety of Aleozen(R) at 10 and 30 days after its administration and PTSD in the study population after one year of inclusion. Results: No statistical differences were noted between the two groups in term of baseline characteristics including age, sex and the ISS score. After 90 days of follow-up, and according to the CAPS-5 scale, 85 patients (42.5%) of the population study showed PTSD. Concerning primary endpoint, less PTSD were seen in intervention group compared to placebo group (38.8% versus 61.2% respectively; p<0.001). During the study, no adverse events were noted. Conclusion: Results of this work suggest the potential preventive effects of an herbal supplement on PTSD for traumatic patient in emergency. Further confirmatory studies are needed.

sponsor and funder have no role in study design; collection, management, analysis, and interpretation of data; writing of the report; and the decision to submit the report for publication, including whether they will have ultimate authority over any of these activities.
Ethical approval: The study was approved by the local ethics committee (The Ethics Committee of the Faculty of Medicine of Sousse) Ethical review: A written informed consent was obtained from all participants.
Human and animal rights statement: All procedures performed in the study were in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 Helsinki Declaration and its later amendments or comparable ethical standards.
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Introduction:
Trauma-related nightmares and sleep disturbance are common symptoms of post-traumatic stress disorder (PTSD) [1]. It's a debilitating psychopathological response to a traumatic event which can severely compromise quality of life. In recent years, the increasing of estimated prevalence of PTSD can be due partly to the improvement of the standardized evaluation procedure. [1] One of the first epidemiological studies of PTSD was "Mental health in general population" survey which was carried in metropolitan France between 1999 and 2003 including 36,000 people. This study revealed that PTSD instantaneous prevalence (last month) was 0.7% of the population with nearly equal average between men (45%) and women (55%) [2]. Same result was reported in the European study of the epidemiology of mental disorders [3]. Moreover, several psychiatric and physical comorbidities may occur with PTSD, particularly mood disorders, anxiety, and substance abuse [4]. Nevertheless, its severity is worsened by the high risk of suicidal behavior [5]. Moreover, one of the first published studies that assessed the relation between exposure to war, PTSD and mortality was conducted among Vietnam veterans in the United State. Veterans with elevated level of PTSD risk have a highest mortality risk (adjusted hazard ratio = 2.34, 95% confidence interval: 1.24, 4.43) [6].
Definition of PTSD was conflictual; Barrois defines this traumatic neurosis as "a group of psychological disorders that arise after a longer or shorter latency, following a very intense emotional shock" [7].
Anxiolytic and/or sedative phytotherapy may represent an alternative symptomatic treatment for anxiety states which are not too severe. Thus, in its recommendations for out-patient management of anxiety disorders in adults, the ANAES (Agence Nationale d'Accréditation et d'Evaluation en Santé) supports the use of a phytotherapy preparation, Euphytose, based on dry extracts of Crataegus, Balloia, Passiflora, Valeriana, Cola and Paullinia [8].
The burden of several diseases associated with psycho traumatic disorders highlight the importance of prevention and primary care which remains under-identified, particularly in Tunisia. So, several therapeutic protocols have been proposed over the years. The antidepressant treatment (yet first-line treatment in all international guidelines) represents only 30% of PTSD treatment and the remaining 70% include anxiolytics, hypnotics and herbal medicine. Several meta-analyzes have evaluated the efficacy of antidepressants, but data for herbal medicine remains very poor. We tested Aleozen ® , a balanced and synergistic . CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) combination of plant extracts growing in Tunisia that helps treat states of stress, insomnia, and anxiety [9][10][11].
This study aims to evaluate the efficacy and the tolerance of Aleozen ® ) versus placebo in patients with high risk of developing PTSD.

Methods:
A double-blind, randomized placebo-controlled clinical trial was conducted from June 2018 to September 2018. The study was approved by the Ethics Committee; it was recorded in the ClinicalTrials.gov register.
The treatment was provided by Aleonat Laboratories, Tunisia.

Population:
Patients, over 18 years of age, who had been exposed to a traumatic event (a violent mechanism such as assault, public road or domestic accident) meeting criterion A of the definition of PTSD according to DSM-V [10] were included.
PDI is a 13-item self-report scale for determining a person's emotional distress reactions, at the time of a traumatic event and in the minutes and hours after it. People with severe peritraumatic distress are at risk of developing post-traumatic stress disorder. A score greater than 15 indicates significant dissociation [12].
The PDEQ has been validated in French and uses the English version. It is a self-administered questionnaire to retrospectively assess the dissociative elements of consciousness at the time of the trauma and in the following minutes in 10 items. A score greater than 15 indicates significant dissociation [13]. L. Crocq is a self-administered questionnaire of 20 questions, exploring the five aspects cognitive, affective and mental state immediately after the event; A score greater than 50 indicates significant dissociation [14].
. CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted January 13, 2022. ; Individuals with serious trauma admitted to the intensive care unit, patients with psychiatric illness or using psychotropic medication, non-cooperating patient and non-consenting patients were not included.
About the sample size to estimate the minimum sample size, we used the following formula n = [3.84 * ((P * Q) / i2)] with P = the prevalence of PTSD = 0.15, Q = 1-P = 0.85, i = 0.05, or 195 patients.
We supposed that the prevalence of PTSD was about 15% according to the study of Van Zuiden et al where the prevalence of the PTSD was found= 18,44% and in a second reason to a study carried out in our department (not yet published) including patients with the same criteria as our current study (patient at high risk of manifesting PTSD) the prevalence was found to be 14% [15].

Study design:
Participants were randomized to 10 days double-blind treatment with ALEOZEN ® or placebo.

Data collection:
Data was collected by medical staff practicing at Sahloul emergency department. On the first day of admission to the emergency room, a pre-tested questionnaire with three items was used for data collection.
The first item treated demographic characteristics such as age, sex or medical history.
The second one included the elements of physical examination; traumatic lesions and the injury severity score (ISS) [16]. In the third part we tried to assess the severity of the traumatic event using the PDEQ [13], the PDI scale [12] and the Immediate stress questionnaire (L. Crocq) [14].
The follow-up of the patient was carried on the 10th day, 1month, 3month and one year after the study inclusion via a telephone consultation. These calls were made by a researcher who was blind to the treatment taken. Double-blind anonymity was issued after statistical analysis of all data. The main objective was to confirm the diagnosis of PTSD according to the . CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

(which was not certified by peer review)
The copyright holder for this preprint this version posted January 13, 2022. ; https://doi.org/10.1101/2022.01.12.22268879 doi: medRxiv preprint Clinically Administered PTSD Scale for DSM-5 (CAPS-5 scale) [17] at the third month. At the 10 th day, telephone call was made ensuring the safety and the adherence to the treatment; at one month, we wanted to evaluate its safety and effectiveness. At 3 months, the calls were made to evaluate PTSD. At one year after inclusion to the study, the purpose of the telephone call is to evaluate the progress of patients admitted to each group and assess the progress of patients according to their post-traumatic stress disorder (figure 2). We adopted score CAPS-5 >32 to retain PTSD diagnosis [18]. CAPS-5 is a self-administered 20-item questionnaire that evaluates the 20 symptoms of PTSD according to the CAPS-5. A provisional diagnosis can be obtained by considering any item with a score of 2 or more as present, and then adhering to CAPS-5 instructions that require at least: 1 item B (questions 1-5), 1 item C ( questions 6-7), 2 items D (questions [8][9][10][11][12][13][14], and 2 items E (questions [15][16][17][18][19][20]. A threshold of 38 at CAPS-5 seems reasonable to suggest the presence of PTSD until psychometric studies are available.

Endpoints of the study
The primary endpoint was to assess improvement of PTSD at 90 days after the beginning of Aleozen therapy. The secondary objectives were to evaluate the safety of Aleozen ® at 10 and 30 days after its administration and efficacy of PTSD after one year of inclusion. We evaluated also the improvement of symptoms severity of PTSD (reduction of the CAPS-5 score by 50%), and necessity of psychiatric follow-up, use of additional psychotropic or anxiolytic treatment or adverse events.

Statistical analysis
The data was entered and analyzed using the Statistical Package for Social Sciences (SPSS).
Categorical variables were reported as count and percentages. Quantitative variables were expressed in terms of means and standard deviation. For the comparison of the qualitative variables, we used the Chi2 test and for the comparison of the quantitative variables, Student's and ANOVA test. Variables having p-value of less than 0.05 were considered as significant at 95% confidence level.
Ethical review: A written informed consent was obtained from all participants.

Results:
We included 200 patients in our study: 100 patients in each group. The characteristics of participants are shown in table1. No statistical differences were noted between the two groups in term of age, sex and the ISS score. Overall, 8.5% of the participants were lost to follow-up.
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Patients with PTSD were referred either to psychiatrists or to a psychologist working in the emergency rooms according to the patient's choice.
A decrease in the total rate of CAPS-5 by 50% was observed mostly among patients in the Aleozen ® group (51% VS 29%, p=0.004).
When comparing the CAPS-5 scale between the two groups; no significant differences were noted 10 and 30 days after inclusion. However, a significant difference appeared in day 90 The comparison between the 4 items of CAPS 5 showed almost similar results between the two groups after 10 days and 30 days of inclusion. However, a significant difference was found after 90 days and one year of follow up, particularly between B, C and D items.
When observing the triggering mechanism of ED admission, a significant benefit of Aleozen® was noted in traffic accident and assault among the two groups (p=0.025 and 0.006 respectively).
The variation of CAPS-5 in relation to the admission has been shown in table 2. Observation a comparable variation at 30 days against two significant differences in the variation of the CAPS at 90 days and one year The comparison between the 4 items of CAPS5 showed almost similar results between the two groups of studies after 10 days, 30 days and one year of inclusion. However, after 90 days and one year of follow up, the differences were significant. (Table 3) The difference between the 30-and 90-day CAPS-5 items, compared with the 10-day results, showed significant difference between the group of patients who received a placebo and the group who received the Aleozen ® always after 90 days of inclusion in items B, C and D (table   4).
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The copyright holder for this preprint this version posted January 13, 2022. ; https://doi.org/10.1101/2022.01.12.22268879 doi: medRxiv preprint After one year following up, and according to the CAPS-5 criteria, PTSD was diagnosed among 12% of placebo group and 6% from Aleozen ® group. These patients were referred to psychiatrists or psychologist. Five patients from placebo group were put on anxiolytics.
During the study, no adverse events were noted.

Discussion
In this randomized, double-blind, prospective study, our result strongly suggest benefits of a herbal medicine administration early after trauma on prevention of PTSD or reduction of subsequent PTSD symptom severity in emergency department in high-risk patients.
We showed in our study that prescribing Aleozen ® in emergency department for high risk patients for developing PTSD can prevent this stress disorder. For screening high risk patient we used 2 tools: PDI [12] and PDEQ scales. Those tools assessed peri-traumatic distress and peri-traumatic dissociation. While searching in the literature, we have found that they are the two main risk factors that were associated with development of PTSD.  [23] showed that this scale is a strong psychometric tool for making a provisional diagnosis, to quantify the severity of PTSD symptoms and monitor clinical change over time witch make it the gold standard.Thus, it's important to understand neurobiological mechanisms involved in generating PTSD to provide challenges for treatment. In the first studies in patients with PTSD, researchers showed autonomic reactivity in response to trauma-related signs, such as increased heart rate and skin conductance [24,25]. Moreover, many pharmacological challenge trials with yohimbine (an α 2-adrenergic receptor antagonist) demonstrated excessive neurochemical then behavioural reaction in relation with central noradrenergic hyper-reactivity in PTSD [26,27]. As we understand that the pathophysiology . CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. . CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

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The copyright holder for this preprint this version posted January 13, 2022. Griffonia simplicifolia [42,43], the main component of aleozen, rich in 5 Hydroxytryptophan precursor of serotonin regulating sleep and mood, and Gentiana lutea [44][45][46] with its presence of xanthones is inhibitory of MonoAmine Oxidase, therefore antipsychotic.
Rhodiola rosea [47,48], Crataegus oxyacantha [49], Eschscholtzia californica [50] and Melissa officinalis [51] are mild sedatives, Zinc and Vitamin B6 are nutrient for the nerve  42.83 in the 10 th day to 17.05 after 90 days (reduction was high than 50%) [15]. Second, in a Double-blind, randomised, placebo-controlled study to evaluate the efficacy and safety of a fixed combination containing two plant extracts (Crataegus oxyacantha and Eschscholtzia californica) and magnesium in mild-to-moderate anxiety disorders, Hanus and colleague approved that on whatever parameter (Hamilton total score, Hamilton somatic score, or VAS) . CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

(which was not certified by peer review)
The copyright holder for this preprint this version posted January 13, 2022. ; https://doi.org/10.1101/2022.01.12.22268879 doi: medRxiv preprint the percentage of patients responding to treatment was always significantly greater in the study drug group than in the placebo group (58% vs 43%, p=0.008). [53] Furthermore, one of the advantages of the study is the homogeneity of the sample with nearly demographic and clinical characteristics in both randomized groups, allowing a valid comparison without selection bias.
In our study, the treatment was administered between the first and the third day after trauma in an arbitrary manner. The optimal time to initiate preventive therapy remains under discussion. Besides, considering the unknown effects of chemoprophylaxis in peoples who would recover naturally after being exposed to a traumatic situation [54], it looks unjustified to provide all patients with preventive treatment. Therefore, targeting only those at high risk of developing PTSD would be a more effective strategy. Indeed, there is promising evidence that quantitative measures can predict the onset of PTSD [55]. Among the strengths of this study is that it had included patients, using PDI, PDEQ and L-CROQ scales, with scores greater than predictive level for PTSD occurrence [19].
Limitations: There were still several limits in our study; mainly it was a prospective monocentric research. Furthermore, the originality of our study is the herbal medicine treatment that was safe and we did not report any major treatment-related side effects during and after the study. Future multicentric confirmatory studies are needed.

Conclusion:
Through the different results of our study, we have been able to demonstrate that early administration of an herbal supplement prevents the occurrence of PTSD and lowers the severity of symptoms in patients with high risk for development of PTSD.  is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

(which was not certified by peer review)
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(which was not certified by peer review)
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(which was not certified by peer review)
The copyright holder for this preprint this version posted January 13, 2022. ; https://doi.org/10.1101/2022.01.12.22268879 doi: medRxiv preprint . CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted January 13, 2022. ; https://doi.org/10.1101/2022.01.12.22268879 doi: medRxiv preprint is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted January 13, 2022. ; https://doi.org/10.1101/2022.01.12.22268879 doi: medRxiv preprint Figure 2: Study protocol . CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted January 13, 2022. ; https://doi.org/10.1101/2022.01.12.22268879 doi: medRxiv preprint Figure 3: Evolution of the mean of CAPS-5 between Aleozen and Placebo groups over time P<0,005 * . CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted January 13, 2022. ; https://doi.org/10.1101/2022.01.12.22268879 doi: medRxiv preprint