Tony Marteau

Sitou Afanou

David Sodoyer

Sébastien Ambellouis

Fouzia Boukour

Audio Events Detection in Noisy Embedded Railway Environments

Keywords: Audio event detection, Abnormal event, Transport environment, Railway, Deep learning, CRNN

Ensuring passengers' safety is one of the daily concerns of railway operators. To do this, various image and sound processing techniques have been proposed in the scientific community. Since the beginning of the 2010s, the development of deep learning made it possible to develop these research areas in the railway field included. Thus, this article deals with the audio events detection task (screams, glass breaks, gunshots, sprays) using deep learning techniques. It describes the methodology for designing a deep learning architecture that is both suitable for audio detection and optimised for embedded railway systems. We will describe how we designed from scratch two CRNN (Convolutional Recurrent Neural Network) for the detection task. And since the creation of a large and varied training database is one of the challenges of deep learning, this article also deals with the innovative methodology used to build a database of audio events in the railway environment. Finally, we will show the very promising results obtained during the experimentation in real of the model.

INTRODUCTION

Surveillance in the railway field is an expensive task. It requires deploying huge resources, both human and material, to ensure the safety of passengers. A whole framework dedicated to this task must be deployed: CCTV cameras and microphones, patrol and surveillance agents, barriers, etc. Nowadays, most of autonomous surveillance systems still require a human operator. With the recent image and signal processing techniques as neural networks (NN) and deep learning (DL), a robust surveillance automation becomes possible. The automation's aim is to help railway operators by reducing security issues by detecting an event very early and allowing the prompt intervention of the railway police.

Developing audio and video algorithms to detect critical events is not a new research action. But with recent NN innovations, this research area has grown very quickly these last years. Moreover, smart video based event recognition is an active research field but is more difficult inside the railway vehicle due to occlusion issues. In this context, analysing audio environment of a railway has yield promising results in the past [START_REF] Pham | Audio-video surveillance system for public transportation[END_REF][START_REF] Zouaoui | Embedded security syst. for multi-modal surveillance in a railway carriage[END_REF] with classical machine learning techniques. In this paper, we present a work in line with this question: how to detect some critical events by analysing audio environment inside a train ?

We propose to design an event detection system based on NN and DL techniques, that will be based on existing audio equipment's in actual commercial trains. We aim at increasing the capabilities of the actual surveillance systems with automatic detection and identification of some abnormal sounds.

The automatic sound classification and recognition are two active areas of study [START_REF]Computational Analysis of Sound Scenes and Events[END_REF][START_REF] Purwins | Deep learning for audio signal processing[END_REF][START_REF] Abeßer | A review of deep learning based methods for acoustic scene classification[END_REF] and are present in various fields of application as speaker recognition [START_REF] Ravanelli | Speaker recognition from raw waveform with sincnet[END_REF], speech emotion classification [START_REF] Zhang | Cooperative learning and its application to emotion recognition from speech[END_REF][START_REF] Xie | Speech emotion classification using attention-based lstm[END_REF], urban sound analysis [START_REF] Salamon | Fusing shallow and deep learning for bioacoustic bird species classification[END_REF], audio surveillance of roads [START_REF] Foggia | Audio surveillance of roads: A system for detecting anomalous sounds[END_REF], acoustic scene classification [START_REF] Drossos | Unsupervised adversarial domain adaptation based on the wasserstein distance for acoustic scene classification[END_REF], event detection [START_REF] Turpault | Sound event detection in domestic environments with weakly labeled data and soundscape synthesis[END_REF] and localisation [START_REF] Adavanne | Sound event localization and detection of overlapping sources using convolutional recurrent neural networks[END_REF]. It aims at detecting the onset and offset times and labelling for each sound event in an audio sequence.

The prolific research is due to advances of NN and DL that has deeply changed the way to design and use automatic detection systems, for both sound or image stream. In this paper, we study sound classification algorithms to deal with abnormal audio events recognition. As previously cited, several research have already been conducted between 2005 and 2015 : The European research projects BOSS, the french projects SURTRAIN and D ÉGIV [START_REF] Pham | Audio-video surveillance system for public transportation[END_REF][START_REF] Zouaoui | Embedded security syst. for multi-modal surveillance in a railway carriage[END_REF]. These works did not use DL and NN because the computing power of computers did not allow us to consider on-board setup. Recently, Laffitte et al. showed the way and presented studies on the automatic detection of screams and shouts in subway train using deep neural networks (DNN) [START_REF] Laffitte | Deep neural networks for automatic detection of screams and shouted speech in subway trains[END_REF][START_REF] Laffitte | Assessing the performances of different neural network architectures for the detection of screams and shouts in public transportation[END_REF].

A supervised DNN requires a large amount of data for the training task of the model. Obtaining a database combining both quantity, thousands of data, and quality, for which all the "event to be recognised" are precisely labelled become a complex paradigm. Some audio databases are publicly available in the scientific community like databases from different challenges of Detection and Classification of Acoustic Scenes and Events (DCASE) [1] but the embedded railway environment is not generally considered. This is understandable since it is difficult to collect huge amount of recordings in a train. Indeed, railway is a highly regulated environment, where very strict rules must be followed, in particular concerning fire risks assessment, electromagnetic compatibility, vibrations and personal data protection. Therefore, only certified on-board computers can be used in trains. These computers have limited computing resources for heat dissipation considerations.

The present paper addresses two problems. the first problem is dealing with the build of a railway synthetic database dedicated to abnormal audio events detection. This database has been built by mixing sound patterns and real embedded railway background sounds. The second problem is focusing on the design of convolutional and recurrent neural network for abnormal events detection trained from this database. In the introduction section we present some stud-ies on audio classification and detection. The second section present an original database dedicated to the abnormal events detection in railway environment. Two architectures of convolutional and recurrent neural network are presented in the third section. The following sections are dedicated to the experiments description and detection results respectively. Finally the last section presents the conclusions.

A railway database for abnormal events detection

An embedded railway environment is a very specific place where new acoustic constraints have to be considered. This acoustic environment is very noisy and not stationary. It is a mixture of many acoustic sources emitted from mechanical, electrical and electronic sub-systems working simultaneously and also emitted by passengers. In this context we propose to build a dedicated database by mixing railway background and abnormal event sounds. Both are presented in the following sections and are followed by a description of the mixing method we use.

Railway background sounds

Railway background sounds have been recorded during technical rolling on board of multiple (suburbans, regional and high speed) SNCF train to create variability and make our system less specific. The mobile capture equipment have been placed in the middle and at the tail of the train. Six hours of background sounds have been recorded. The audio signal has been recorded on a single 32 bits channel and has been sampled at 44.1kHz. These background sounds are a mixture of sounds of the engines, sounds of friction of the wheels on the rails, sounds of air conditioner, commercial audio messages etc. These railway background signal is clearly a polyphonic and not stationary background signal. s caisses ?

Abnormal events and additional sounds

Four types of abnormal sounds events to detect have been chosen: gunshots, screams, glass breaks and sprays. The samples of these class sounds are extracted from Freesound website [START_REF] Font | Freesound technical demo[END_REF] in order to check the audio content all the samples have been listen before incorporating them into the final dataset. These abnormal sound are recorded on a 32 bits mono channel signal and sampled at 44.1kHz.

In a commercial train, other operation sounds as buzzers, door opening/closing, passengers conversations etc. appear. Because, these sounds are not recorded during technical rolling, we added all these additional sounds from an other railway audio dataset.

The duration distributions of abnormal events and additional sounds are presented in table 1. Table 1: Duration distribution of the abnormal events sequences and additional sounds.

Database samples generation process

Here we detail how we mix the background, abnormal and additional sounds to generate one audio sequence of our new database. The duration of each generated sound sequence is 10 seconds and below is the workflow we follow to process each sound sequence in the dataset:

1. Selection of a background sound randomly in the background dataset. The gain of the audio sample is selected randomly between 0 and -10 dB to create variability without introduce audio saturation. 2. Selection of 0 up to 3 abnormal events to detect. The temporal localisation is fixed randomly within the 10 seconds of background. Overlapping of samples is allowed and a random gain between -5 and -15 dB is applied. 3. Choice randomly of the presence or not of an other abnormal events (repeated for the three other abnormal sounds). These other events can occur when dedicated events take place. In order to make the system more reliable against these others events, necessary for a correct identification, the detector learn to consider all the other sounds as patterns not to be detected. The temporal localisation is randomly set in the 10 seconds background. Overlapping of samples allowed and a random gain between -5 and -15 dB is applied. 4. Choice randomly the presence or not of an other normal events. The temporal localisation and the gain are choosen randomly in same interval conditions.

The labels are generated at the same time than the integration of the samples of the events to be detected. The labeling is a One Hot encoding labeling: each sequence associated to an event to detect has its own label tensor. Each component of this tensor is initialized to 0, except for the frames when a event to detect occurs where the element is set to 1. The length of this label vector is equal to the length of output model. For example, the Figure 1 shows an overview of the spectrogram and its label. In this sequence a 2-seconds scream sample is inserted at the 7.5th second of the background file, the start timestamp will be 7.5 and the end timestamp will be 9.5.

Abnormal event detection

The algorithm has to label each abnormal event that appears during one 10 seconds sequence. All the sequence is consider as the input of our CRNN. We adopt a One-vs-All (OvA) strategy to predict the labels. One CRNN is viewed as a binary classifier designed and trained for one event to detect: scream, gun shot, glass break and spray. We can take into account the polyphonic detection problem ie. the cases where several events appear at the same time. Finally by using dedicated "less complex" networks, we can expect faster detection by using multi-processors capacity of the computer. Each CRNN consists in extracting the feature map of each sequence (convolution layers) and to analyze the temporal coherence of the frequency activity (recurrent layers). Finally, it computes one event activity probability for every frames of the sequence. The final detection is done by applying a threshold σ = 0.5.

Model architecture

We propose two models based on the Convolutional Recurrent Neural Network (CRNN) developed in the recent papers [START_REF] Lim | Rare sound event detection using 1D convolutional recurrent neural networks[END_REF][START_REF] Cakir | Convolutional recurrent neural networks for polyphonic sound event detection[END_REF][START_REF] Adavanne | Sound event detection using spatial features and convolutional recurrent neural network[END_REF][START_REF] Adavanne | Sound event detection in multichannel audio using spatial and harmonic features[END_REF]. These studies show that the combination of convolutional and recurrent layers allows to jointly capture the invariance in frequency domain and to model short and long term temporal dependencies.

The first model consists in the following layers : two convolutional, two Gated Recurrent Unit (GRU) [START_REF] Cho | On the properties of neural machine translation: Encoder-decoder approaches[END_REF] and three fully connected (FC) layers. GRU is prefered to Long Short Term Memory (LSTM) to reduce the number of parameters and avoid the vanishing gradient problem. In many works in audio events detection, MEL coefficients (MFCCs), are generally used as input features [START_REF] Laffitte | Assessing the performances of different neural network architectures for the detection of screams and shouts in public transportation[END_REF]. In [START_REF] Cakir | Convolutional recurrent neural networks for polyphonic sound event detection[END_REF], the input of the network is a log time-frequency representation of the data in MEL band energies over frames. In our work, we use directly the magnitudes of the spectrum [START_REF] Huzaifah | Comparison of time-frequency representations for environmental sound classification using convolutional neural networks[END_REF] and let the first layers optimise the extraction of higher level parameters. N spectra are computed on each 10 seconds sequence of the database.

The basic version of this first model (CRNN 1) is defined as follows (fig. 2a):

-1 convolutional layer. It is composed of 32 filters that use k × 15 kernel.

Here k is the total number of filters in the time-frequency representation. We use a stride of 4 samples to reduce the dimensionality of the resulting feature map. We use a stride rather than pooling to obtain better computational performance [START_REF] Springenberg | Striving for simplicity: The all convolutional net[END_REF]. The convolutional layer is activated by a ReLu function. -1 convolutional layer. This second convolutional layers is composed of 32 filters that use 32 × 15 kernel, a stride of 1 and is activated by a ReLu function. -2 layers of GRU. GRUs are used to extract temporal information from the feature map output of the second convolutional layer. Each GRU layer has 32 units. -3 FC layers. The 2 first layers are respectively 128 and 64 neurons layers activated through a ReLU function. The last one is a single neuron layer activated through a Sigmoid function. These layers gradually reduce the size of the output and are distributed over the time. The last layer is computing the activity probability for each class.

The second model (CRNN 2) is inspired by the network architecture of [START_REF] Adavanne | Sound event detection using spatial features and convolutional recurrent neural network[END_REF]. For this configuration, the convolution operation of the first and the second layer does not integrate all the frequencies as before i.e. it uses a 3 × 15 kernel, 3 along the frequencies range as in [START_REF] Adavanne | Sound event detection using spatial features and convolutional recurrent neural network[END_REF]. A third convolutional layer is added and a maxpooling operation is used after each convolutional layer. More precisely, the basic version of this second model (CRNN 2) is defined as follows (fig. 2b): -1 convolutional layer. The layer is composed of 32 filters that use 3 × 15 kernel. As in CRNN 1 we use a stride of 4 samples to reduce the dimensionality of the feature map. A max-pooling of 5 × 1 is applied to reduce the dimension output along the frequencies range. -1 convolutional layer. The layer is composed of 32 filters with a 3 × 15 kernel and a stride of 1. It is followed by a 2 × 1 max-pooling. -1 convolutional layer. The layer is composed of 32 filters with a 3 × 3 kernel, a stride of 1 and is followed by a 2 × 1 max-pooling. -2 layers of GRU. GRUs are used to extract temporal information from the feature-map of the third convolution. Each layer has 32 units. -3 FC layers. The 2 first layers are activated through a ReLU function, and the last one through a Sigmoid function. These layers gradually reduce the size of the output and are distributed over the time as in CRNN 1. The last layer is computing the activity probability for each class.

For both network, the last FC layer is not providing one probability for all frames. Because of the use of a 4 samples stride for the first layer of each network, the length of the output vector is divided by 4 i.e. it is equal to N/4.

Evaluation

The experiments consist firstly in evaluating the influence of the units number of the GRU and the structure of the convolutional layers for CRNN 1 and CRNN2. Secondly, we compare the performances of both architectures. For this both study the evaluations have been realised with the synthetic mixture described in section 2.3. Finally, in the last run, we present a preliminary result for the portability and the feasibility of detection in real conditions of the railway environment.

Features extraction description

The input features of the networks is the module of the complex-valued spectra computed on a T seconds audio signal x(t). The spectra x(f, n) are calculated by a Fast Fourier Transform using a sliding Hamming windows of 200 samples and a 60% overlap: f and n denote respectively the frequency and the frame index. Finally, the input of the network is a matrix composed of N magnitude vectors |x(f, n)|. In our experiments, T = 10s, the sampling rate is f s = 44.1kHz and the input is 101 × 5511 matrix composed of 5511 vectors of k = 101 frequencies.

Evaluation procedure in synthetic mixtures case

We evaluate both CRNN architectures by modifying their parameters.

In a first step, we study the influence of the number of units per recurrent layer: 0 (simple convolutional network), 32 (the basic CRNN described previoulsly) and 64 units per recurrent layers. For these three cases, the number of convolution filters is fixed to 64. This step is realised for the architecture CRNN 1.

In a second step, we test the influence of the filters number per convolutional layers for two configurations: 32 filters (the basic CRNN described previoulsy) and 64 filters. In both cases, the units number of GRU layers is fixed to 32. This step is realised for the architecture CRNN 1 and CRNN 2.

CRNN is trained and tested independently for each event using dedicated built synthetic database. In total, we generated for each event 11000 10 seconds sequences: 7000 sequences for training, 2000 for validation, and 2000 for testing. This corresponds to 30 hours of sounds for each class.

For learning phase, we use a 0.01 learning rate and the Adam optimizer for binary crossentropy loss function. Early stopping is triggered after twenty iterations without loss improvement on validation database. A batch normalisation, a 0.2 dropout and a layer normalisation are applied on each convolutional or recurrent layer during the training phase.

The test phase consist in presenting the 2000 sequences of 5511 spectra to compare the N predictions with the N truth labels for each sequence. With a sequence length of 5511, the length of output vector of the CRNN is equal to N = 1375. The evaluation is made by computing accuracy, precision, recall rates and F 1score [START_REF] Mesaros | Metricsfor polyphonic sound event detection[END_REF] for the 2000 × N = 2750000 predictions. The rates are calculated as:

accuracy = TP+TN TP+FP+TN+FN (1a) F 1score = 2 • precision • recall precision + recall (1b) precision = TP TP + FP (2a) recall = TP TP+FN (2b)
where TP, FP, TN and FN are respectively the number of true positive, false positive, true negative and false negative predictions.

Detection description in real environment

To check the performance of our CRNN architectures under the real conditions of the railway environment, we carried out tests at the SNCF Technicentre des Ardoines, in Vitry sur Seine. There, we were able to access an Ile-de-France region suburban train (Z2N train) operating on lines C and D. The train used for the test was stationary for maintenance reasons. The train, however, had its engine running, and many trains were near which produced a huge amount of noise. We set up the system inside the train and used only one IP camera. When the train is set ON, the system launched automatically with the onboard computer. It assigned itself a fixed IP address and started its autotests. The system was able to connect to the IP camera and start reading the audio stream from the IP camera. Some abnormal events were played using a speaker placed at different locations in the train:

1. Speaker at 2m from the microphone, and we play 3 different samples 2. Speaker at 4m from the microphone, and we play 3 different samples 3. Speaker at 6m from the microphone, and we play 3 different samples The audio stream is captured and stored in a FIFO memory of 10 sec refreshed every 0.5 seconds. The detection tests was performed using the models learned with the synthetic mixture presented in 2.3 (without new learning phase). In this real context the evaluation consisted in checking the detection of the corresponding event by monitoring logs files of the system. For these experiments only two critical abnormal events have been tested: the scream and the gunshot events.

Results in synthetic environment

The table 2 and the table 3 present the results of the experimentation plan described in the section 4.2.

For both tables T arget and BG refer respectively to the event class and the background.

The first conclusion is that both architectures yield good results in our railway environment with an accuracy over 90%.

On the one hand, regarding the impact of the recurrent layers (table 2), we can observe that the performance decrease without recurrent layers for all rates. It confirms that we need to take into account the temporal evolution of spectral patterns extracted by the convolutional Layers. The GRU layers do not benefit to the spray events that has really complex spectral-temporal structure. For the three other classes, recurrent layers improve target recall: 12% for Scream and up to 92% for Gunshot. On other hand, the number of units in recurrent layers does not influence significantly the quality detection.

The table 3 presents the effect of the number of filters on the performance. It is difficult to highlight a major improvement w.r.t the number of filters used. Nevertheless, for CRNN 1, 32 or 64 filters yield quite similar rates and for CRNN 2, increase the number of filters seems to severely decrease performance for all events except for scream.

Results in real environment

The results in Table 4 present the number of detected events for scream and Gunshot. In general manner, all events are correctly detected. However, it appears clearly that the detection rate depends on the distance between the source and the microphone. The sensibility effect is reduced by increasing the number of microphones. In this case, the microphones have to be distributed in the railway vehicle insuring that the distance between passengers and one microphone is less that 6m.

CONCLUSIONS

In this paper, we present a new railway audio database and two CRNN architectures designed for abnormal audio event detection. Our evaluation show that using a kernel shape of the same size as the number of frequency bands (CRNN 1) yield better rates. As in [START_REF] Cakir | Convolutional recurrent neural networks for polyphonic sound event detection[END_REF][START_REF] Adavanne | Sound event detection using spatial features and convolutional recurrent neural network[END_REF], the detection results show that catching the temporal structure of the spectrum improves the performance rates. Increasing the number of filters has a weak impact on the detection performance only for CRNN 1. The complexity of the CRNN 1 and the number of parameters are lower than for CRNN 2. It seems to be a quite promising embedded solution for real railway conditions.

Acknowledgement

We would like to thank Helmi REBAI and Martin OLIVIER for strongly contributing to the advancement of this study.

Fig. 1 :

 1 Fig. 1: Spectogram 10 log 10 |x(f, t)| of an audio sequence of 5511 frames (10 sec) with the label vector associeted in red line.

Fig. 2 :

 2 Fig. 2: The two basic CRNN architectures.


Vincent Roumy

Lastenia Ruiz

Juan Celidonio

Ruiz Macedo

Andrea-Luz Gutierrez-Choquevilca

Jennifer Samaillie

Arévalo Leonor

Wilfredo Ruiz Encinas

Hivelli Mesia

Ricopa Ericka

Céline Cotrina

Sevser Rivière

Sahpaz

Leonor Arévalo Encinas

Wilfredo Ruiz Mesia

Hivelli Ericka

Ricopa Cotrina

Céline Rivière

Sevser Sahpaz

Simon Bordage

Guillaume Garçon

Jean Dubuisson

Sebastien Anthérieu

Karin Séron

Thierry Hennebelle

Viral hepatitis in the Peruvian Amazon: Ethnomedical context and phytomedical resource

Keywords: Antiviral activity, Hepatitis C, Peru, Medicinal plant, Traditional use

à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche

Introduction

Since 2017, viral hepatitis B and C have been considered as a major public health concern affecting 325 million people and leading to around 1.5 million deaths every year [START_REF] Lima | Global Hepatitis Report[END_REF]. Approximately 1.5-2 million people are newly infected with viral hepatitis C every year, bringing the total number of the population chronically ill to 71 million in 2019 (for 150 million infected people), and causing around 400 000 deaths [START_REF] Carrat | Clinical outcomes in patients with chronic hepatitis C after direct-acting antiviral treatment: a prospective cohort study[END_REF][START_REF] Petruzziello | Global epidemiology of hepatitis C virus infection: an update of the distribution and circulation of hepatitis C virus genotypes[END_REF][START_REF] Séron | Natural products and hepatitis C virus; natural antimicrobial agents[END_REF]. The estimated HCV prevalence in Latin America was around 1.4% in 2018 (MINSA, 2018), with higher prevalence in tropical area such as Peruvian Amazonia, where this study was carried out. Contrary to hepatitis B, whose prevalence has reduced over the past decade, hepatitis C cannot be easily prevented by vaccination or available treatments and it became one of the most common causes of death among Amerindian populations. Moreover, the Peruvian health sector policy promotes community-based and self-supported health care services in the isolated indigenous communities. Therefore, traditional medicine remains the first recourse for treating health problems but still needs scientific evaluation (PAHO, 2019;[START_REF] Mejia | Plantas medicinales de uso popular en la Amazonia Peruana[END_REF]. In this context, attention has been paid to plant species used in traditional medicine against hepatitis and associated liver diseases in order to contribute to the "Materia medica for infectious diseases in the Amazonian district of Loreto" (Consensus Statement on Ethnopharmacological Field Studies -ConSEFS [START_REF] Heinrich | Best practice in research: consensus statement on ethnopharmacological field studies-ConSEFS[END_REF]. This study took place in the northern region of Peru called Loreto with a high level of both biodiversity and traditional knowledge. The bioprospecting was multisite and performed in Iquitos city surroundings or in Quechua/Jivaro communities along the Pastaza River. These 2 different areas were chosen to obtain more extensive data. The first one, located around Iquitos, is characterized by mixedblood native people's ethnomedical system including neighbouring Amerindian knowledge and more "conventional" medicines. The second one consisted of the riverside indigenous populations of the Pastaza, mainly composed of Quechua, Jivaro (Shuar, Ashuar...) and Candoa (Candoshi, Shapra) groups. In this more remote area, hepatitis is a major concern and herbalism is still the most effective treatment [START_REF] Duke | Amazonian ethnobotanical dictionary[END_REF]FORMABIAP and AIDESEP, 2008). Despite many centuries of colonisation and contemporary petroleum or forestry exploitations, the Peruvian Amazonian territory, nearly 60% of the country's area, is still inhabited by remote indigenous populations. The Pastaza River Wetland is located in Loreto and covers 3.8 million hectares, the largest tropical area of Peru. The Pastaza River begins in the Ecuadorian Andes and the mixture of this spring water with the black one from the flooded forests contributes to a very high and peculiar biodiversity. This area includes over 300 indigenous communities mainly composed of Quechua-speaking population (more than 6000 people) mixed with around 7500 Jibaroan speakers (C. [START_REF] Germaná | 10 years in the Abanico del Pastaza, nature, cultures and challenges in the Northern Peruvian Amazon[END_REF]. Their subsistence economy relies on slash-and-burn horticulture, hunting, fishing and gathering, which contributes to the maintenance of large forest areas. Their elementary conception and exchange with their natural environment is based on the animism. It consists in the attribution of a common subjectivity or a "soul" to every being, including plants or animals and lead to a social structuration of the relations between human beings and natural species [START_REF] Descola | Beyond nature and culture[END_REF]. Sickness in many ways reveals a disruption affecting the individual's soul and its relation with otherness, humans or non-humans. Accordingly to this belief, although this population uses many medicines, ill people mostly attend a Shaman ("yachak" in Quechua or "curandero" in Spanish; [START_REF] Roumy | Amazonian plants used by Peruvian Quechua and Mestizo people to treat malaria: evaluation of their activity[END_REF].

Materials and methods

Ethnopharmacological survey

The ethnobotanical study was conducted during humanitarian missions from June to August 2013, 2014 and 2016.The survey was carried out with 32 informants of various cultural origins from the surroundings of Iquitos (capital city of Loreto), and in 15 riverine Quechua communities of the Pastaza River from Santa Ana (river mouth of Pastaza) to Sabalo Yaccu (upstream remote community situated 250 km North, Fig. 1). Cultural heterogeneity of informants permitted to gather exhaustive data on local phytotherapies. Close attention was paid to the plant therapy used against hepatitis including associated remedies (e.g. special plant diets, symptomatic treatment for jaundice and liver-related ailments). These biomedical uses of plant-derived remedies belonged to different categories according to the International Classification of Primary Care (ICPC), mainly infections and gastrointestinal disorders [START_REF] Staub | Classifying diseases and remedies in ethnomedicine and ethnopharmacology[END_REF].

Information presented here was compiled through general conversations, informal interviews, rainforest walks and other activities shared with local people (harvesting, weeding, hunting, nursing...) adopting a "participant-observation" methodology. Depending on their willingness, origin and availability, the contribution of each informant was so variable that we preferred not to take the numbers of citations into account here (number of citation are just mentioned Table 1).

Plant material

The plants collected were vouchered, deposited and identified at the Herbarium of the Universidad Nacional de la Amazonia Peruana by the botanist Juan Celidonio Ruiz Macedo, (UNAP, Iquitos) and according to regional floras [START_REF] Duke | Amazonian ethnobotanical dictionary[END_REF]Rutter, 1990;Velasquez, 1990). This project was realized in accordance with the Universidad Nacional de la Amazonía Peruana guidelines (Laboratorio de Investigación de Productos Naturales Antiparasitarios de la Amazonia LIPNAA, UNAP) pertaining to ethnopharmacological studies, and ethical approvals with signature from each informant were obtained before investigations.

Plant samples were dried, powdered and extracted three times with methanol for 24 h and filtered (10 g of powdered plant in 100 mL of MeOH). Extracts were taken to dryness at 40 °C under vacuum and weighed. The processing of the plants performed in this study was different from the traditional preparations (Table 1). Therefore, it was not an exact replication of the traditional knowledge but it was admitted that methanolic plant extracts provided more consistent antimicrobial activities [START_REF] Murphy Cowan | Plant Products as Antimicrobial Agents[END_REF]. Moreover, methanol also provided a more complete extraction, including fewer polar compounds, and was more representative of the traditional preparations than other chemical solvents.

Cytotoxicity on HepG2 cells

Methanolic dried extracts were suspended in DMSO to obtain an original concentration of 120 mg/mL. Cells were subcultured in 96-well plates with 3500 cells/well in 100 μL of growth medium (Dulbecco's Modified Eagle Medium, DMEM, supplemented with 10% foetal bovine serum 50 U/mL penicillin and 50 μg/mL streptomycin). After 24 h of incubation (in humidified atmosphere, 5% CO2 at 37 °C), the medium was discarded and replaced with 5 dilutions of the plant extracts in DMEM (at 240, 120, 60, 30, and 15 μg/mL) in quadruplicate. Cellular growth control was performed using medium with 0.2% DMSO. Then, after an incubation period of 48 h, the medium was discarded and replaced with DMEM containing 0.5 mg/mL MTT (3-( 4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide). After 1 h 30 of incubation at 37°C and 5% CO2, the water-insoluble formazan was dissolved in 100 μL DMSO and the absorbance was measured at 550 nm using a UV-spectrometer (Tecan, Spark 10M). The cytotoxicity of the crude extracts and standard (camptothecin: IC50: 0.9 ± 0.1 μg/mL) was determined by comparing the absorbance of treated cells with the absorbance of control cells cultured in 0.2% DMSO. Data were expressed as a percentage of inhibition calculated according to the formula (% Cell viability = Abs treated cells x 100 / Abs control cells) and analysed by linear regression.

Anti-HCV activity

We used HCV cell culture system (HCVcc) to perform the antiviral screening. A modified HCV strain (JFH1, genotype 2a) containing cell culture-adaptive mutations [START_REF] Delgrange | Robust production of infectious viral particles in Huh-7 cells by introducing mutations in hepatitis C virus structural proteins[END_REF][START_REF] Goueslain | Identification of GBF1 as a cellular factor Rrequired for Hepatitis C virus RNA replication[END_REF] was used. JFH1 was kindly provided by T. Wakita (National Institute of Infectious Diseases, Tokyo, Japan).

The viral stocks were produced in human hepatoma Huh-7 cells. For antiviral assay, Huh-7 cells, 6000/well, seeded in 96-well plate the day before infection were inoculated with HCVcc at a multiplicity of infection (MOI) of 0.8 during 2 h at 37°C in the presence of plant extracts at 25 μg/mL. Epigallocatechin-3-gallate (EGCG, Calbiochem) from green tea extract, an HCV entry inhibitor, was used as a control, inhibiting here 99.5% of HCV infection at 25 μg/ml [START_REF] Calland | -)-Epigallocatechin-3-gallate is a new inhibitor of hepatitis C virus entry[END_REF]. Then, the inoculum was removed and cells were overlaid with fresh medium containing plant extracts at 25 μg/mL for 28 h at 37°C. After 30 h, cells were fixed with ice-cold methanol and were subjected to immunofluorescent detection of E1 envelope protein as described [START_REF] Rouillé | Subcellular localization of hepatitis C virus structural proteins in a cell culture system that efficiently replicates the virus[END_REF]. Nuclei were stained with 1 μg/mL of DAPI (4′,6-diamidino-2-phenylindole), and infected cells were detected by immunofluorescent labeling of E1 envelope glycoprotein (A4 epitope). Confocal images were recorded using an automated confocal microscope IN Cell Analyzer 6000 (GE Healthcare Life Sciences) using a 10× objective with exposure parameters 405/450 nm and 561/610 nm. Six fields per well were recorded. Each image was then processed using the Colombus image analysis software (Perkin Elmer) [START_REF] Calland | Polyphenols Inhibit Hepatitis C Virus Entry by a New Mechanism of Action[END_REF]. The ratio of infected cells over total cells represents the infection rate. Infection rates in DMSO controls were expressed as 100%.

Results

Ethnobotanical study: local uses of medicinal plants in case of "hepatitis"

The ethnobotanical survey led to the collection and identification of 35 plant species providing 45 extracts. All the remedies were not specifically indicated in case of viral hepatitis infection but rather against hepatic injury and associated symptoms (Table 1). In such cases of infectious diseases with digestive symptoms, useful medicine against hepatitis does not necessarily have antiviral activity. Moreover, it is relevant to show how food and medicinal plant are interrelated. This ethnobotanical survey with related publications indicated that it is usual in Peruvian Amazon to find dietary and other restrictions with plants, such as sexual abstinence, or social seclusion [START_REF] Sanz-Biset | Plant use in the medicinal practices known as "strict diets" in Chazuta valley (Peruvian Amazon)[END_REF]. This dietary restriction called "dieta" in Spanish and "sasina" in lowland Quechua consists of stopping food like meat, fat, eggs, salt, sugar, spices, alcohol, until healing, and progressive reestablishment of normal diet with boiled plantains, manioc and fish. During this survey, restricted diets were mentioned for chronic or important diseases such as hepatitis and associated digestive affections. This practice also depended on plant species, preparation and administration route, or particular uses. Stronger diets may also be observed for "teaching plants", such as Aspidosperma excelsum, Hura crepitans, Jatropha curcas, Mansoa alliacea, Maytenus macrocarpa, Phyllanthus sp., Pouteria caimito. Indeed, these species are also used in apprenticeships of traditional medicine that need a ritual gathering. These particular and well-structured traditional medicinal practices are usually characterized by an important symbolic significance [START_REF] Jauregui | Plantas con madre". Plants that teach and guide in the shamanic initiation process in the East-Central Peruvian Amazon[END_REF].

In this context, a comparative study of local uses allowed an a posteriori classification of most of the plant remedies in broadly 3 interconnected categories (A, B, C):

-A: Preparations with low and accurate dosing for targeted therapy against aetiology of liver diseases. These remedies were usually characterized by a narrow therapeutic range, associated with a special food diet and strictly contraindicated in combination with "modern medication": Aspidosperma excelsum, Hura crepitans, Jatropha curcas, Mansoa alliacea, Maytenus macrocarpa, Phyllanthus sp., Pouteria caimito, Pseudelephantopus spiralis. Most of these species were considered as "teaching plants"as mentioned above.

-B: Preparations with flexible dosing usually indicated to alleviate symptoms of hepatitis and liver injuries such as jaundice, functional digestive disorders, inflammation, ascites...: Bixa orellana, Cajanus cajan, Carica papaya seed, Crescentia cujete, Curcuma longa, Desmodium adscendens, Eryngium foetidum, Gossypium barbadense, Malachra ruderalis, Physalis angulata, Senna reticulata, Tessaria integrifolia, Urera baccifera.

-C: Preparations used without particular dosing as substitutes for food or "functional food" in order to replace contraindicated unpalatable nutrients such as animal proteins and lipids, or to drain the body: Costus erythrocoryne, Gynerium sagittatum, Cecropia ficifolia, hearts of palms (Arecaceae such as Astrocarium spp., Oenocarpus bataua), preparations called "agua de tiempo" ("drunk at any time" e.g.

Scleria microcarpa, Carica papaya leaves, Persea americana).

It should be noted that a same species could belong to different categories depending on its preparation.

B

The bark decoction is drunk against viral infections such as HIV (once a day) but the treatment mustn't exceed one month, associated with strict restrictions (food diet a , sexual abstinence), without sun exposure (risk of blindness AP: aerial parts, B: stem bark, Br: branch, EP: entire plant, Fr: fruit (G: green), HP: heart of palm (interior part of the trunk), L: leaves, R: root., NC: not classified (plant adulteration). a Special diet: dietary restrictions without meat, fish, salt, spices, fat, sweet, alcohol, unpalatable foods. b " Agua de tiempo": which means "water at any time", can be drunk all day long.

Ethnomedical study: The Amerindian situation with respect to illness and hepatitis

Despite favourable weather conditions, the daily life reality of Amerindian people living in rainforests is often a perpetual fight for good health. In fact indigenous people suffer from many difficulties related to their particular environment, socioeconomic context, history, culture, or governmental disregard and social discrimination, which results in a reduction of their life expectancy [START_REF] Montenegro | Indigenous health in Latin America and the Caribbean[END_REF]. This high mortality depends on various risk factors whose control could improve the management of diseases such as hepatitis as we described below:

Physical and material factors:

-Location of population. Geographic and rural isolation of indigenous communities contribute to increasing diseases morbidity by different ways: low access to health facilities (in case of emergency such as malaria crisis, ante/post-natal care, infected ascites due to hepatitis, etc.), lack of safe drinking water, dangerous work (woodcutting, hunting, fishing), hostile environments (predators, animal bites, drowning, wounds causing tetanus, mosquito or insect bites causing infections such as malaria), and other rural lifestyles that can expose populations to higher health risks. Solutions should consist in the development of transport logistics (boats with motors and fuel dedicated to emergencies) and protection devices (lyophilized antiserums, mosquito nets, clothing and safety equipment such as gloves and rubber boots).

-Evictions from ancestral lands. The Amerindian way of life is directly linked to their natural resources and this equilibrium can be destabilised by the intervention of modern society through oil or gas industries, dam or road building, woodcutting, trafficking in protected species, and other projects leading to the deforestation and dispossession of indigenous territories. In the oil concessions 1AB/192, including a part of the Pastaza river basin, the surface area deforested or covered by crude oil spills was estimated at 10 538 ha [START_REF] Martínez | Impacts of petroleum activities for the Achuar people of the Peruvian Amazon: summary of existing evidence and research gaps[END_REF]. These environmental dislocations lead to brutal changes of daily life and disturb subsistence activities such as hunting, fishing, harvesting and collecting clean water from springs. It also weakens the indigenous cohesion and identity of the community. Moreover it deeply affects individual health with stress, diseases, hunger and poverty. It would seem important to moderate the external access to the rainforest and its exploitation.

-Material goods from modern society. Even though technology and new medications from the industrial world can be useful for indigenous communities, some imported goods remain deleterious for health e.g. fake or outdated medicines, strong spirit ("aguardiente", i.e. fermented juice of sugar cane), expired or "synthetic food" (freeze-dried milk), batteries and other harmful pollutants. Numerous cases of diseases due to the environmental impact of industries were reported in this area: acute cases of poisoning, cutaneous cancer, allergies, spontaneous abortions, malaria (due to the creation of large standing water basins leading to the proliferation of mosquitoes), leishmaniasis (because of proliferation of Phlebotominae in timber farms); this has contributed to increasing mortality in the neighbouring communities [START_REF] Martínez | Impacts of petroleum activities for the Achuar people of the Peruvian Amazon: summary of existing evidence and research gaps[END_REF][START_REF] San Sebastián | Exposures and cancer incidence near oil fields in the Amazon basin of Ecuador[END_REF], 2002). Environmental exposure and human health parameters studied in the communities along the Pastaza river revealed high and toxic concentrations of mercury (41% of inhabitants with hair mercury level > 10μg/kg, and 4% of fish were considered as toxic with [Hg] > 0.5 μg/g). Similar results were obtained for barium, lead and chlorides as consequences of the petroleum exploitations [START_REF] Webb | Deforestación, explotación petrolera y salud humana en la cuenca del río Pastaza: informe corto sobre el mercurio y los hidrocarburos aromáticos policíclicos[END_REF]. It should be pointed out that an average of 2 spills a year are officially reported in Pastaza river, and that the high level of heavy metal contamination of the soils suggested other unreported leaks [START_REF] Osinerg | Derrames reportados: 1998-2006: informes preliminares de derrame o fuga de petr óleo crudo o derivados, remitidos por Oxy-Per ú y Pluspetrol del Norte S[END_REF]. Protection of these areas from foreign intrusion, and shifting of policies in the direction of environmental sustainability, with reparation of the past damages and prevention of further destructions, should be considered as parts of a reasonable health policy for these areas (e.g. water borehole drilling and water treatment systems).

-Housing and living area. In the Amazonian communities, an indigenous family usually lives in the same house, in quite a limited area, and sometimes shares the same mosquito net during the night. This proximity can be reinforced during a travel, usually by boat, or a stay in town, and enhances the spread of contagious diseases. More resources could be given to local trainings on prevention in health and safety.

-Community infrastructure. Waste disposal (sludge, septic and solid waste), safeguard water resources, electricity for cold chains (permitting conservation of vaccines, venom antiserum, interferon against hepatitis), collective transport dedicated to emergency usually lacked in isolated areas, aggravating the impact of infectious diseases. Assistance for the development of these defective or nonexistent infrastructures is still lacking (only 1 motorised medical boat was available for 2-5 communities).

Socio-cultural factors:

-Children's schooling and health education. School or health and safety training are the initial steps for limiting the spread of illnesses, particularly for current contagious diseases such as syphilis, AIDS, hepatitis and tuberculosis. In remote villages or poor neighbourhoods, the language barrier and environmental hostility block the achievement of these pedagogic supports and contribute to worsening the health situation of these underprivileged areas. Intensive bilingual trainings at school on prevention in health would sustainably reduce the incidence of contagious diseases among the younger population.

-Cultural practices among Amerindian populations. Some particular cultural practices can enhance the likelihood of transmission of infectious diseases as hepatitis or exacerbate symptoms of illnesses, e.g. ways of feeding (without cutlery, irregular schedule and supply of food depending on weather conditions) or drinking (for instance "masato" is a usual Amazonian drink prepared from masticated manioc, usually mixed with water from the river, and distributed in the same cup to every visitor or members of a ceremony), delousing, sexual practices (sometimes polygamy), tattooing, piercing or lip perforations with unsterilized tools [START_REF] Coimbra | Hepatitis B epidemiology and cultural practices in Amerindian populations of amazonia: the tupi-mondé and the xavante from Brazil[END_REF]. This point highlights once again the need for bilingual trainings on prevention in health, with supplying of preventive goods like vitamin supplements, condoms, disinfectants etc.

-Cultural impact of modern society. Despite numerous projects for Amazonian conservation, and even thought traditional knowledge is cumulative and dynamic, the modern society contributes to accelerating the changing value, which implies a rapid loss of the "pre-existing know-how" and traditional therapies [START_REF] Heinrich | Ethnopharmacological field studies: a critical assessment of their conceptual basis and methods[END_REF]. Ethnographic surveys, especially those dedicated to ethnomedicine, ethnobotany, ethnoscience and other types of written testimonies or audio-video recordings are good ways to preserve traditional knowledge, especially when they are performed by the members of indigenous communities themselves.

-Employment or income. Indigenous citizens living near a city or an industrial establishment may have opportunities to get a paid work, but employment rates and incomes are usually lower for them than for unskilled workers [START_REF] Rakibul | Cultural and socio-economic factors in health, health services and prevention for indigenous people[END_REF]. This disparity is all the more detrimental to these new employees as they live far from their plentiful forest, leading to gradual poverty and health decline. Moreover, numerous cases of prostitution and sexual abuse are observed for indigenous women living near such establishments, with introduction of new diseases by the foreign workers, e.g. HIV and hepatitis [START_REF] Martínez | Impacts of petroleum activities for the Achuar people of the Peruvian Amazon: summary of existing evidence and research gaps[END_REF]. The compensation per indigenous employee should be normalized, avoiding any conflict of interest, and child prostitution or sexual abuse should be severely punished.

-Lack of data. Low access of health brigade and other assistance mandated for vaccination campaigns, disease eradication or prevention, epidemiologic survey, emphasised by cultural Amerindian specificities (e.g. poor written languages, absence of birth or death registrations, impossibility to gather all the members of a community) raise the difficulty of data compilation required for the improvement of health implementation plans. In this context, epidemiologic data on hepatitis are all the more incomplete as for this infectious disease the diagnosis assessment is logistically impossible and sick persons usually live in seclusion. New census campaigns aiming at completing national registrations and epidemiological data, with routine screening for viral diseases such as HIV and hepatitis, should be more widely undertaken.

Physiological and medical factors:

-Genetic determinism. Genetic and epigenetic heritage with poor diversity, due to the long-time isolation of these communities, makes these populations more sensitive to new diseases emerging from the modern society (e.g. viral or bacterial diseases like the flu or whooping cough were introduced by foreigners). Immunological studies exhibited the negative effects of homozygosity on health of Amerindians which are often infested with intestinal macroparasites. These chronical exposures lead to the overexpression of gene encoding for Ig,E, and results in a decrease of defence against other infections such as hepatitis [START_REF] Salzano | Parasitic load in South American tribal populations[END_REF]. Moreover, fieldwork data collection also showed that those isolated populations are more predisposed to develop side effects with some modern medication (intolerance or hyper-reactivity to vaccines, antibiotics, psychotropics), which can overcomplicate the cure of diseases [START_REF] Hurtado | The epidemiology of infectious diseases among South American Indians: a call for guidelines for ethical research[END_REF]. Epidemiological monitoring and research on indigenous susceptibility to drug and disease must be carefully considered and registered. Specific study of local iatrogenic effects and plant-drug interactions should require more attention from both researchers and medical staff.

-Discrepancy of healing concepts. Modern medicine is based on universal scientific practices adapted to large populations with standard ethical regulations, while traditional Amazonian medicines are influenced by various popular beliefs (here related to the animistic ontology, [START_REF] Descola | Beyond nature and culture[END_REF][START_REF] Luna | Vegetalismo: Shamanism among the Mixed-blood native people Population of the Peruvian Amazon[END_REF] and depend on both practitioner and patient culture [START_REF] Lévi-Strauss | L'efficacité symbolique[END_REF]. In the case of the traditional medicine of Quechua people from Loreto, different aetiological categories of illnesses may be distinguished such as: "Unkuy", due to natural causes (excess of water, wind, heat...); "Pahu", relating to supernatural origins such as spirits from the forest; and "Wiruti" that referred to an invisible dart or a smoke blown by a shaman for therapy and sorcery in case of human conflicts [START_REF] Roumy | Amazonian plants used by Peruvian Quechua and Mestizo people to treat malaria: evaluation of their activity[END_REF]. The traditional curer can also include some "modern" aetiological factors such as the inter-or intra-specific contagiousness of diseases. In this context, modern medication is usually locally underestimated or proscribed and considered as primarily preventative, whereas traditional medicines are "curative". For example, administration of tablets and other pharmaceutical forms are often contraindicated with traditional medications and considered as ineffective against the aetiology of the disease. Compliance with prescriptions and medical advice must be explained and supervised while always considering possible interactions with local medicine.

These complex interactions between different medical systems may sometimes create a mutually beneficial relationship but also generate medical incompatibilities leading to the loss of medication compliance and treatment efficacy, especially for chronic diseases such as viral hepatitis.

More specific factors affecting viral hepatitis:

In case of hepatitis B or C in Amazonian communities, other aggravating factors of morbidity are present such as: the need for health centres or medical devices for diagnosis (the existing ones are far away and expensive), the non-assessment of vaccination due to the household head willing or for administrative reason (undocumented patient), the local unawareness of transmission routes, the cost and conservation of medication or vaccines, the non-respect of nutritional-hygienic precautions, the material or cultural incompatibilities with condom protection, etc.

The clinical context is also a major determinant for the spread of disease. Healthy carriers may contaminate their relatives without wariness [START_REF] Alberti | Hepatitis C viraemia and liver disease in symptom-free individuals with anti-HCV[END_REF]. Patients with associated cutaneous infections are more likely to be contaminated by viral hepatitis (31.5% of inhabitants have dermatoses in Peruvian Amazon; [START_REF] Coimbra | Hepatitis B epidemiology and cultural practices in Amerindian populations of amazonia: the tupi-mondé and the xavante from Brazil[END_REF][START_REF] Gutierrez | Skin diseases in the Peruvian Amazonia[END_REF]. Clinical observations also exhibited that cumulated liver damages due to other diseases such as malaria and other parasitoses also exacerbated the hepatitis symptoms, and mutually, chronic viral hepatitis may be a risk factor for severe malaria (an overall prevalence of HBsAg of 23.8% have already been reported in Vietnamese patients hospitalized for malaria ; [START_REF] Barcus | Hepatitis B infection and severe Plasmodium falciparum malaria in Vietnamese adults[END_REF]. These factors and clinical data could be better controlled through bilingual trainings on transmission of viral infections in indigenous communities. For all the above reasons or aggravating factors, and despite casual medical supports, hepatitis is still one of the first causes of illness and mortality in Amazonia. Nonetheless, this analysis of indigenous situation will permit a better adjustment of health strategy in these communities and other tropical areas. It also justified the ethnopharmacological work dedicated to the collect of cultural data for their preservation and the improvement of local treatments.

Results of biological assays and interpretation.

In vitro assays revealed interesting activities (> 50% inhibition of HCV infection at 25 μg/mL) for 18 plant extracts corresponding to 15 species: Bixa Orellana (L), Euterpe precatoria (R), Scleria microcarpa (R and AP), Astrocaryum jauari (R), Phyllanthus stipulatus (R and AP), Oenocarpus bataua (Fr), Cecropia ficifolia (L), Pouteria caimito (L), Persea americana (L), Physalis angulata (APGFr), Chamaesyce thymifolia (R and AP), Mansoa alliacea (EP), Maytenus macrocarpa (B), Curcuma longa (R), Caesalpinia pulcherrima (L). The same assays showed that 6 other extracts had low activities (30% to 50% of HCV inhibition): Aspidosperma excelsum (B), Gossypium barbadense (Fl), Hura crepitans (B), Jatropha curcas (L), Malachra ruderalis (AP), Senna reticulata (Fl) (see Table 2; Fig. 2). Moreover, the remedies from category A (preparations with low and accurate dosing as targeted therapy against hepatitis) were moderately more active than plants from categories B or C (80% of activity, > 30% inhibition at 25 μg/mL, for category A, instead of 37.5% or 47.1% for categories B or C), which attested the previous classification assumed in the ethnobotanical study (Fig. 3) and corroborated biological activities described in previous study for "teaching plants" from category A [START_REF] Jauregui | Plantas con madre". Plants that teach and guide in the shamanic initiation process in the East-Central Peruvian Amazon[END_REF]. Huh-7 cell nuclei number analysis showed that only 4 of the most active plants were also toxic on Huh-7 cells: Persea americana (L), Physalis angulata (APGFr), Curcuma longa (R) and Aspidosperma excelsum (B), which demonstrated a potential toxicity of these species. Nevertheless, this narrow therapeutic range did not forbid the use of species like Curcuma longa or Physalis angulata which have specific toxicity against hepatoma cell lines (HepG2 or Huh-7) without effects on healthy human cells [START_REF] Salama | Hepatoprotective effect of ethanolic extract of Curcuma longa on thioacetamide induced liver cirrhosis in rats[END_REF]. Moreover, cytotoxic assays realised on HepG2 cells (see Table 2), did not reveal important growth inhibitions except for Curcuma longa (as previously cited) and Aspidosperma excelsum (a species carefully used for the antimalarial activity of its indole alkaloids, and characterized by a moderate anti-HCV activity).

Discussion

Phytochemical analysis

Bibliographical review exhibited less studies on anti-HCV plant properties than on anti-HBV. However, some publications related the antiviral activity of plant extracts and natural products extracted from plants on HCV in vitro [START_REF] Séron | Natural products and hepatitis C virus; natural antimicrobial agents[END_REF]. These compounds target the different steps of the viral infectious cycle, entry, replication, cell-to-cell spread and assembly. Our groups have recently identified dehydrojuncusol from Juncus maritimus as a new inhibitor of HCV replication [START_REF] Sahuc | Dehydrojuncusol, a natural phenanthrene compound extracted from Juncus maritimus is a new inhibitor of hepatitis C virus RNA replication[END_REF]. Interestingly, the target of the molecule is the viral NS5A protein, which is one of the main target of the drugs used in hepatitis C therapy. In this study, some similarities were observed between plant extract already described in literature for their anti-HBV activities, and plant extracts with anti-HCV capacity identified here (Phyllanthus spp, Euterpe precatoria, Curcuma longa are active against both hepatitis virus). A search in the literature indicated that phytochemical antiviral agents of some of the 15 most active plants have already been characterized but the majority of them are unknown, as described below:

Bixa orellana (leaves): No antiviral activity had been reported, but antimicrobial terpenes and phenolics compounds have already been identified from leaves without observed toxicity [START_REF] Stohs | Safety and efficacy of Bixa orellana (Achiote, Annatto) leaf extracts[END_REF]. Moreover, hepatoprotective activity had been characterized in rats with liver damage induced by CCl4 [START_REF] Ahsan | Hepatoprotective activity of methanol extract of some medicinal plants against carbon tetrachloride induced hepatotoxicity in albino rats[END_REF].

Euterpe precatoria (roots): Stem extract E. precatoria previously tested exerted a reduction in HBV replication at 100 mg/mL (measured by PCR, [START_REF] Quintero | Inhibition of hepatitis B virus and human immunodeficiency virus (HIV-1) replication by Warscewiczia coccinea (Vahl) Kl. (Rubiaceae) ethanol extract[END_REF]. Regarding the root of this species, antioxidant and hepatoprotective flavonoids (quercetin, catechin, epicatechin, rutin and astilbin) were isolated. Catechin, epicatechin and rutine did not show any activity against HCV, whereas quercetin activity against HBV has already been described (partial inhibition of HBsAg secretion at 0.2 mmol/mL, [START_REF] Galotta | Antioxidant and cytotoxic activities of 'açaí' (Euterpe precatoria Mart.)[END_REF][START_REF] Calland | -)-Epigallocatechin-3-gallate is a new inhibitor of hepatitis C virus entry[END_REF][START_REF] Yang | Cytotoxicity on HepG2 cells IC50 (µg/mL) ± SD Bixa Orellana (L) 6.5 ± 2.6 88.6 ± 6.7 196.5 ± 9.1 Euterpe precatoria (R) 6.6 ± 2.9 90.4 ± 13.5 163.6 ± 17.8 Scleria microcarpa (R) 7.7 ± 2.5 85.1 ± 4.8 > 250 Astrocaryum jauari (R) 7.7 ± 1.4 76.5 ± 4.1 87.4 ± 10.0 Phyllanthus stipulatus (R) 8.7 ± 2.9 85.2 ± 19.6 192.6 ± 10.3 Oenocarpus bataua (Fr) 9.6 ± 6.0 61.2 ± 13.1 109.2 ±[END_REF]. Therefore, the presence of quercetin could partially explain anti-HCV activity observed for the root of this species.

Scleria microcarpa and Astrocaryum jauari: No previous data on compounds and biological activity of these plants have been published.

Phyllanthus stipulatus (roots or aerial parts): Despite absence of previous study of this species the genus Phyllanthus is one of the most active botanical group against viral hepatitis all over the world. Lignans and gallic derivatives isolated from various species of Phyllanthus (P. amarus, P. multiflorus, P. tenellus, P. virgatus) were characterized by anti-HBV activities. Other previous study showed that Phyllanthus urinaria grown in different areas in China exhibited different anti-HBV activity, nonetheless this species is now commercially produced in China for clinical use [START_REF] Huang | Screening of 25 compounds isolated from Phyllanthus species for anti-human hepatitis B virus in vitro[END_REF]. Additionally, liquid chromatography coupled to mass spectrometry (LC-IT-MS n ) of P. stipulatus showed presence of phenolic compounds as quercetin and gallic acid derivatives potentially involved in anti-HCV and antioxidant activities [START_REF] Da Fontoura Sprenger | Characterization of four Phyllanthus species using liquid chromatography coupled to tandem mass spectrometry[END_REF].

Oenocarpus bataua (fruits): Antioxidant phenolic compounds have been identified using UPLC-API-IT-MS n (stilbenes, hydroxycinnamic acid, procyanidin and anthocyanin derivatives) but anti-HCV activity has never been described before [START_REF] Rezaire | Amazonian palm Oenocarpus bataua ("patawa"): Chemical and biological antioxidant activity -Phytochemical composition[END_REF].

Cecropia ficifolia (leaves): This species has never been studied for its chemical compounds or biological activity but other species of the genus Cecropia (C. palmata and C. obtusa leaves) have been described to contain anti-HCV triterpenic compounds as oleanolic acid [START_REF] Schmidt | Some triterpenic compounds in extracts of Cecropia and Bauhinia species for different sampling years[END_REF][START_REF] Kong | Oleanolic acid and ursolic acid: Novel hepatitis C virus antivirals that inhibit NS5B activity[END_REF].

Pouteria caimito (leaves): Antioxydant proanthocyanidin and anti-inflammatory terpenes (spinasterol) have already been identified in leave extract, but no antiviral activity has been mentioned before [START_REF] França | Potential radical-scavenging activity of Pouteria caimito leaves extracts[END_REF].

Persea americana (leaves): Antiviral activity of the infusion of P. americana leaves against herpes simplex virus (HSV-1), adenovirus (AD3…), and human immunodeficiency virus 1 (HIV-1) have been previously reported, and may be induced by synergistic effect between flavonoids, chlorogenic acid, and other unidentified compounds [START_REF] De Almeida | Flavonol monoglycosides isolated from the antiviral fractions of Persea americana (Lauraceae) leaf infusion[END_REF][START_REF] Wigg | In-vitro virucidal and virustatic anti HIV-1 effects of extracts from Persea Americana Mill, (Avocado) leaves[END_REF]. Nevertheless, the leave extract toxicity observed in this study was also previously demonstrated on livestock and mice (on myocardium or mammary gland, at 60-100 mg/kg and caused by the fatty acid "persin"), prohibiting the use of this plant by pregnant/nursing woman and patients with heart weakness [START_REF] Oelrichs | Isolation and identification of a compound from avocado (Persea americana) leaves which causes necrosis of the acinar epithelium of the lactating mammary gland and the myocardium[END_REF].

Physalis angulata (aerial parts with green fruits): No previous study reported antiviral activity of this plant species. Even if cytotoxicity was observed in this screening on HepG2 cells, previous data showed that cytotoxic effect did not affect normal mouse liver cells. Furthermore, hydroethanolic extract has been shown to possess potent anti-inflammatory and anti-hepatoma activities due to steroid compounds as physalins, and flavonol glycosides such as myricetin derivatives [START_REF] Wu | Antihepatoma activity of Physalis angulata and P. peruviana extracts and their effects on apoptosis in human Hep G2 cells[END_REF][START_REF] Chiang | Antitumor agent, physalin F from Physalis angulata L[END_REF][START_REF] Ismail | A novel cytotoxic flavonoid glycoside from Physalis angulata[END_REF].

Chamaesyce thymifolia (roots or aerial parts): Moderate antiviral activity on HSV-1 and bovine viral diarrhea virus (BVDV) with low selectivity has been described for phenolic compounds (quercetin and galloyl esters of glucose derivatives) isolated from the aerial parts, but antiviral activity against hepatic viruses has never been studied [START_REF] Amaral | Antiviral investigation on the flavonoids of Chamaesyce thymifolia[END_REF][START_REF] Lin | Antioxidant and antiviral activities of Euphorbia thymifolia L[END_REF]. Even if anti-inflammatory and hepatoprotective activities have been characterized for this species, dermatotoxicity (due to phorbol derivatives) and low selectivity observed for this plant do not allow chronic or important use of this remedy [START_REF] Mali | A review on phyto-pharmacological potentials of Euphorbia thymifolia L[END_REF][START_REF] Muthumani | Phytopharmacological activities of Euphorbia thymifolia Linn[END_REF].

Mansoa alliacea (entire plant): Even if this well-known traditional remedy is rich in organosulfur compounds with anti-inflammatory and anti-infectious properties, its organoleptic properties and its cytotoxic naphthoquinones may limit the recommended dosage [START_REF] Itokawa | Cytotoxic naphthoquinones from Mansoa alliacea[END_REF][START_REF] Pires | An overview about the chemical composition and biological activity of medicinal species found in the Brazilian Amazon[END_REF][START_REF] Towne | Effect of Mansoa alliacea (Bignonaceae) leaf extract on embryonic and tumorigenic mouse cell lines[END_REF].

Maytenus macrocarpa (bark): Terpenic derivatives from the leaves (di and triterpens) exhibited marginal cytotoxic activity and antiviral activity against HIV (triterpenonic acids), but no antiviral activity against hepatic viruses has already been published [START_REF] Chavez | Sesquiterpene polyol esters from the leaves of Maytenus macrocarpa[END_REF][START_REF] Piacente | Triterpenes from Maytenus macrocarpa and evaluation of their anti-HIV activity[END_REF].

Curcuma longa (roots): The polyphenolic compound "curcumin" has been shown to inhibit HCV entry and replication, and HBV replication, and is considered to be a potentially important chemo-preventive agent against a variety of cancers, including liver cancer, without observed toxicity [START_REF] Kim | Curcumin inhibits hepatitis C virus replication via suppressing the Akt-SREBP-1 pathway[END_REF][START_REF] Salama | Hepatoprotective effect of ethanolic extract of Curcuma longa on thioacetamide induced liver cirrhosis in rats[END_REF]. This study and previous data demonstrated that Curcuma longa could be used as a complementary therapy for HCV, while considering low oral bioavailability of curcumin, it should be administered with piperin (from Pieraceae or other botanical families; [START_REF] Shoba | Influence of piperine on the pharmacokinetics of curcumin in animals and human volunteers[END_REF].

Caesalpinia pulcherrima (leaves): Quercetin flavonoids and isoflavons were responsible of antiinflammatory and antiviral activities (against HSV or adenovirus), but no previous data were recorded on hepatic viruses [START_REF] Akram | Antiviral potential of medicinal plants against HIV, HSV, influenza, hepatitis, and coxsackievirus: a systematic review[END_REF][START_REF] Chiang | In vitro antiviral activities of Caesalpinia pulcherrima and its related flavonoids[END_REF]. Nonetheless, a slight toxicity on brine shrimp had been reported (26% mortality at 1000 µg mL -1 ), that could limit the recommended dosage for pregnant women or young children [START_REF] Chanda | Brine shrimp cytotoxicity of Caesalpinia pulcherrima aerial parts, antimicrobial activity and characterisation of isolated active fractions[END_REF][START_REF] Rao | Anti-inflammatory activities of flavonoids isolated from Caesalpinia pulcherrima[END_REF].

The drug extracts characterized by lower anti-HCV activity than species mentioned above may have other interests against hepatitis. For instance, they may be efficient against other hepatitis viruses (non-evaluated in this study) or may reduce liver degeneration and fibrosis, thereby contributing to alleviate hepatitis disease as observed for the biological activities of species cited below: -Anti-oxidant and/or anti-inflammatory properties: Crescentia cujetes [START_REF] Parvin | Evaluation of in vitro antiinflammatory and antibacterial potential of Crescentia cujete leaves and stem bark[END_REF], Eryngium foetidum [START_REF] Paul | Eryngium foetidum L.: A review[END_REF], Jatropha curcas Leaves [START_REF] Thomas | Therapeutic biology of Jatropha curcas: A Mini Review[END_REF], Senna reticulata [START_REF] Navarro | Proanthocyanidin characterization, antioxidant and cytotoxic activities of three plants commonly used in traditional medicine in Costa Rica: Petiveria alliaceae L., Phyllanthus niruri L. and Senna reticulata Willd[END_REF], Tessaria integrifolia [START_REF] Peluso | Studies on the inhibitory effects of caffeoylquinic acids on monocyte migration and superoxide ion production[END_REF], Urera baccifera [START_REF] Gindri | Genotoxic evaluation, secondary metabolites and antioxidant capacity of leaves and roots of Urera baccifera Gaudich (Urticaceae)[END_REF]. -Hepatoprotective activities: Desmodium adscendens [START_REF] François | Safety of Desmodium adscendens extract on hepatocytes and renal cells. Protective effect against oxidative stress[END_REF], Cajanaus cajan [START_REF] Ahsan | Hepatoprotective activity of methanol extract of some medicinal plants against carbon tetrachloride induced hepatotoxicity in albino rats[END_REF], Carica papaya (leaf and seed: [START_REF] Pandit | Ameliorative effect of leaves of Carica papaya in ethanol and antitubercular drug induced hepatotoxicity[END_REF][START_REF] Adeneye | The aqueous seed extract of Carica papaya Linn. prevents carbon tetrachloride induced hepatotoxicity in rats[END_REF]. -And other pathway as fighting hepatitis symptoms (diuretic against ascites, choleretic against icterus, digestive …) or improving bioavailability of antiviral remedies.

Nonetheless, some of the selected species have previous toxicological reports that could contraindicate their dispensation for chronic use in case of pregnancy or childhood: Carica papaya: Seeds extract caused concentration-dependent tocolysis of uterine strips rats [START_REF] Adebiyi | Tocolytic and toxic activity of papaya seed extract on isolated rat uterus[END_REF].

Aspidosperma excelsum: Toxicity of the bark methanolic and dichloro-methane extracts were observed on brine shrimp (LC50: 764 and 85 µg/mL, [START_REF] Desmarchelier | Studies on the cytotoxicity, antimicrobial and DNA-binding activities of plants used by the Ese'ejas[END_REF].

Gynerium sagittatum: The high content of isoflavonoids makes this plant root an important source of phytoestrogen that could contraindicated the chronic use of the plant in case of pregnancy [START_REF] Benavides | Flavonoids and isoflavonoids from Gynerium sagittatum[END_REF]. Jatropha curcas: Leaf extract exerted cardiovascular effects (beta-blockers) on guinea pigs. Moreover, the presence of the toxin toxalbumin (curcin) may contraindicate a chronic use of this plant by pregnant women or young children [START_REF] Thomas | Therapeutic biology of Jatropha curcas: A Mini Review[END_REF]. Pseudelephantopus spiralis: An important cytotoxicity of leaf extract has already been described on VERO cells and macrophage (CC50 :1-5 µg/mL) and may contraindicate its chronic use [START_REF] Girardi | Evaluation of antiplasmodial and antileishmanial activities of herbal medicine Pseudelephantopus spiralis (Less.) Cronquist and isolated hirsutinolide-type sesquiterpenoids[END_REF]. Socratea exorrhiza: The leaf extract cytotoxicity evaluated in this study on HepG2 cells (IC50: 39.3 ± 18.3 µg/mL) highlighted potential side effects of this drug in chronic use.

Conclusion

This study exposed the results of a bioprospecting leading to the collect and identification of 35 plants with assessment of their antiviral or cytotoxic activities, and may contribute, with the ethnomedical analysis of indigenous situation, to a better adjustment of the health strategy against hepatitis in Peruvian Amazon. 45 plant extracts were tested for antiviral activity against HCV with counting of cellular death in case of toxicity and cytotoxicity evaluation in HepG2 cells. In vitro assay revealed interesting activity of 18 extracts (> 50% infection inhibition at 25 µg/mL) with a selectivity index > 2 for 15 of them. Lower antiviral activities were also observed for 6 other drug extracts (30% to 50% infection inhibition at 25 µg/mL).

Result analysis showed an interesting global level of activity (60% of plant extract inhibited more than 30% of HCV virus at 25 µg/mL). This supported the traditional medicinal uses of these plants and highlighted the relevance of the ethnobotanical approach. Moreover, drugs from the category A were more active against HCV than plants from categories B or C, that reinforced the tripartite drug classification assumed during the survey, and permitted to justify the cautious but efficient use of these plants against viral hepatitis. Nonetheless, drugs from other categories (B or C) may also occasionally have anti-HCV activities and are still helpful to prevent collateral effects of hepatitis. On the other hand, bibliographical report and cytotoxicity evaluations permitted to refine the therapeutic indications regarding to patient's vulnerability (in case of pregnancy or childhood), specially for Socratea exorrhiza leaves and Gynerium sagittatum aerial parts, which were usually indicated for daily use (category C) whereas potential toxicity had been characterized. The bibliographic study of the 15 most active plants (> 50% inhibition at 25 µg/mL) also indicated that only 2 of them have already been studied for antiviral activity against hepatitis (Curcuma longa and Socratea exorrhiza), suggesting new avenues to be followed for the 13 other species. More generally, these results will permit to improve the medical administration of the different plant species regarding their traditional uses and considering their antiviral or cytotoxic activities, with a better understanding of their preparation and posology in case of acute hepatitis (punctual administration), liver dysfunctions (regular treatment), or incorporated in a diary food diet against digestive troubles.

Fig. 1 :

 1 Fig. 1: Maps of the North of Peru (Loreto) with studied areas.

Fig. 1 :

 1 Fig. 1: Maps of the North of Peru (Loreto) with studied areas.

Fig. 2 :Fig

 2 Fig. 2: Antiviral activity of plant extracts (HCV inhibition > 30% at 25 µg/mL) and cytotoxicity (% of viability at 25 µg/mL)

Acknowledgements

Members and contributors of the scientific humanitarian association ARPIA (Association for Research on Indigenous Amazonian Pharmacopeia); F.M. Lambert (Commission du Développement durable et de l'Aménagement du territoire); CPER Alibiotech; C. Mori and C.

Güivinsinti; Luis and Aurelio Chino (presidents of Indigenous Quechua Federations), the members of Quechua community "Sabalo Yacu" who contributed to the building of a traditional medical centre in the Alto Pastaza.

Conflict of interest

The authors declare that they have no conflict of interest.

Supplementary data