Serena Ivaldi
email: serena.ivaldi@inria.fr

Pauline Maurice
email: pauline.maurice@loria.fr

Waldez Gomes

Jean Theurel
email: jean.theurel@inrs.fr

Liên Wioland

Jean-Jacques Atain-Kouadio
email: jean-jacques.atain-kouadio@inrs.fr

Laurent Claudon
email: laurent.claudon@inrs.fr

Hind Hani
email: hind.hani@univ-lorraine.fr

Antoine Kimmoun
email: a.kimmoun@chru-nancy.fr

Jean-Marc Sellal
email: jm.sellal@chru-nancy.fr

Lien Wioland
email: lien.wioland@inrs.fr

Bruno Levy
email: b.levy@chru-nancy.fr

Jean Paysant
email: jean.paysant@univ-lorraine.fr

Serguei Malikov
email: s.malikov@chru-nancy.fr

Bruno Chenuel
email: b.chenuel@chru-nancy.fr

Nicla Settembre
email: n.settembre@chru-nancy.fr

Using exoskeletons to assist medical staff during prone positioning of mechanically ventilated COVID-19 patients: a pilot study

Keywords: Exoskeletons, Prone Positioning, COVID-19, Human Factors, DHM

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Introduction

The COVID-19 pandemic has stressed healthcare systems worldwide as never before, with significant consequences for clinical management, including rationing of care, and facing a limitation of capacity and resources of Intensive Care Units (ICU) to safely maintain a high number of patients on mechanical ventilation during the surge. Prone positioning (PP), i.e. when a patient is repositioned from a supine position (i.e., lying on his/her back) to lie on a prone position (i.e., on his/her front side), is known to improve oxygenation and ventilatory mechanics in Acute Respiratory Distress Syndrome (ARDS) patients who require mechanical ventilatory support [START_REF] Guérin | Prone positioning in severe acute respiratory distress syndrome[END_REF]. PP is therefore largely used during the COVID-19 pandemic. For instance, in the Hospital of Nancy, the ICU performed 116 PP maneuvers in the first 10 days of the outbreak, which is equivalent to the number of maneuvers they usually perform in a whole year. Although turning a patient into the prone position is not an invasive procedure, it is complex and has Using exoskeletons to assist medical staff during prone positioning of mechanically ventilated COVID-19 patients: a pilot study many potential complications requiring adequate and well-trained healthcare staff (Fig. 1). It is also an exhausting and very timeconsuming task. Namely, PP procedure requires the medical staff to remain with their torso bent forward for several minutes, thus causing load and potential injuries in the low back [START_REF] Hoogendoorn | Flexion and rotation of the trunk and lifting at work are risk factors for low back pain: results of a prospective cohort study[END_REF]. In addition, obesity-related complications have been identified as risk factors of severe COVID-19 [START_REF] Caussy | Prevalence of obesity among adult inpatients with COVID-19 in France[END_REF], and patients weighting up to 150kg are common in ICUs. Obesity makes it even more difficult and physically demanding for caregivers to manipulate the patient's body when he/she is curarized.

Fig. 1. The PP maneuver executed on a patient simulator (a manikin) at the Simulation Center of the University Hospital of Nancy (France). Although small differences in the practice of PP can happen from one hospital to another, the maneuver is substantially standardized (video tutorial: https://www.youtube.com/watch?v=X-qyeN3e8iU). A: A doctor positioned behind the head of the patient secure the head to avoid extubation and coordinate the whole procedure. Four teammates are distributed on both sides of the patient to reposition and turn the patient. B: The 4 teammates lift the patient and position him/her on one side of the bed. C: The 2 teammates on the right place a clean bed linen. D: The 2 teammates on the left pivot the patient temporarily on the side using the old linen. E: The patient is rotated toward vent until in prone position, lying on his/her abdomen. F: The 4 teammates lift the patient to position him/her at the center of the bed and add pillow underneath him/her.

Musculoskeletal injuries and back pain generated by the repetition of strenuous tasks are well-known in industrial scenarios [START_REF] Hoogendoorn | Flexion and rotation of the trunk and lifting at work are risk factors for low back pain: results of a prospective cohort study[END_REF], and many back-support systems have been proposed to alleviate the problem. Given the similarity of the postures, robotics assistance used in industry might also be useful to assist caregivers. In the specific case of PP maneuver, collaborative robots or mobile manipulators are not suitable, mainly due to limited robot payload and lack of available space in the ICU. Motorized beds that can help with manual repositioning of patients (e.g., Hospidex toto with inflatable air cells and Vendlet V5S with motorized bars) do exist, but they cannot fully replace the work of the caregivers and require substantial time and financial resources to be put in place. Conversely, occupational exoskeletons [START_REF] De Looze | Exoskeletons for industrial application and their potential effects on physical work load[END_REF] appear as less invasive for the current practice, easy to set up, cheap, and compatible with the caregivers' work in the ICU. Exoskeletons are wearable devices generally aimed at supporting physical tasks by generating appropriate force on one or multiple human joints. Recently, there has been increasing interest in employing exoskeletons for workplace ergonomics, to reduce physical workload [START_REF] De Looze | Exoskeletons for industrial application and their potential effects on physical work load[END_REF] and risks of developing musculoskeletal disorders [START_REF] Theurel | Occupational Exoskeletons: Overview of Their Benefits and Limitations in Preventing Work-Related Musculoskeletal Disorders[END_REF]. Exoskeletons can be active (motorized) or passive, in which case mechanical elements such as springs store and restore energy, transferring the load from one body part to another. Previous studies generally agree on the efficiency of passive back-support exoskeletons (designed to assist with the back and hip muscles) to reduce lumbar muscular activity and perceived exertion/discomfort, particularly during operations involving trunk flexion/extension in the sagittal plane [START_REF] Theurel | Occupational Exoskeletons: Overview of Their Benefits and Limitations in Preventing Work-Related Musculoskeletal Disorders[END_REF]. However, the magnitudes of the reduction observed in back muscle activity when using those systems differ substantially from one study to another [START_REF] Alemi | Effects of Two Passive Back-Support Exoskeletons on Muscle Activity, Energy Expenditure, and Subjective Assessments During Repetitive Lifting[END_REF][START_REF] Frost | Plad (personal lift assistive device) stiffness affects the lumbar flexion/extension moment and the posterior chain emg during symmetrical lifting tasks[END_REF][START_REF] Graham | Effectiveness of an on-body lifting aid at reducing low back physical demands during an automotive assembly task: Assessment of EMG response and user acceptability[END_REF][START_REF] Koopman | Effects of a passive exoskeleton on the mechanical loading of the low back in static holding tasks[END_REF], likely because of considerable disparities in protocols (e.g. populations, tasks, postures, exoskeleton designs). This results in confusion regarding the magnitude of assistance to be expected when using a back-support exoskeleton. Besides, while occupational exoskeletons are deployed in the industrial sector [START_REF] De Looze | Exoskeletons for industrial application and their potential effects on physical work load[END_REF], there are very few reports of their use in the

2

Exploratory Study to Select the Most Suitable Exoskeleton at the Hospital Simulation Center

Preliminary visual video analysis of PP maneuvers revealed that the medical staff can assume postures with forward trunk bending up to 45 degrees with raised arms straight forward, exert traction to the trunk bending up to 20-30 degrees, and hold prolonged static postures with the trunk bent forward up to 60 degrees. To confirm the visual observation, we recorded the whole-body kinematics of one physician (M, 35 years old, 175cm) performing the PP maneuver, using the Xsens MVN inertial motion capture system (Xsens, Enschede, the Netherlands, capture rate: 240Hz). Postural analysis with the AnyBody biomechanical simulation software (AnyBody Technology, Aalborg, Denmark) revealed that when operating at the side of the patient, the physician spends approximately 40% of the maneuver time with the torso bent more than 20 degrees forward; when operating behind the head of the patient to secure the head and avoid extubation, the physician maintains a static posture with important flexion of the trunk for several minutes. The precise angle of flexion, in this case, depends on the height of the patient's bed, his/her location relative to the bed, and the doctor's height. Even when not associated with load manipulation, such postures cause mechanical load in the lower back [START_REF] Hoogendoorn | Flexion and rotation of the trunk and lifting at work are risk factors for low back pain: results of a prospective cohort study[END_REF]. For such postures, exoskeletons for lumbar support can help, but to be used in ICU they have to match many usability constraints, such as being lightweight and unburdensome. Based on this analysis, and with the urgency of the situation in mind, we identified four commercial exoskeletons that were already available in our teams and that could meet the requirements: Corfor (Corfor, France), Laevo v1 (Laevo, Netherlands), BackX (SuitX, USA), and CrayX (German Bionics, Germany). Corfor is a passive soft exoskeleton (also known as exosuit), Laevo and BackX are passive rigid exoskeletons based on springs, while CrayX is an active exoskeleton, employing electrical actuators.

Methods -Five experienced PPT volunteers performed 11 PP maneuvers, consisting of Prone to Supine (PS) and Supine to Prone (SP) gestures, with a 100kg manikin. All participants filled a questionnaire to retrieve human factors (Questionnaire A, Supplementary Materials). Two of the participants (M, 30 and 35 years old) alternatively tried all 4 exoskeletons at the manikin's side. After testing each exoskeleton, these 2 subjects answered an evaluation and acceptance questionnaire (Questionnaire B, Supplementary Material) adapted from [START_REF] Wioland | Processus d'acceptabilité et d'acceptation des exosquelettes: évaluation par questionnaires[END_REF], consisting of 19 items on a 5-point Likert scale with a neutral option, to evaluate the perceived effort, safety, comfort, efficacy, installation, intention to use. They also reported on their perceived efforts using an effort evaluation questionnaire (Questionnaire C, Supplementary Material) and on their experience in a semi-directed interview conducted by one experimenter. To analyze the kinematics and dynamics of the maneuver, we replayed the participant's wholebody motion recorded with the Xsens motion capture system with a Digital Human Model (DHM) using the Dart physics engine.

The DHM has 43 degrees of freedom and it is scaled based on the participant's height and mass using average anthropometric coefficients. We used a hierarchical velocity quadratic programming (QP) controller based on the OpenSoT library [START_REF] Penco | Learning Robust Task Priorities and Gains for Control of Redundant Robots[END_REF] to retarget the participant's upper-body motion directly in the Cartesian space (the lower-body was mainly fixed during the PP maneuvers).

In the absence of physiological measures, we used the DHM computed joint torques as a surrogate measure of the efforts performed by the participants during the experiment to investigate the effect of the exoskeleton. We used the DHM L5/S1 flexion/extension joint torques estimated with the dynamic simulation to compare the lumbar effort exerted by the participant with and without the exoskeleton. When no exoskeleton is worn, the joint torque exerted by the human is directly retrieved from the simulation. However, when the participant is equipped with the exoskeleton, the net torque exerted at the L5/S1 joint to counter the dynamics and gravity effects on the upper-body is a sum of the human-generated torque and of the exoskeleton assistive torque: 𝜏 "#$% = 𝜏 '()*+ +𝜏 ,-. . The net torque is retrieved from the DHM simulation, but in order to estimate the human torque, the assistive torque 𝜏 ,-. provided by each exoskeleton is needed. To compute this torque, one needs the details about the mechatronics design of the platform. Since the Laevo was unanimously perceived as the most suitable exoskeleton to help in PP maneuvers, we computed 𝜏 ,-. for the Laevo only, using the Laevo empirical calibration curve published by Koopman et al. in [START_REF] Koopman | Effects of a passive exoskeleton on the mechanical loading of the low back in static holding tasks[END_REF] and the maximum torque (40 Nm) reported in the Laevo user manual. The dynamics analysis was performed with a simplified simulation, which has limitations. The effects of the Laevo weight (~2.5kg) and of external loads were ignored in the torque estimation. That is, we ignored the efforts associated with manipulating the manikin, and only focused on the postural efforts. This simplification matters only at the lateral position, since the external load at the head position was only associated with maintaining the endotracheal tube, and was not significant.

Results -In comparison with the non-assisted situation, both participants perceived a reduction of physical effort for all exoskeletons except the CORFOR. Table 1 reports the questionnaire's scores (mean±stdev) for each construct. All the exoskeletons scored positively in terms of perceived safety and comfort, and the participants did not notice a change in their performance, positive or negative, while using the exoskeleton. Laevo was the easiest to install and had the highest and only positive score in the intention to use. Both participants reported that CrayX was too cumbersome to wear in an ICU, while the mechanical design of BackX was unpleasantly hindering several arm movements of the PP maneuver. Both were restricting the range of motion for some gestures. CORFOR was not helpful. Conversely, they were satisfied with Laevo in terms of perceived assistance during bent postures, easiness of equipment, and freedom of movements. Importantly, they mentioned that Laevo was not modifying their movements during the PP maneuver, which was confirmed by the analysis of the kinematic data [START_REF] Settembre | The use of exoskeletons to help with prone positioning in the intensive care unit during COVID-19[END_REF]. Kinematic analysis -We first verified whether the motion of the L5/S1 joint in the sagittal plane (low-back flexion/extension motion) was affected by the use of any of the 4 exoskeletons. We manually segmented each trial into 2 segments: Prone to Supine (PS) and Supine to Prone (SP) positioning. We compared the low-back flexion angle for the PS and SP, for all 4 exoskeletons and without exoskeleton: the joint angle profiles are overall similar for all conditions; their minimum and maximum values are similar (typical profiles in Supplementary Material). The median value of the back-flexion angle of the participant across one trial did not vary significantly from one condition to another (Fig. 3). Minor variations from one condition to another can be explained by small differences in the manikin's position on the bed and the overall maneuver performed by the team. These results suggest that the range of motion of the L5/S1 flexion/extension joint during the PP maneuver was not affected by the use of any of the exoskeletons.

Dynamic analysis -We estimated the human L5/S1 flexion torque during the PP maneuver, both with and without the Laevo (Fig. 4). When using Laevo for the SP and PS maneuvers, the low back torque medians were reduced by 11.3% and 13.0% respectively. Those results, though limited to one participant, suggest that wearing the Laevo may reduce the human low-back torque during PP maneuvers, which agrees with the subjective report of the participants. This result also agrees with the 15% reduction of the L5/S1 moment observed in [START_REF] Koopman | Effects of a passive exoskeleton on the mechanical loading of the low back in static holding tasks[END_REF] for a similar static forward bending task.

Quantification of the LAEVO's assistance at the hospital simulation center

To complement the preliminary results described in the previous section, we performed a subsequent experiment to probe into the physiological effects of the Laevo during PP maneuvers. For sanitary reasons, this experiment was also conducted at the Hospital Simulation Center.

Methods -

The same 2 volunteers were equipped with an ECG sensor (Delsys Trigno ECG Biofeedback, 2 channels, bandwidth: 30Hz, sampling rate 4370 sa/sec with onboard Butterworth bandpass filter 40/80 dB/Dec) and 12 surface EMG sensors (Delsys Trigno, EMG sampling rate 4370 sa/sec). Surface electrodes were positioned on the skin according to Seniam protocol recommendations after abrasion and cleaning with alcohol. The muscle activity was recorded bilaterally in erector spinae longissimus (ESL), erector spinae iliocostalis (ESI), trapezius ascendens (TA), biceps femoris long head (BF); and laterally on the right side for rectus abdominis at T10 level (RA), rectus femoris (RF), gluteus maximus (GM) and tibialis anterior (TAL). The participants performed the PP maneuver without and with the Laevo exoskeleton, positioned successively at the head and at the side of the manikin. The physiological signals were also recorded during a control condition, where the subject was standing still and silent. For all the conditions, we recorded a single repetition lasting 4 minutes.

Results -Heart rate:

The instantaneous heart rate was computed offline by the Delsys EMGWorks software with its proprietary Template Matching method. Fig. 6 compares the instantaneous heart rate across conditions for the first participant. Table 2 summarizes the median heart rate value for both participants. Overall, we did not observe any significant change in the heart rate when using the exoskeleton. For participant 1, we observed a slight decrease in the median heart rate value in both positions (head: -3.66%; side: -6.07%), whereas for participant 2 the median heart rate either increased (head: +4.85%) or decreased (side: -6.61%).

It must be noted that both participants are confirmed athletes, which explains their low basal heart rates. The weak differences in heart rate do not indicate a particular physiological gain when using the Laevo for such short tasks (4 minutes). For longer tasks and/or more repetitions of PP maneuvers results may differ, and a more thorough evaluation is needed to confirm. EMG: The raw EMG signal was rectified and filtered through an RMS filter with a 100ms window, then by a 4th order Butterworth low pass filter with a 10Hz cut-off frequency. Table 3 displays the change in the RMS value of the EMG signal when using the Laevo, compared to the baseline with no exoskeleton. We observed a general decrease of the activity of the back-extensor muscles (except ESI which was not affected and the head condition for P2) and the hip extensor muscles, when using the Laevo, probably depending on the task, the position in the team and the individual characteristics. Conversely, the activity of the antagonist trunk and leg muscles and of the lower-leg muscles remained similar when using the Laevo. Although our results are strongly limited by the small number of participants and the absence of repetitions of the task, they suggest a promising physiological gain brought by the Laevo. This gain is coherent with the subjective evaluation retrieved in the questionnaires, and endorsed the use in the ICU. Furthermore, those results are in line with the changes in muscle activity observed in other studies where the Laevo was tested in similar static forward bending tasks [START_REF] Koopman | Effects of a passive exoskeleton on the mechanical loading of the low back in static holding tasks[END_REF][START_REF] Bosch | The effects of a passive exoskeleton on muscle activity, discomfort and endurance time in forward bending work[END_REF].

Implementation in Real-life Conditions in the ICU of the University Hospital of Nancy

Building upon these promising pilot results, and given the urgency associated with the COVID-19 crisis, we proceeded to test in realistic conditions to demonstrate the feasibility of using Laevo in a COVID-19 ICU situation.

Methods -

The same 2 participants were each equipped with a Laevo complying with the drastic hygiene rules of the ICU during the outbreak. Each participant wore the Laevo over a single-use scrub suit and underneath the personal protective equipment (long sleeve gown) covering the entire body to reduce the risk of contamination. That way the exoskeleton was not in contact with the patient nor with the contaminated environment. During a typical 3-hour shift, the participants performed 10 PP maneuvers on 10 ICU patients; each participant was positioned 3 times at the head and 7 times at the patient's side, respectively. After each maneuver, the participants reported their perceived effort using a Borg-CR10 scale (questionnaire D part 1, Supplementary Material). Cardiac activity was monitored with a Holter-ECG during the whole shift in the ICU. At the end of the shift, they reported on the use of the exoskeleton (questionnaire D part 2, Supplementary Material), then filled the extended version (22items) of the exoskeleton evaluation questionnaire (questionnaire B, Supplementary Material), and reported on their experience in an interview. Concomitantly, the PPT colleagues filled a questionnaire to report on their experience working alongside people equipped with exoskeletons (questionnaire E, Supplementary Material).

Results -Both physicians perceived a physical relief in the low back during bent postures, particularly when working at the patient's head, and indicated an intention to adopt such technology after the pilot study. ECG data analysis was inconclusive due to the multiple biases in this real-life condition (e.g., elevated stress of the participants due to the COVID-19 context) and the frequency of multiple gestures performed during the PP maneuver [START_REF] Brugada | Investigation of palpitations[END_REF]. Overall, Laevo was positively evaluated in terms of physical relief (questionnaire score: 4.2±0.4): both participants reported that the perceived general fatigue at the end of the shift was reduced when using Laevo. Importantly they both said they would use Laevo again without hesitation for future shifts where they would be positioned at the patient's head (effort score on Borg-CR10 scale: head: 1.8±0.4; side: 3.2±0.7). The participants found the Laevo comfortable (questionnaire score: 4.5±0.5), except when walking, which is a well-known issue of Laevo v1 [START_REF] Baltrusch | The effect of a passive trunk exoskeleton on metabolic costs during lifting and walking[END_REF] that was improved in recent versions. Laevo did not prevent or constrain their usual gestures and activity in the ICU (Fig. 7). Accordingly, their teammates did not notice any particular changes in the practice, and no physical or psychological side effects were observed. Those results are important for a future adoption of Laevo in the current practice, because the positive attitude of the co-workers is fundamental for the acceptance of new technology at work [START_REF] Allienne | Ethical and Social Considerations for the introduction of Human-Centered Technologies at Work[END_REF].

5

Use of the Exoskeleton in the ICU of the University Hospital of Nancy in the second wave of pandemic

Two additional Laevo v2.5 were provided to the medical staff, for a total of 4 exoskeletons since October 2020 (two v1 and two v2.5). At the present day (January 26, 2021) the 4 exoskeletons have been tested by more than 60 healthcare workers (medical doctors and nurses). Preliminary feedback from the medical staff confirms that the exoskeletons are useful to reduce the physical effort and back pain. Laevo v2.5 is preferred because of the walking feature (a lever enables to unblock the exoskeleton and walk normally). The adoption of the devices has been variable: some physicians are regular users (i.e., they ask to use the exoskeleton when they have to perform one or more PP), others are neutral or even sceptic.

The characteristics of the second wave of COVID-19, interestingly, have determined some differences in the use of the exoskeletons. The number of daily PP maneuvers performed in the ICU has been highly variable but generally lower than the in the first wave, sometimes even zero: so, the exoskeletons have not been used on a regular basis. The lower number of PP and the staff rotations made it possible for physicians to significantly reduce the number of PP per day: so, the exoskeleton was really not used as a personal equipment but shared among a pool of users and for few PP. While this could seem a positive point (it can help a larger group of users), it introduced a fundamental problem which is the repetitive act to calibrate and fit the exoskeleton to each person's body at each use. This caused a major obstacle to the regular use: several users reported that they considered it "difficult to adjust" and that the fitting took too much time. When physicians only have 1 PP to perform, for example, the benefit brought by the exoskeleton is not worth the time and frustration they experience when they have to wear it and adjust it. Interestingly, this problem is even bigger for Laevo v2.5, since the physicians must change the metallic bars corresponding to their size, an operation that is considered also difficult.

Overall, while it is clear that the exoskeleton is helping the physicians, its use as a shared tool (and not as a personal equipment) is preventing its adoption.

Conclusion

Our pilot study demonstrated that back-support exoskeletons are readily feasible to deploy in an ICU to physically assist the medical staff engaged in PP maneuvers, even in the dreadful sanitary context of the COVID-19 pandemic. Despite the many limitations of our exploratory study, carried out rapidly due to the urgency of the situation and with limited availability of physicians heavily busy in the hospital, we were able to demonstrate the feasibility and potential usefulness of using a back-support exoskeleton to assist during PP maneuvers in the ICU in less than two weeks. Though only 2 physicians used the passive exoskeleton Laevo during the PP maneuvers in the ICU in April 2020, they both perceived a physical relief in the low back during bent postures, particularly when working at the patient's head, and indicated an intention to adopt such technology after the pilot study. EMG and ECG analysis in the lab study confirm the help of the device. Four Laevo exoskeletons have been used in the ICU of the Hospital of Nancy since October 2020: tested by more than 60 people, they have been used regularly only by a small group of physicians. Their adoption is currently mainly limited by the use as a shared tool, while they should be a personal equipment. This is not surprising: it is known that the performance and physical assistance provided by an exoskeleton are not the only criteria for its adoption, and other factors, individual or organizational, are also critical [START_REF] Wioland | Processus d'acceptabilité et d'acceptation des exosquelettes: évaluation par questionnaires[END_REF]. Nevertheless, the positive feedback from the regular users of the exoskeleton is very encouraging: we are currently evaluating the use of the exoskeletons in the ICU as a personal equipment with a small group of volunteers.

The future adoption of this technology in the current practice will require more thorough studies quantifying the physiological benefits for a larger participant group size, with more repetitions of the maneuver to match the real use of the exoskeleton over an entire work shift, and evaluating the effects in a longitudinal study. By reporting the experimental protocol and all the materials, we encourage other teams to replicate our study, help the medical staff of other hospitals, and to share feedback on experience among interested worldwide intensive care units.

… times per month ▢_______

B. Acceptance evaluation of an exoskeleton for prone positioning

This questionnaire is an extract from a larger questionnaire1 that was developed by INRS to investigate the acceptance of exoskeletons introduced in an industrial context. Some questions have been adapted to our specific use case.

This questionnaire must be filled by the physicians equipped with the exoskeleton to receive physical assistance during the prone positioning maneuver. It is used to evaluate the exoskeletons in the simulated study and in the real-life conditions. Questions marked with an asterisk are to be filled only after using the exoskeleton in real-life condition, i.e., in the ICU. Reverse questions are marked with R.

Questionnaire for exoskeleton evaluation

Question Answer

Participant ID Exoskeleton type

❏ Laevo ❏ Corfor ❏ CrayX ❏ BackX
Instructions: In the questionnaire you will find a series of statements about your experience with the exoskeleton. For each statement that follows, please give your opinion by checking the corresponding box on a five-point Likert scale.

C. Evaluating the overall effort of a prone positioning maneuver with/without an exoskeleton

This questionnaire must be filled after realizing a prone positioning (PP) maneuver with or without an exoskeleton. It provides a subjective evaluation of the amount of physical effort and discomfort perceived while executing the PP. It provides a surrogate measure of standardized quantitative measures of efforts, such as surface EMGs placed over muscles of interests, whenever obtaining such measures is not possible. For example, using surface EMGs in the ICU was not possible for sanitary reasons.

Questionnaire for evaluation of the overall effort

Question Answer

Participant ID Exoskeleton type

❏ Laevo ❏ Corfor ❏ CrayX ❏ BackX ❏ none
Instructions: This questionnaire is to be completed after a PP maneuver with or without an exoskeleton. Put a circle on the relevant areas in the double-sided images, as well as a number, as per instructions. A D. Questionnaire for physicians using the exoskeleton in the ICU This questionnaire must be filled by the physicians equipped with the exoskeleton and using them in the ICU for PP maneuvers. The first part must be filled at the end of each PP maneuver (i.e., if the physician performs 20 maneuvers, he/she will fill the sheet 20 times, one after each PP). The second part must be filled at the end of the work-shift, for example in the changing room of the medical staff to avoid contamination. It must be noted that since the first batch of questionnaires are filled in the ICU following each PP, the paper sheets are considered "potentially contaminated" and therefore should rest untouched in a safe place for 3-4 days before manually processing them by the experimenters, in order to reduce any risk of contamination by direct skin contact.

Questionnaire to evaluate the exoskeleton in the ICU

Instructions: This questionnaire is to be completed by volunteers equipped with exoskeletons throughout their working hours. The first part must be filled after each PP maneuver. The second part must be filled at the end of their working hours. Four commercial exoskeletons were used in the exploratory study at the Hospital Simulation Center: Corfor (Corfor, France), Laevo v1 (Laevo, Netherlands), BackX (SuitX, USA), and CrayX (German Bionics, Germany). It must be noted that in this study we are not claiming a comprehensive comparison of the different exoskeletons that could have been helpful. At the time of the study (which was during the 1 st wave of the COVID-19 pandemic) it was not possible for us to rapidly purchase more exoskeletons to evaluate, and it is possible that other passive or active exoskeletons for low-back support which exist on the market could have been helpful for our task. Rather, we have an empirical proof of feasibility at least for one exoskeleton: it can be used in the ICU to assist in Prone Positioning, it is well perceived and possibly beneficial. The device was reported as not helpful for the specific PP maneuver.

1st PART : TO FILL AFTER EVERY PP

Figure 3 -evaluation of the Laevo exoskeleton by the PP team. We only had three Laevo v1, one for each size (small, medium, large). Each exoskeleton was attributed to the participants according to their height and following the recommendations of the manufacturer. The Laevo exoskeleton was immediately perceived helpful and intuitive. One participant reported a slight discomfort on the sternum during back flexion and on the thighs during walking, noticing it would be better to unlock it to walk normally. This issue was solved in later versions of the Laevo exoskeleton, such as the v2.5 that was purchased and used in the 2 nd wave of the pandemic in the ICU (since October 2020).

Figure 4 -BackX worn by one participant. The arrows point to the metallic arcs that are constraining the arms during the PP gestures: the participants pointed out that this was one of the main reasons for not choosing to use this exoskeleton for this particular gesture. The BackX was perceived similar to the LAEVO in terms of assistance, but both participants reported that the metallic curved bars from the sternum to the hip were preventing several arm movements necessary to complete the PP maneuver, and as such they felt they could not execute the entire maneuver with this equipment in real conditions.

Figure 5 -CrayX worn by two participants. The arrows highlight the external parts of the exoskeleton that add constraints to the workspace. The CrayX was considered too cumbersome to be used around patients, and very difficult to tune. Both participants perceived it required additional concentration effort during back flexion to enable the active support, which they couldn't improve even after changing the sensitivity parameter. Furthermore, one participant reported a critical discomfort due to a part of the CrayX applying a force on the dorsum.

III. ANALYSIS OF MOTION AND LUMBAR EFFORT WITH DIGITAL HUMAN MODEL

We replayed the participant's whole-body motion recorded with the Xsens motion capture system with a 43 DoFs Digital Human Model (DHM) using the Dart physics engine. The DHM consists of 19 rigid bodies linked together by 18 compound joints, for a total of 43 DoFs (11 for the back and neck, 9 for each arm including the sternoclavicular joint, and 7 for each leg), plus 6 DoFs for the free-floating base. Each DoF is a revolute joint controlled by a single actuator. The length of each segment of the DHM was scaled to match the participant's body segment length, while the DHM inertial parameters were scaled based on the participant's height and mass using average anthropometric coefficients.

To retarget the participant's upper-body motion in the Cartesian space, we used a hierarchical quadratic programming (QP) controller based on the OpenSoT library, which computes velocity commands to animate the DHM. The QP objective function consisted of the following tasks and priorities:

• level 1 (top priority tasks): balance (center of mass) position task, feet position task (fixed);

• level 2: Cartesian trajectory tracking of the pelvis and thoracic spine segments (position and orientation), of the right and left shoulder, elbow and wrist (position only), and of the head orientation; where the reference trajectories for the tracking tasks were the 3D positions and orientations of the Xsens avatar's body segments.

After retargeting the participant's upper-body motion, we used the DHM L5/S1 flexion/extension joint torques estimated with the dynamic simulation to compare the lumbar effort exerted by the participant with and without the exoskeleton. The pipeline for this processing is represented in the following Figure.

IV. KINEMATIC ANALYSIS

We analyzed the motion of the L5/S1 joint in the sagittal plane during the use of the four exoskeletons in the Prone to Supine (PS) and Supine to Prone (SP) positioning. The following figure displays typical profiles of low-back flexion angle of one participant, for the PS and SP, for all four exoskeletons and without exoskeleton. The joint angle profiles are overall similar for all conditions; variations from one condition to another can be explained by small differences in the manikin's position on the bed and intrinsic variability in the entire maneuver performed by the team. V. DYNAMIC ANALYSIS We estimated the human L5/S1 joint torque during the Prone to Supine (PS) and Supine to Prone (SP) positioning, with and without the Laevo exoskeleton, using our DHM simulation.

When the participant is equipped with the exoskeleton, the net torque exerted at the L5/S1 joint to counter the dynamics and gravity effects on the upper-body is a sum of the human-generated torque and of the exoskeleton assistive torque: 𝜏 "#$% = 𝜏 '()*+ +𝜏 ,-. . In order to estimate the human torque, the assistive torque 𝜏 ,-. provided by each exoskeleton is needed. To compute this torque, one needs the details about the mechatronics design of the platform.

We performed this computation only for the Laevo exoskeleton, since it was the one unanimously perceived by the participants as the most suitable candidate for use during PP maneuvers. To study the quantification of the Laevo's assistance, we recorded physiological measures on the participants executing PP maneuvers with and without the Laevo exoskeleton. The participants were equipped with an ECG sensor (Delsys Trigno ECG Biofeedback, 2 channels, bandwidth: 30Hz, ECG sampling rate 4370 sa/sec with onboard Butterworth bandpass filter 40/80 dB/Dec) and 12 surface EMG sensors (Delsys Trigno, EMG sampling rate 4370 sa/sec). The following figure shows the sensors placement on the participant's body, which was done according to Seniam protocol recommendations after abrasion and cleaning with alcohol.

Fig. 3 .

 3 Fig. 3. Lumbar spine flexion angle of participant 1 performing the PP maneuvers. Boxplots represent the distribution of angle across time, both for the prone to supine (PS), and supine to prone (SP) maneuvers.

Fig. 4 .

 4 Fig. 4. Lumbar flexion torque of participant 1 performing the PP maneuvers. Boxplots compare the torque at L5/S1 with and without the Laevo, estimated with the DHM simulation.

Fig. 6 .

 6 Fig. 6. Boxplots representing the distribution across time of the heart rate of participant 1 with and without the Laevo, measured during a control resting condition and while executing the PP maneuver at the head and at the side of the simulated patient.

Fig. 7 .

 7 Fig. 7. Laevo used in the ICU. A and B: the exoskeleton is worn on clothes, hidden below the medical gown. C: the exoskeleton assists the physician positioned behind the head of the patient during a PP maneuver.

  exoskeleton is easy to set up A The use of the exoskeleton 2 Overall, I find the exoskeleton easy to use A 3 I find that I can easily perform my movements with the exoskeleton A 4 I find that I can easily move and walk with the exoskeleton A 5 I find that I control my gestures as I wish with the exoskeleton A 6 R I find that the exoskeleton prevents me from working the way I want A 7 I find that I easily got used to working with the exoskeleton A 8 R I find that using the exoskeleton requires an extra effort of concentration A My performance with the exoskeleton 9 I find that the speed of my work with the exoskeleton is … B 10 * I find that the quality of my work with the exoskeleton is … B 11 I find that my effectiveness with the exoskeleton is … B 12 I find that the productivity of the team with the exoskeleton is … B My health and safety 13 R Overall, I find that my physical efforts with the exoskeleton are … B 14 R Overall, I find that with the exoskeleton, my fatigue is ... B 15 I feel safe working with the exoskeleton. A My feeling with the exoskeleton 16 R I feel nervous when I use the exoskeleton. A 17 R I feel worried when I use the exoskeleton. A 18 I feel confident when I use the exoskeleton. A 19 R I find I annoy my colleagues when I use the exoskeleton. A Future use 20 If I have a choice, I am thinking of using or continuing to use the exoskeleton in the next months A 21 * I find that over the course of the day I have adapted to the exoskeleton A 22 * I find that using the exoskeleton during the day has been beneficial A

  on this image on the areas where you felt physical effort during PP maneuvers [with the exoskeleton]. A Put a circle on this image on the areas where you felt physical effort becoming annoying during the PP maneuver [with the use of the exoskeleton], and a number next to each circle (1-10) to indicate the severity (1= no effort, 10= a lot of effort). A Only if you used the exoskeleton Could you indicate on this image the areas where you feel a new redistribution of physical effort, which you do not feel without the exoskeleton? Put a circle on the areas where you feel new efforts, and a number next to each circle (1-10) to indicate how much (1= no effort, 10= a lot of effort). B Could you indicate on this image the areas where you feel discomfort caused by the exoskeleton? Put a circle over the areas of discomfort, and a number next to each circle (1-10) to indicate how much (1= no discomfort, 10= a lot of discomfort).

Figure 1 :

 1 Figure 1: the four commercial exoskeletons used in the exploratory study

Figure 6 -

 6 Figure 6-In the study conducted at the Hospital Simulation Center, the motion of one physician executing the PP maneuver was captured with the Xsens MVN suit (Step 1). We used the whole-body kinematic estimation of the Xsens MVN software (Step 2) as an input to our dynamic simulation with a Digital Human Model (Step 3). The analysis of motion and estimation of human lumbar effort are based on this dynamic simulation.

Figure 7 -

 7 Figure 7-Lumbar spine flexion angle of one participant performing the PP maneuvers at the Hospital Simulation Center.

  Based on the Laevo empirical calibration curve published by Koopman et al. and on the Laevo user manual which specifies that its set of springs provides a maximum torque of 40 Nm, we used the following model to estimate 𝜏 /*,0. : 𝜏 /*,0. = 1 𝑘 3 + 𝑘 % 𝜃 𝑘 3 + 𝑘 % 𝜃 -𝑘 /.77 , 𝜃 ´> 0 𝜃 ´< 0 where 𝜃 is the back flexion angle, 𝑘 3 = -80 3𝑁𝑚 ⁄ and 𝑘 % = are constants that encode the spring linearity in its range of operation from 20 to 50 degrees (with the maximum assistance of 40 Nm at 50 degrees), and 𝑘 /.77 = 10𝑁𝑚 represents frictional losses which introduce hysteresis in the system (numerical values of the model's coefficients were set so that the model matches the calibration curve in [9] as closely as possible). The following figure displays the estimated joint torque and lumbar flexion angle across time for one PP trial for one participant.

Figure 8 -

 8 Figure 8 -Estimation of the human lumbar flexion torque and lumbar flexion angle of one participant performing the PP maneuvers at the Hospital Simulation Center. The torques are estimated with the DHM simulation. The plot reports one PP trial across time.

Figure 9 -

 9 Figure 9 -ECG and surface EMG placement for the quantification of Laevo's assistance during a PP maneuver. A: EMG sensors on ESL, ESI, TA, BF and GM. B: ECG sensor with 2 electrodes and EMG sensors on RA, RF and TA.

Figure 10 -

 10 Figure 10 -Boxplots representing the distribution across time of the EMG signal for 8 right side muscles of one participant with and without the Laevo exoskeleton, during a control condition (i.e., resting) and executing the PP maneuver at the head and at the side of the patient simulator at the Hospital Simulation Center.

Table 1 :

 1 Questionnaires scores for the 4 exoskeletons tested in the Simulation Center.

		Corfor	Laevo	BackX	CrayX
	Reduction of physical effort	3.0±0.0	4.0±0.0	4.0±0.0	4.0±0.0
	Perceived safety and comfort	4.37±0.7	4.5±0.5	4.0±1.1	3.8±1.0
	Easiness to install	3.5±2.1	4.5±0.7	1.5±0.7	1.5±0.7
	Intention to use	3.0±0.0	4.5±0.7	2.5±0.7	3.0±0.0

Table 2 :

 2 Median across time of heart rate value (in bpm) for the 2 participants (P1 and P2) performing a PP with and without the Laevo exoskeleton.

		Head No Exo	Head Laevo	Side No Exo	Side Laevo
	P 1	47.55	45.80	51.69	48.55
	P2	64.88	68.03	71.95	67.19

Table 3 :

 3 Changes in RMS value of the EMG signal when using the Laevo, compared to the baseline without exoskeleton, for both participants (P1 and P2) and both positions (in %).

		TA	ESL	ESI	GM	RF	BF	RA	TAL
	P1 Head	-35.24 -11.94 -0.70	-30.10	+0.35	-16.23 -1.20 -1.83
	P2 Head	+2.20 +5.15	+0.42	+4.48	NA	-20.38 -0.12 +5.60
	P1 Side	-14.75 -8.81	-1.04	-11.51	-5.80	-52.69 +0.32 -9.73
	P2 Side	-4.81 -11.42 -1.84	+7.71	NA	-33.87 -0.48 -19.84
	NA=not available because of a technical problem with the sensor reading.

nd PART: FILL WHEN YOUR WORK WITH THE EXOSKELETON IS FINISHED

	Question Left forearm / hand Right shoulder / arm		❏ .. ▢_______ Answer ❏ 10 = very annoyed ▢_______
	Participant ID Right forearm / hand		▢_______
			Answer: _________
	n. PP of the day With exoskeleton? Compared to the "normal" situation (no exoskeleton), did you find the new situation more physically demanding? II. EXOSKELETONS USED IN THE EXPLORATORY STUDY	Yes / No ❏ 1 = much less ❏ …
			❏ 5 = identical
	Position?		Head / Side ❏ …
	Your perception during the PP maneuver	❏ 10 = much more
	Answer: _________ Please note for each anatomical zone your perceived effort during your last PP maneuver according to Borg's Perceived Effort Rating Scale (Borg CR10). Compared to the "normal" situation (no exoskeleton), did you find the new situation more ❏ 1 = much less cognitively demanding (for example, you had to pay more attention...)? ❏ …
	0 nothing at all		❏ 5 = identical
	0.5 extremely weak / very, very slight 1 very weak / very slight 2 weak / slight 3 moderate		❏ … ❏ 10 = much more ❏
	4 5 strong / severe 6 7 very strong / very severe If you had the choice, would you use an exoskeleton yourself in the next few months if the current sanitary situation continued? 8 9 10 extremely strong / very, very severe	Answer: _________ ❏ Strongly disagree ❏ Disagree ❏ Neither agree nor disagree ❏ Agree
			❏ Strongly agree
	Neck	▢_______
	Your perception about the PP maneuver during COVID-19
	Lower back On a scale of 1 (not stressful at all) to 10 (very stressful), if you performed the PP maneuver during the COVID-19 outbreak, how much ▢_______ "stressful" was it …?
	Legs … physically	▢_______	▢_______
	… cognitively (pay attention)		▢_______
	Left shoulder / arm How often do you practice the PP maneuver every day during the COVID-19 outbreak? ▢_______
	… times per day		▢_______
	Left forearm / hand … times per month	▢_______	▢_______
	How many times have you practiced the PP maneuver today? Right shoulder / arm Do you have any comments on your experience today working alongside people equipped ▢_______	(free comment)
	with exoskeletons?
	Right forearm / hand Are there things you noticed about the use of and the work with the exoskeleton? ▢_______
	Please note for each anatomical zone your perceived effort during your last PP maneuver according to Borg's Perceived Effort Rating Scale
	(Borg CR10). 2 Question 0 nothing at all		Answer
	0.5 extremely weak / very, very slight Participant ID 1 very weak / very slight
	2 weak / slight Exoskeleton type 3 moderate	❏ LAEVO
	4
	Between when you started to work and now, how many times have you 5 strong / severe
	practiced the PP maneuver today? 6
	7 very strong / very severe
	Have you systematically used the exoskeleton to perform the PP maneuver? Yes / No 8
	9
	10 extremely strong / very, very severe
	Neck		▢_______
	Lower back		▢_______
	Legs		▢_______
	Left shoulder / arm		▢_______

If not, why did you remove it?

In how many of the total PP maneuvers out of the total PP maneuvers did you use the exoskeleton?

_______ (number) out of __________ (total)

In total, how long did you keep it?

Wioland L., L. Debay, J.-J. Atain-Kouadio (2019) Processus d'acceptabilité et d'acceptation des exosquelettes: évaluation par questionnaires. Références en santé au travail, TF

274, n. 160, pp. 49 -76. Available at: http://www.inrs.fr/dms/inrs/CataloguePapier/DMT/TI-TF-274/tf274.pdf

Supplementary Material

I. QUESTIONNAIRES

In this supplementary material, we report the entire set of questionnaires that we used during our study, to facilitate reproducibility and replication of our results by other teams.

A. Human factors related to prone positioning

This questionnaire must be filled by any participants to the prone positioning (PP) study before doing any PP maneuver in simulation or in the ICU. It is necessary to retrieve relevant information such as experience with the PP in the hospital before and during the COVID-19 pandemic, history of back pain that is medically relevant for the study, attitude towards the exoskeleton technology. A semi-directed interview to gain a deeper knowledge of the expectations of the participants is complementary: the experimenter can ask, for example, if the participants have already seen or used an exoskeleton, a robot or a prosthetic device, and get insights into their past experience.

Questionnaire on human factors

Instructions: This questionnaire is to be completed once, before the start of any experiment. Did you change any settings after removing the system (for example, after using the restroom)? If so, specify when and why.

Did you unhook the thighs pads to walk? If yes, how many times?

Did the exoskeleton prevent you from making one or more movements? If yes, can you list them.

Did something unexpected happen? If so, can you describe it? Do you have any comments about your experience today as a physician equipped with an exoskeleton? Are there things you noticed while using and working with the exoskeleton?

(free comment)

E. Questionnaire for colleagues in the ICU

This questionnaire must be filled by colleagues of the physicians equipped with the exoskeleton. The first part is filled before the normal work-shift. The second part is filled at the end of the work-shift, when they have had the experience of working with colleagues wearing the exoskeleton. The questionnaire can be filled outside the ICU, for example in the medical staff changing rooms, thus avoiding any risk of contamination by paper.

Questionnaire for colleagues in the ICU

Instructions: you are going to work alongside people equipped with exoskeletons. We need to get some information about your perceptions. This is important for our research to evaluate the impact of introducing such a tool in a hospital setting during COVID-19. The questionnaire consists of two parts: the first, to be completed just before you start working with your colleagues equipped with exoskeletons in the ICU; the second, at the end of your working day. Please fill the two parts outside the ICU.


Layany Carolyny

Mourão Mulvaney

Francisco Humberto Xavier-Júnior

S Rodrigues

Didier Stien

Marques Silmara

Vera Allegretti

Garcia Lúcia

Sapotaceae P Royen

Alice M S Rodrigues

Allegretti

Vera Lúcia

Antimicrobial and anthelmintic activities of the ethanolic extract, fractions and isolated compounds from Manilkara zapota L. P. Royen (Sapotaceae)

Keywords: Traditional medicine Meso-and Southern America, phytochemistry, anthelmintic, antimicrobial, chromatography. Highlights Charged Aerosol Detector Compound 1: chlorogenic acid Compound 2: myricetin-3-O-β-D-glucopyranoside Compound 3: myricitrin Compound 4: mearnsitrin Compound 5: germanicol Compound : βamyrin Compound 7: α-amyrin Compound 8: germanicol acetate COSY: Correlation Spectroscopy EEMz: dry ethanolic extract ESI: Electrospray interface F: fraction obtained from High polarity insoluble fraction HMBC: Heteronuclear Multiple Bond Correlation HPFMz: High polarity insoluble fraction HRMS: High Resolution Mass Spectroscopy HSQC: Heteronuclear Single Quantum Coherence LPFMz: lower polarity fraction Mz: Manilkara zapota PVP: polyvinylpyrrolydone TOF: Time-of-flight

published or not. The documents may come

INTRODUCTION

Infectious diseases caused by bacteria, viruses, fungi, and parasites are the greatest cause of death, disability, and social and economic difficulties for millions of people worldwide [START_REF] Ni | Emerging and neglected infectious diseases: Insights, advances, and challenges[END_REF] . This problem is especially high in developing countries, due to poor availability of drugs and the emergence of widespread drug resistance [START_REF] Ayukekbong | The threat of antimicrobial resistance in developing countries: causes and control strategies[END_REF] .

Infections caused by microorganisms such as Staphylococcus aureus can promote disorders in the skin and mucosa, toxic shock syndrome, endocarditis, osteomyelitis, and infections of the urinary and intestinal tracts. [START_REF] Snowden | A comparison of the anti-staphylococcus aureus activity of extracts from commonly used medicinal Plants[END_REF] . Additionally, dermatophyte fungi are also able to invade the keratinized tissues of animals and humans [START_REF] Salakshna | A cohort study of risk factors, clinical presentations, and outcomes for dermatophyte, nondermatophyte, and mixed toenail infections[END_REF] . Among them,

Trichophyton rubrum is most widespread, representing 80% of clinical isolates [START_REF] Zhan | Phylogeny of dermatophytes with genomic character evaluation of clinically distinct Trichophyton rubrum and T-violaceum[END_REF] .

Candida albicans is another important opportunistic pathogenic fungus that causes candidiasis responsible for a variety of conditions in humans, ranging from superficial cutaneous lesions to generalized infection [START_REF] Zida | Anti-Candida albicans natural products, sources of new antifungal drugs: A review[END_REF] .

In the same way, helminthiases caused by intestinal nematodes and transmitted via the soil are the most common and persistent neglected parasitic infections, especially in developing countries. Strongyloides stercoralis is the main neglected geohelminths responsible to infect 30-100 million people in the world [START_REF] Lustigman | A research agenda for helminth diseases of humans: the problem of helminthiases[END_REF][START_REF] Olsen | Strongyloidiasis -the most neglected of the neglected tropical diseases?[END_REF] . The autoinfection ability of S. stercoralis leads to syndromes of persistent infection that can result in chronic illness with various clinical manifestations. In immunocompromised patients, the uncontrolled multiplication of the parasite (hyperinfection) and the potentially fatal distribution of larvae in various organs represents a major concern in terms of public health [8; 9] .

Therefore, the discovery of new compounds with chemical structures and mechanisms of action different from those current antimicrobial and anthelmintic drugs are urgently need. This fact is due to the increased incidence of new and reemerging infectious diseases, the ineffectiveness of treatments using broad-spectrum anthelmintics, and especially the increasing inefficacy of the drugs employed in the current therapy against pathogenic microorganisms and intestinal parasites [10; 11] .

Manilkara zapota (L.) (Synonyms: M. zapotilla, M. achras, Mimusops manilkara, Achras zapota), commonly known as sapoti, is a member of the Sapotaceae family and it represents a potential natural resource to treat infectious process [START_REF] Quattrocchi | Crc world dictionary of plant names: Common names, scientific names, eponyms, synonyms, and etymology[END_REF] . In folk medicine, the leaves are used to treat coughs, colds, and diarrhea, the crushed seeds are used as a diuretic and febrifuge, and the husks have astringent and febrifuge activities [START_REF] Ganguly | Evaluation of antinociceptive and antidiarrhoeal properties of Manilkara zapota leaves in Swiss albino mice[END_REF] . In the literature, M. zapota has been reported to exhibit antioxidant properties [14; 15] , as well as analgesic [START_REF] Jain | Evaluation of analgesic activity of Manilkara zapota (leaves)[END_REF] , antimicrobial [17; 18] , antihyperglycemic, and hypocholesterolemic activities [19; 20] , due the presence of phenolic acids, flavonoids, tannins (mainly in the immature fruit), triterpenes, and saponins (mainly in seeds) compounds [19; 21-23] . Despite these important properties, the bioguided phytochemical study of the compounds from the M. zapota leaves has not yet been developed. In this sense, the goal of this work was to characterize the phytochemistry of the ethanolic extract, fractions, and isolated compounds from the Manilkara zapota L. P. Royen leaves and to evaluate their antimicrobial action and, for the first time, their anthelmintic activity.

MATERIALS AND METHODS

Materials

Leaves of Manilkara zapota (Mz) were collected from the orchard of the Ivermectin and chlorogenic acid (95%) were puchased from Sigma-Aldrich (St. Louis, MO, USA) and used as a control drug and an analytical standard, respectively, in the anthelmintic activity assays. Polyvinylpyrrolidone (PVP-K30) was obtained from Synth (São Paulo, Brazil). Oxacillin, itraconazole, and fluconazole from Molekula (Dorset, UK) were used as control drugs in the antimicrobial activity assays. All solvents employed in the study were HPLC or UPLC grade.

Bioactive compounds of the M. zapota

Ethanolic extract of the M. zapota

The ethanolic extract was produced by turbolization. 900 g of the triturated and dried leaves of M. zapota were dispersed in 3 L of ethanol under constant stirring at 16,000 rpm for 10 min using a mechanical disperser (Ultra Turrax T50, IKA Works Inc., Wilmington, NC, USA) at 25 °C. The suspension was filtrated in nylon filter to remove non-soluble particles. Afterthat, the residue was re-extracted with 3 L of ethanol and filtrated. The extracts were combined and the solvent was evaporated under vacuum until complete dryness, resulting in the dry ethanolic extract (EEMz, 96.47 g).

2.2.2.

Fractions of the M. zapota 30 g of the EEMz was suspended in 300 mL of hexane/ethyl acetate mixture (1:1) and extracted in the Ultra Turrax system for 5 min at 16,000 rpm. Lower polarity fraction (LPFMz) and the high polarity insoluble fraction (HPFMz) were obtained. These fractions were gently separated by centrifugation at 5,000 g for 15 min and posteriorly dried under vacuum in order to produce 16.25 g and 11.44 g of LPFMz and HPFMz, respectively.

Isolation of the M. zapota compounds

1.5 g of the HPFMz was purified by flash chromatography using a Combi Flash ® Rf + were isolated and chracterized.

Identification of the components by chromatography analyses

Thin layer chromatography

Thin layer chromatography analyses were performed using aluminum plates coated with silica gel 60 F 254 (Merck, Darmstadt, Germany). The lower polarity compounds were elueted with hexane: ethyl acetate (70:30) and ethanolic anisaldehyde was used as stain.

For the high polarity compounds, the mobile phase was ethyl acetate: formic acid: glacial acetic acid: water (52:11:11:26) and a solution of diphenylboryloxyethylamine and polyethyleneglycol (NP/PEG) was used as the developer. UV detection was performed at 254 and 366 nm.

Gas chromatography-mass spectrometry (GC-MS)

A gas chromatograph (Agilent, model 6890, Santa Clara, CA, USA) coupled to an Agilent 5975 mass spectrometer was used to identify compounds present in LPFMz.

The analyses were performed using an HP-5MS capillary column (30 m × 0.25 mm, 0.25 µm) (J&W Scientific, Santa Clara, CA, USA). The column temperature was increased from 110 ºC to 280 ºC during 26 min, using a heating rate of 5 ºC/min. The injector and detector were maintained at 250 ºC and 300 ºC, respectively. The carrier gas was helium at a flow rate of 1 mL/min. The mass spectra were obtained using an ionization energy of 70 eV and m/z from 40 to 600 a.m.u.. Injection of 1 µL of sample was made in split mode (1:30). The compounds were identified by comparison of the fragmentation patterns with data in the NIST 11 spectral library and data reported in the literature.

HPLC analyses

The analytical profiles were obtained with a Dionex

Structural elucidation of the HPFMz

Optical rotation

The optical rotation was measured in methanol (MeOH) using an Anton Paar MCP 300 polarimeter (Graz, Austria) equipped with a 35 μ cell with a length of 100 mm.

1 H and 13 C NMR and 2D-NMR spectra

NMR analyses were performed in a Bruker (Billerica, MA, USA) 500 MHz spectrometer equipped with a 1 mm inverse detection probe. The samples were dissolved in deuterated methanol (CD 3 OD) in 5 mm tubes. The chemical shifts (δ, in ppm) were reported based on the signal for tetramethylsilane (TMS).

High resolution mass analyses

High

Antimicrobial activity

The antimicrobial activities of the EEMz, HPFMz, LPFMz, and isolated flavonoids 2, 3, Laboratory Standards Institute (CLSI) [24; 25] . Previously, the samples were solubilized with dimethyl-sulfoxide (DMSO). Then, the solutions were placed in 96-well microplates, diluted in a growth medium. Lastly, the pathogen suspension was added, resulting in concentrations ranging from 512 to 1 μ /m . The microplates were for incubated at 37 ºC for 24 h (bacteria), 48 h (yeast), and 5 days (dermatophyte).

and
Oxacillin ( EEMz, HPFMz, LPFMz, F 2, F 3 , and compounds 1 (chlorogenic acid) and 3 (myricitrin)

were analysed. In order to increase the solubility in the culture medium, samples were dispersed in polyvinylpyrrolidone (PVP-K30) at 1:4 ratio. The assays were performed in 24-well plates containing RPMI 1640 medium (2 mL/well) supplemented with gentamicin (50 mg/L) and amphotericin B (2.5 mg/L). Two parasites were transferred to each well and the plates were incubated under 5% CO 2 at 37 ºC for 72 h. 50, 100, 200 and 400 μ /m were analysed. Positive control was treated under the same concentrations of ivermectin, while negative control only fresh medium containing PVP was used. At 6, 24, 48, and 72 h, the motility and survival of the worms were evalueted and morphological changes was observed by DM500 microscope (Leica, Wetzlar, Germany). All the tests were performed in triplicate and survival curves were plotted using Prism 5.0 software (GraphPad, San Diego, CA, USA).

Statistical analyses

All analyses were performed in triplicate. Statistical significance was determined by the Mantel-Cox test, using GraphPad Prism 5.0 software. The differences between the survival curves were considered statistically significant when the p-value was smaller than 0.001.

RESULTS AND DISCUSSION

Bioguided studies of the antibacterial, antifungal, and anthelmintic (against S.

venezuelensis) activities of the extract, fractions, and isolated compounds obtained from the leaves of M. zapota was firstly developed in this work. EEMz was obtained with a yield of 10.71% while LPFMz and HPFMz showed yields of 54.16% and 38.13%, respectively.

Identification of compounds in the LPFMz fraction

GC-MS analyses revealed a predominance of pentacyclic triterpene compounds

showing typical fragmentation patterns of ursane and oleane skeletons, with retention times between 40 and 47 minutes corresponding at 75% of the all LPFMz fraction (Fig. 1). Table 1 shows the compounds identified from their fragmentation patterns, compared to data obtained from the NIST 11 library. The presence of long chain hydrocarbons and sterols in the aerial parts of M. zapota has been reported previously [21; 26] and was confirmed here by the presence of compounds A, B, and C.

Please, insert here the Figure 1 and Table 1 Compound 5 presented a base peak at m/z 204, accompanied by the ions with m/z 189 and 177, which were characteristic of the compound germanicol (olean-18-en-3β-ol) [START_REF] Oe | Occurrence and sources of triterpenoid methyl ethers and acetates in sediments of the cross-river system, southeast Nigeria[END_REF] (Fig. 2). Peaks at m/z 218, 203, and 189 were associated with retro-Diels-Alder type fragmentation of ring C of olean-12-enes and ursan-12-enes. These characteristic peaks were observed for compounds 6 and 7 f β-α-amyrin, respectively.

T p f β-amyrin show greater abundance of the peak at m/z 203, compared to the peak at m/z 189 wh h α-amyrin analogues show similar abundances of these peaks [27; 28] . The mass spectra of triterpene esters reflect the fragmentation pattern of the main skeleton, with smaller ions derived from the acid portion [START_REF] Fingolo | Triterpene esters: Natural products from Dorstenia arifolia (Moraceae)[END_REF] . Hence, the fragmentation pattern of compound 8 corresponded to that expected for germanicol acetate (m/z 468), with the fragments characteristic of germanicol (at m/z 204 and 169), together with fragments at m/z 453 and 408 reflecting the loss of methyl and acetyl groups, respectively, and a fragment at m/z 43 corresponding to COCH 3 [START_REF] Oe | Occurrence and sources of triterpenoid methyl ethers and acetates in sediments of the cross-river system, southeast Nigeria[END_REF] .

Please, insert here the Figure 2 Compounds 9 and 10 p α-β-amyrin acetates, respectively, with fragments peaks at m/z 218, 203, and 189 characteristics of amyrins and fragments with loss of the acetyl group [START_REF] Oe | Occurrence and sources of triterpenoid methyl ethers and acetates in sediments of the cross-river system, southeast Nigeria[END_REF] . Compound 11 was identified as lupeol acetate, with peaks at m/z 453, 408, 365, 218, 189, and 43 [START_REF] Naumoska | Analysis of triterpenoids and phytosterols in vegetables by thin-layer chromatography coupled to tandem mass spectrometry[END_REF] . Amyrins, amyrin acetates, and lupeol acetate have been reported in leaves of M. zapota [14; 16] . However, germanicol and its acetate were firstly reported in the leaves of M. zapota in the current study. However, the current study is the first time that germanicol and its acetate have been reported in M. zapota leaves.

Identification of compounds isolated from HPFMz

Four compounds from HPFMz can be isolated and purified by preparative HPLC.

Compounds 1 (3.5 mg), 2 (0.7 mg), 3 (13.8 mg), and 4 (4.6 mg) showed retention times of 2.01, 3.51, 4.14, and 5.26 min, respectively, detected in the HPLC analysis (Fig. 3).

The structures of the compounds were determined based on the one-and twodimensional NMR ( 1 H, [START_REF] Ganguly | Evaluation of antinociceptive and antidiarrhoeal properties of Manilkara zapota leaves in Swiss albino mice[END_REF] C, COSY, HSQC, and HMBC), high-resolution mass spectrometry, and optical rotation analyses.

Please, insert here the Figure 3 Compound 1 presented the molecular formula C 16 H 18 O 9 , with a mass of 354. Its structure was identified as that of chlorogenic acid (Fig. 4). The high-resolution negative mode mass spectrometry analysis resulted in extraction of the [ -H] -ion with m⁄z 353.0872. The structural elucidation data corroborates Fayek et al. (2013) [START_REF] Fayek | New triterpenoid acyl derivatives and biological study of Manilkara zapota (L.) Van Royen fruits[END_REF] , who isolated this compound from the fruit of M. zapota. Other earlier studies have also been identified chlorogenic acid in the fruit and seeds of M. zapota [31; 32] . It could be inferred from the 1 H NMR spectra of substances 2, 3, and 4 that they possessed similar base nuclei and belonged to the flavonoid class of compounds (Fig. 4). The pair of doublets at δ .18 (± 0.8) (J = 2. δ ± 0.5 1H) and δ .35 (± 0.8) (J = 2.1 ± 0.4 1H) could be attributed to the 5,7,9,10-tetrasubstituted ring A, while the presence of two singlets at δ 6.95 (± 0.8) was indicative of a flavonol with a 1' 3' 4' 5'-tetrasubstituted ring B, corresponding to myricetin [START_REF] Shen | Polar constituents from sageretia thea leaf characterized by HPLC-SPE-NMR assisted approaches[END_REF] .

Please, insert here the Figure 4 Considering the sugars linked to C-3 of the aglycones, compound 2 showed the p f β-D-glucopyranoside unit, due to the high coupling constants (J HH )

involving each consecutive pair of H-1, H-2, H-3, H-4, and H-5 protons of the sugar, varying between 8.8 and 11.9 Hz, which is characteristic of protons in axial positions.

For compounds 3 and 4, the same saccharide unit was attached to C- reported in other plants and identified as myricetin-3-O-β-D-glucopyranoside [34; 35] . The absence of a smaller value of J HH between the H-3′′ H-4′′ p f m h was not a 3-O-galactoside [START_REF] Castillo-Munoz | Flavonol 3-O-glycosides series of Vitis vinifera Cv. Petit Verdot red wine grapes[END_REF] . The literature confirms the presence of myricetin-3-O-β-D-galactopyranoside in species of the Sapotaceae family [START_REF] Baky | A review on phenolic compounds from family Sapotaceae[END_REF] .

Compound was identified as myricetin-3-O-α-L-rhamnopyranoside, also known as myricitrin. This compound has also been identified in previous studies using the leaves of M. zapota [21; 33; 37] .

3

In vitro antimicrobial activity

MIC of the extracts, fractions, and isolated flavonoids of M. zapota are shown in Table 2. These results showed that EEMz, HPFMz, LPFMz were potentially active against C.

albicans ( I = 1 μ /m ). In the case of T. rubrum, EEMz and HPFMz presented moderate activity, while LPFMz was ineffective. HPFMz showed high activity against Please, insert here the Table 2 It has been found that the presence of terpenoids and glycosidic flavonoids in crude extracts plays an important role in determining their antimicrobial activity [START_REF] Cowan | Plant products as antimicrobial agents[END_REF] . The presence of such compounds in the extract and the higher polarity fraction could provide an explanation for the observed antimicrobial action against the pathogens tested. The results obtained to C. albicans corroborate previous studies using aqueous extract of leaves collected in India [START_REF] Osman | Antimicrobial investigation on Manilkara zapota (l.) p. Royen[END_REF] and various Candida species [START_REF] Otari | Green phytosynthesis of silver nanoparticles using aqueous extract of Manilkara zapota (L.) seeds and its inhibitory action against Candida species[END_REF] . The antimicrobial properties of M. zapota extracts against S. aureus have been observed previously [39; 41] . These activities may be related to the m m x f α-βamyrin detected in the LPFMz that possesses known antimicrobial activity [START_REF] Fernandes | Triterpene esters and biological activities from edible fruits of Manilkara subsericea (Mart.) Dubard, Sapotaceae[END_REF] .The presence of one or more sugar units at different positions of the aglycone can increase solubility and enhance the inherent medicinal properties of flavonoid compounds [START_REF] Ren | Synthesis of flavonol 3-O-glycoside by UGT78D1[END_REF] .

Taken together, all the samples originated from the leaves of M. zapota presented antimicrobial activities (of different magnitudes). These findings demonstrate the high antimicrobial potential of the leaves of M. zapota in combating three important human pathogenic microorganisms.

In vitro anthelmintic activity assays

The anthelmintic effect of EEMz, HPFMz, LPFMz, F 2 , F 3 , and isolated flavonoids (compounds 1 and 3) were evaluated by the survival percentage and cuticular integrity of female (L4) S. venezuelensis parasites. The positive control (with addition of PVP) presented 100% survival of the parasites. All samples 4 μ /m w h after 24 h to the parasites (Fig. 5). For EEMz, no survival of parasites at 72 h was 1 2 μ /m (F . 5A). H F z w h 2 μ /m f 48 h although survival at 72h have been occurred for 100 and 5 μ /m p 5.5% and 72%, respectively (Fig. 5B). F 2 was lethal at all concentrations tested after 72 h of incubation (Fig. 5C), while F 3 was only lethal at the two highest concentrations (Fig. 5D). The parasites exposed to chlorogenic acid 2 μ /m w h h f 24 h, while after 72 h there was no survival at any concentration (Fig. 5E). At 72 h, there was no survival of the parasites exposed to myricitrin (compound 3) at 400 and 2 μ /m . H w h f h 11.1 1.1% were determined to 100 and 50 μ /m p y (F 5F). I w p h treatment had statistically significant difference of the control (p <0.0001).

Please, insert here the Figure 5 Ivermectin (pharmacological control) reduced the motility of the organisms, but there was survival of all the individuals after 72 h. Similar behavior to infective larvae (L3) of S. stercoralis 25 μ /m f m h w h of the parasite motility and survive during 6 days [START_REF] Boonmars | In vitro antiparasitic activity of extracts of Cardiospermum halicacabum against third-stage larvae of Strongyloides stercoralis[END_REF] . The lower polarity fractions were also tested (data not shown) but showed unsatisfactory and imprecise results at all concentrations evaluated due the formation of insoluble clusters in the culture medium.

A 1 μ /m chlorogenic acid was lethal in the first 24 h, while EEMz and F 2 caused total mortality at the end f h m (F . ). A 2 μ /m y chlorogenic acid was lethal after 24 h. However, there was no survival for any of the samples tested after 72 h. Images of the parasites after 72 h of incubation are shown in Fig. 7. Parasites exposed to myricitrin (Fig. 7B) exhibited distortions and cuticular wrinkling. Those treated with F2 (Fig. 7C) showed distortions, depressions, and alterations in cuticular integrity, suggestive of direct cytotoxic effects on the morphology of the worms. The parasites exposed to ivermectin (Fig. 7D) exhibited reduced motility, but the integrity of the cuticular surface remained similar to that of the positive control (Fig. 7A).

Please, insert here the Figure 6 and7 Thus, the first study describing the direct action of extracts, fractions, and isolated compounds obtained from M. zapota leaves on adult females of S. venezuelensis have been presented in this work, highlighting the potential anthelmintic activities, especially of the ethanolic extract, F 2 , and chlorogenic acid. The chemical analyses showed that both the ethanolic extract and the fractions contained glycosidic flavonoids as well as phenolic acids and their derivatives, suggesting that the effective anthelmintic action against the nematode was due to the presence of these compounds in the leaves of M.

zapota.

Various studies of plant species have been undertaken in the search for new treatments for veterinary and human parasitosis. Free-living females of Strongyloides ssp. exposed to a methanolic extract of Lawsonia inermis have been analyzed taking into account the presence of distortions ,and longitudinal and transverse cracks [START_REF] Ismail | Comparison between the effect of Lawsonia inermis and flubendazole on Strongyloides species using scanning electron microscopy[END_REF] . Sarojini et al.

(2012) [START_REF] Sarojini | Correlation between the phytochemical constituents and anthelmintic activity of Lawsonia inermis leaf extracts[END_REF] reported strong anthelmintic action of L. inermis extract due to the presence of glycosides, terpenoids, and flavonoids. Carvalho et al. (2012) [START_REF] Carvalho | The anthelmintic effect of plant extracts on Haemonchus contortus and Strongyloides venezuelensis[END_REF] described the action of extracts of Piper tuberculatum, Lippia sidoides, and Mentha piperita against

Haemonchus contortus

Ndhlala et al. (2016) [START_REF] Ndhlala | Antimicrobial, anthelmintic activities and characterisation of functional phenolic acids of Achyranthes aspera linn.: A medicinal plant used for the treatment of wounds and ringworm in east africa[END_REF] described the chemical composition and anthelmintic activity of extracts of Achyranthes aspera against the free-living nematode Caenorhabditis elegans. The aqueous and acetone extracts of A. aspera exhibited strong activity, with UHPLC-MS/MS analyses revealing the presence of rutin, chlorogenic acid, and genistein, which were associated with the activity.

Comparison of the analogous flavonoids revealed a close relationship between their structures and antiparasitic activity. The presence of substituents (hydroxyl, methyl, methoxy, and glycosides) is fundamental to determine its effectiveness. In the case of hydroxyls, the number and pattern of hydroxylations in ring B has been found to have a major influence on the antiparasitic activity of flavonoids [START_REF] Kerboeuf | Flavonoids and related compounds in parasitic disease control[END_REF] . However, the mechanisms of action of flavonoids in helminths remain to be fully elucidated, even if several possibilities have been proposed: an increase in the supply of nitric oxide acting as a neurotransmitter in neuromuscular junctions and causing myoinhibition; nonspecific inhibition of esterases and cholinesterases leading to neuromuscular disorganization of the parasites; increased efflux of calcium causing depolymerization of the microtrabecular network, with consequent disorganization and disaggregation of the myofilaments; and decreased levels of free amino acids and increased levels of glutamic acid, glutamine, citrulline, and gamma-aminobutyric acid (GABA), leading to the release of nitric acid and resulting in neurological alterations [START_REF] Kerboeuf | Flavonoids and related compounds in parasitic disease control[END_REF] . venezuelensis parasites. This study contributes to the scientific basis for the possibility of using plants and compounds of natural origin to combat strongyloidiasis and microbial infections. The development of new therapeutic agents from M. zapota leaves will require further in-depth studies, but nonetheless this plant material appears to offer an effective alternative for the treatment of these diseases that are widespread and difficult to control.

CONCLUSIONS

ACKNOWLEDGMENTS

Compound 3 : 13 C

 313 Yellow powder; R f ~0.8 with orange fluorescence at 366 nm. [α] 25 D : -61° (c 0.1 g / 100 mL, CH 2 OH), λ max : 261, 350 nm; 1 H NMR (CD 3 OD): δ 0.96 (3H, d, J = 6.1, H-") 3.52 (1H , J = 9.2, 6.1, H-5") 3.34 (1H J = 9.2 Hz, H-4") 3.79 (1H dd, J = 9.2, 3.0 Hz, H-3"), 4.22 (1H, dd, J = 3.0, 1.2 Hz, H-2") 5.32 (1H J = 1.2 Hz, H-1") .2 (1H , J = 1.8 Hz, H-6), 6.36 (1H, d, J = 1.8 Hz), 6.95 (2H, s, H-2' H-'); NMR (CD 3 OD): 179.58 (C-4), 166.55 (C-7), 163.15 (C-5), 159.34 (C-2), 158.55 (C-9), 146.84 (C-3' -5') 137.91 ( -4') 13 .25 ( -3), 121.91 (C-1') 1 9.55 ( -2' ') 1 5. 7 ( -10), 103.60 (C-1")

4

 4 were analyzed against S. aureus (ATCC 29213), C. albicans (ATCC 10231), and T. rubrum (SNB-TR1). The broth microdilution technique was used to determine the minimal inhibitory concentration (MIC) according to the guidelines of the Clinical and

  H 22 O 12 , with mass of 478, identified as myricetin-3-O-4'methyl-ether-3-O-α-L-rhamnopyranoside, also known as mearnsetin-3-O-α-Lrhamnopyranoside (mearnsitrin). Take all results together, it is important to highlight that chlorogenic acid, myricetin-3-O-β-D-glucopyranoside, and mearnsitrin compounds in leaves of M. zapota were firstly detected in this work.

S 3 )

 3 . aureus and moderate activity against the other microorganisms. About the flavonoid, compound 2 showed significant activity against S. aureus (MI = 8 μ /m ) promising activity against T. rubrum ( I = 1 μ /m ). y ( mp presented promising activity against the gram-positive bacterium and the dermatophyte

5 (

 5 Antimicrobial, antifungal, and anthelmintic activities of the extract, fractions, and isolated compounds obtained from the leaves of Manilkara zapota were firstly evaluated. Another innovative find of this work was the identification of the compounds 1 (chlorogenic acid), 2 (myricetin-3-O-β-D-glucopyranoside), 4 (mearnsitrin), germanicol), and 8 (germanicol acetate) in M. zapota leaves. Ethanolic extract and fractions showed high antifungal activity against C. albicans. Compounds 3 (myricetin-3-O-α-L-rhamnopyranoside) and 4 presented promising activity against S. aureus while the high polarity fraction and compound 2 exhibited high activity against S. aureus and T. rubrum. The ethanolic extract and F 2 containing phenolic compounds, and chlorogenic acid showed effective antiparasitic action against female (L4) S.

Fig. 1 -Fig. 2 -Fig. 3 -

 123 Fig. 1-Expanded chromatogram profile of the LPFMz Fig. 2-Compounds detected by GC-MS in the LPFMz Fig. 3-HPLC elution of the compounds 1, 2, 3, and 4 in the HPFMz.

Fig. 4 -

 4 Fig. 4-Compounds isolated from HPFMz.

Fig. 5 -

 5 Fig. 5-Anthelmintic activity of M. zapota against S. venezuelensis after 72 h of incubation. Percentage survival of parasites exposed to: EEMz (A), HPFMz (B), F 2 (C), F 3 (D), chlorogenic acid (compound 1) (E), and myricitrin (compound 3) (F). Control (•) 5 μ /m (■) 1 μ /m (▲) 2 μ /m (▼) 4 μ /m (♦).

Fig. 6 -

 6 Fig. 6-Comparison of the anthelmintic activities of the extract, fractions, and compounds obtained from the leaves of M. zapota 1 μ /m (A) 2 μ /m (B). (•) EE z (■) H F z (▲) F 2 (▼) F 3 (♦) h ( mp 1) (○) my ( mp 3) (□).

Fig. 7 -

 7 Fig. 7-Parthenogenetic females of S. venezuelensis after exposure to 4 μ /m f 72 h of incubation. Parasite of the control group with preserved cuticle (A); Parasite exposed to myricitrin, with cuticular wrinkling (arrow) (B); Parasite exposed to F 2 , with cuticular alterations (arrows) (C); Parasite treated with ivermectin, with preserved cuticular integrity (D).

Figure 1

 1 Figure 1 Expanded chromatogram profile of the LPFMz.

Figure 2

 2 Figure 2 Compounds detected by GC-MS in the LPFMz.

Figure 3

 3 Figure 3 HPLC elution of the compounds 1, 2, 3 and 4 in the HPFMz.

Figure 7

 7 Figure 7 Parthenogenetic females of S. venezuelensis f xp 4 μ /m f

  C 21 H 19 O 12 , m/z 463.0877) and [M + H] + , m/z 465.1032 (calculated for C 21 H 21 O 12 , m/z 465.1033).

	Compound 4: Yellow powder; R f ~0.85 with yellow fluorescence at 366 nm [α] 25	D : -
	58° (c 0.1 g/100 mL, CH 2 OH), λ max : 263, 337 nm. 1 H NMR (CD 3 OD): δ 0.95 (3H, d, J
	= 5.7, H-") 3.32 (m H5") 3.33 (m H4") 3.75 (1H	, J = 9.2, 3.5 H-3") 3.88 (3H
	4'-OCH 3 ), 4.22 (1H, dd, J = 3.5, 1.9 Hz, H-2") 5.3 (1H	J = 1.9 Hz, H-1") .13
	(1H, d, J = 2.2 Hz, H-6), 6.27 (1H, d, J = 2.2 Hz), 6.87 (2H, s, H-2' H-'); 13 C NMR
	(CD 3 OD): 168.27 (C-7), 158.77 (C-2), 152.28 (C-3' -5') 139.28 ( -4') 127.42 ( -
	1') 1 9.91 ( -2' -') 1 3. 3 (C-1") 1 . 5 ( -6), 95.85 (C-8), 73.40 (C-5") 72.27
	(C-4") 72.15 ( -3") 72. 5 ( -2") 1. 4 (4'-OCH 3 ), 17.86 (C-"). H E IT F	[
	-H]

1 . ( -6), 94.83 (C-8), 73.34 (C-4") 72.11 ( -3") 72. ( -5") 71.87 ( -2") 17. 4 ( -"). H E IT F [ -H] -, m/z 463.0877 (calculated for -, m/z 477.1039 (calculated for C 22 H 21 O 12 , m/z 477.1033) and [M + H] + , m/z 479.1180 (calculated for C 22 H 23 O 12 , m/z 479.1190).

  All the experiments employing animals were approved by the Ethics Committee for the Use of Animals (CEUA/UNICAMP, protocol no. 4089-1/2015) and complied with the ethical principles for animal experimentation adopted by CEUA, State University of Campinas. Adults (triploid parthenogenetic females) of Strongyloides venezuelensis were isolated from the rodent Bolomys lasiuris and maintained in Rattus norvegicus.

	Infective larvae (L3) of S. venezuelensis were obtained by coproculture. The cultures
	were collected, concentrated and mantained at 28 ºC for 48 h. For the recovery of adult
	female parasites, male SPF Wistar rats were deliberately infected with S. venezuelensis
	(2,000 larvae) during 15 days. Subsequently, rats were euthanasied (by concussion) and
	15 cm of the intestines were removed, longitudinally sectioned, and placed in Petri
	dishes containing RPMI 1640 medium. The parasites leave the intestinal mucosa after 2
	h of incubation at 37 ºC.
	16-. 3 μ /m ) w	f	wh	z	(1 -
	. 3 μ /m )	f	z	( 4-.125 μ /m ) w	mp y	p itive controls for
	antifungal activity. The concentrations of DMSO were less than 0.5 %, thus not toxic
	for the micro-organisms. MIC was determined as the lowest concentration that
	prevented microbial growth.
	2.6 Anthelmintic activity

  3. The signal at δ C 17.6 ± 0.2, assigned to the C-6 methyl carbon of the sugar, together with the set of 479.0820 (calculated for C 21 H 19 O 13 , with m/z 479.0826), giving the molecular formula C 21 H 19 O 13 with mass of 480. The structural elucidation of this compound was

	signals at δ C 71.8 (± 0.2), 72.1 (± 0.4), 73.3 (± 0.1), and 72.0 (± 0.2), enabled its
	classification as a rhamnose.
	HRESITOFMS spectrum of compound 2 presented pseudomolecular ion peaks [M +
	H] + with m/z 481.0986 (calculated for C 21 H 21 O 13 , with m/z 481.0982) and [ -H] -with
	m/z

  presented the molecular formula C 21 H 19 O 13 , with mass of 464, derived of the base formula C 21 H 19 O 12 resulting from the negative mode add [ -H]

-with m/z 463.0877 (calculated for C 21 H 19 O 12 , with m/z 463.0877) and the positive mode ion [M + H] + with m/z 465.1032 (calculated for C 21 H 21 O 12 , with m/z 465.1033). This compound

Table 1 :

 1 GC-MS analysis of the chemical composition of the LPFMz

	666
		Type	RT a (mints)	Compound b	m⁄z	Molecular formula	Peak area (%)
		A	32.76	Squalene	410	C 30 H 50	5.94
		B	36.61	n-Hentriacontane	436	C 31 H 64	2.83
		C	37.35	α-Tocopherol	430	C 29 H 50 O 2	0.86
		5	41.69	Germanicol	426	C 30 H 50 O	14.79
		6	42.13	β-Amyrin	426	C 30 H 50 O	3.89
		7	43.46	α-Amyrin	426	C 30 H 50 O	6.54
		8	44.54	Germanicol acetate	468	C 32 H 52 O	4.84
		9	45.15	β-Amyrin acetate	468	C 32 H 52 O 2	13.90
		10	45.59	α-Amyrin acetate	468	C 32 H 52 O 2
							31.04
		11	46.60	Lupeol acetate	468	C 32 H 52 O 2
	667	a Retention time (mints).

b Compounds listed in order of elution from an HP-5MS capillary column. 668

Table 2 :

 2 Minimum inhibitory concentrations (MICs) of the EEMz, HPFMz, LPFMz, and isolated flavonoids (compounds 2, 3, and 4) obtained from the leaves of M. zapota.

	MIC (µg/mL)

*

Positive control; a fluconazole; b itraconazole; c oxacillin.

CONFLICTS OF INTEREST

The authors declare that they have no conflicts of interest.