Matthieu Furet

Max Lettl

Philippe Wenger

Kinematic analysis of planar tensegrity 2-X manipulators

Keywords: Tensegrity, Kinematics, Singularity, Workspace, Cuspidal

This paper analyzes the kinematics of planar tensegrity manipulators made of two Snelson's X-shape mechanisms in series. The variable instantaneous center of rotation of each mechanism renders the kinematic analysis of the resulting manipulator more challenging. A general formulation of the direct kinematics is set. A method is proposed to solve the inverse kinematic problem in a symbolic way and up to four inverse kinematic solutions are found. The singularities of the manipulator are shown to divide the joint space into two singularity-free components, showing for the first time a planar positioning manipulator that can be cuspidal. The workspace is determined and plotted for different values of the geometric parameters.

Introduction

A tensegrity structure is an assembly of compressive elements (bars) and tensile elements (cables, springs) held together in equilibrium [START_REF] Fuller | Tensile-integrity structures[END_REF], [START_REF] Motro | Tensegrity systems: the state of the art[END_REF]. Tensegrity is known in architecture and art for more than a century [START_REF] Skelton | Tensegrity Systems[END_REF] and is suitable for modeling living organisms [START_REF] Levin | The tensegrity-truss as a model for spinal mechanics: biotensegrity[END_REF]. Tensegrity mechanisms have been more recently studied for their promising properties in robotics such as low inertia, natural compliance and deployability [START_REF] Arsenault | Kinematic, static and dynamic analysis of a planar 2-dof tensegrity mechanism[END_REF], [START_REF] Crane | Kinematic analysis of a planar tensegrity mechanism with presstressed springs[END_REF], [START_REF] Wenger | Kinetostatic Analysis and Solution Classification of a Planar Tensegrity Mechanism[END_REF]. A tensegrity mechanism is obtained when one or several elements are actuated. This work falls withing the context of the AVINECK project involving biologists and roboticists with the main objective to model and design bird necks. Accordingly, a class of planar tensegrity manipulators made of a series assembly of several Snelson's X-shape mechanisms [START_REF] Snelson | Continuous Tension, Discontinuous Compression Structures[END_REF] i.e. crossed four-bar mechanisms with springs along their lateral sides, has been chosen as a suitable candidate for a preliminary planar model of a bird neck, see figure 1. First investigations on the kinematics of such manipulators turn out to be more challenging than expected with interesting properties, which has motivated the work presented in this paper. Snelson's X-shape mechanisms have been studied by a number of researchers, either as a single mechanism [START_REF] Arsenault | Kinematic, static and dynamic analysis of a planar 2-dof tensegrity mechanism[END_REF], [START_REF] Wenger | Kinetostatic Analysis and Solution Classification of a Planar Tensegrity Mechanism[END_REF], [START_REF] Boehler | Definition and computation of tensegrity mechanism workspace[END_REF] or assembled in series [START_REF] Aldrich | Time-energy optimal control of hyper-actuated mechanical systems with geometric path constraints[END_REF], [START_REF] Chen | Analytical Computation of the Actuator and Cartesian Workspace Boundaries for a Planar 2-Degree-of-Freedom Translational Tensegrity Mechanism[END_REF], [START_REF] Bakker | Design of an environmentally interactive continuum manipulator[END_REF]. A planar two-degree-of-freedom manipulator is obtained with a series assembly of two such mechanisms. The manipulator can be driven with tendons threaded through the spring attachment points like in [START_REF] Bakker | Design of an environmentally interactive continuum manipulator[END_REF], or it can be actuated with a rotary motor at a base joint of each mechanism. There are several possible actuation schemes that can involve over-actuation if stiffness needs also to be controlled [START_REF] Aldrich | Time-energy optimal control of hyper-actuated mechanical systems with geometric path constraints[END_REF]. The detailed actuation scheme is not reported here and will be the scope of further work. This paper focuses on the full kinematic analysis of the manipulator, which has never been done before to the best of the authors' knowledge. The variable instantaneous center of rotation of each mechanism renders the symbolic calculation of the inverse geometric problem more challenging, resulting in polynomials of excessive degree if the problem is not tackled with care. A practical method is provided that makes it possible to come up with a quartic polynomial, which turns out to be of minimal degree. The singularities of the manipulator are shown to divide the joint space into two singularity-free components or aspects, showing for the first time a planar positioning manipulator that is cuspidal i.e. that can perform non-singular solution changing motions. The workspace is calculated with its boundaries for different values of the geometric parameters. 2 Derivation of the kinematic equations

Manipulator description and parametrization

The manipulator studied consists of a series assembly of two identical crossedbar mechanisms as shown in figure 2. Each mechanism i has a base bar and an upper bar of length b and two crossed bars of length L. Note that the mechanism assembly condition is L>b. The springs, shown in green in figures 1 and 2, are of length l ij . We need to define a suitable joint variable that describes each mechanism configuration without any ambiguity. In [START_REF] Bakker | Design of an environmentally interactive continuum manipulator[END_REF], the mechanism configuration was chosen as the orientation α i of the upper bar with respect to the base bar, but such a choice is allowed for mechanism motions restricted to -π < α i < π. For a matter of completeness, we are interested in allowing each mechanism to move within its full range. In this case,-2π < α i < 2π and α i is not appropriate to define the mechanism configuration. Let us introduce a line segment of length l i that links the middle points of the top and base bars of each mechanism i (shown in red dotted line in figure 2). The angle between this line and the direction orthogonal to the base bar is referred to as θ i . For the symmetric design considered here, it can be shown that α i = 2θ i and when -π < θ i < π, the mechanism makes a full turn. Assuming that each mechanism remains always in the crossed-bar assembly mode, the manipulator configuration can thus be fully defined with (θ 1 , θ 2 ). Since the mechanism sides define an isosceles trapezoid,

l i (θ i ) = l i1 + l i2 2 = L 2 -b 2 cos 2 (θ i ) (1)

Direct Kinematics

We first want to establish the direct kinematic equations. The base frame is centered at the middle point of the base bar with the x-axis aligned along this bar. The reference point (x, y) is chosen as the middle point of the top-bar (figure 2). Note that any other location on the top bar would not affect the study, with the exception of one of the two extremities. In such a case, indeed, the input/output equations would be the same as in a simple 2R planar manipulator and the kinematic properties of the manipulator studied would not be captured. The direct kinematic equations of a manipulator with n mechanisms in series can be written as for a virtual serial nR manipulator with (varying) link lengths l i and joint angles θ i :

x = n i=1 l i (θ i ) cos( π-2θi 2 + i j=1 2θ j ) y = n i=1 l i (θ i ) sin( π-2θi 2 + i j=1 2θ j ) (2)
For a manipulator made of two mechanisms in series with input variables θ 1 and θ 2 , the direct kinematic equations can be put in the following form :

x = -l 1 (θ 1 ) sin(θ 1 ) -l 2 (θ 2 ) sin(2θ 1 + θ 2 ) y = l 1 (θ 1 ) cos(θ 1 ) + l 2 (θ 2 ) cos(2θ 1 + θ 2 ) (3)
where l 1 and l 2 are defined in [START_REF] Fuller | Tensile-integrity structures[END_REF]. Note that these equations assume that each mechanism is in its crossed-bar assembly-mode.

Inverse Kinematics

We would like now to derive the so-called characteristic polynomial, a univariate polynomial established to solve the inverse kinematics. The direct kinematic equations ( 3) have been derived in their most compact form, namely, the two output variables x and y are expressed as a function of the two input variables θ 1 and θ 2 like in a 2R serial manipulator. However, starting from these equations to derive a characteristic polynomial is not appropriate. Indeed, the square roots appearing in l 1 and l 2 should be first cleared out, with the consequence of raising artificially the degree of the resulting characteristic polynomial and providing spurious solutions. Accordingly, it is better to write x and y as functions of the crossed-bar angles φ 1 and ψ 2 as follows:

x = -b 2 + L cos(φ 1 ) + L cos(ψ 2 + α 1 ) + 1 2 b cos(α 1 + α 2 ) y = L sin(φ 1 ) + L sin(ψ 2 + α 1 ) + 1 2 b sin(α 1 + α 2 ) (4)
In addition, the two loop-closure equations below are derived:

-2Lb sin(α 1 ) sin(φ 1 ) -2Lb(cos(α 1 ) + 1) cos(φ 1 ) + 2b 2 (cos(α 1 ) + 1) = 0 2Lb sin(α 2 ) sin(ψ 2 ) + 2Lb(cos(α 2 ) + 1) cos(ψ 2 ) + 2b 2 (cos(α 2 ) + 1) = 0 (5) A set of four equations in four unknowns is thus available. A univariate polynomial can be obtained upon elimination of three of the four unknowns. There are several ways to do so with a computer algebra software. We have used Maple and its Siropa library [START_REF] Rouillier | [END_REF]. This library was developed in the frame of a collaborative project on the kinematics of parallel manipulators, see e.g. [START_REF] Manubens | Cusp Points in the Parameter Space of Degenerate 3-RPR Planar Parallel Manipulators[END_REF], [START_REF] Moroz | On the determination of cusp points of 3-RPR parallel manipulators[END_REF] for more details on the implementation and use of these tools. It contains specific macro-functions that use efficient algebraic tools such as Groebner bases. Specifically, the Projection function was used to project the system of four equations in order to obtain one single equation in one single variable, chosen here as φ 1 . The half-tangent substitution yields a factored polynomial, one of which defines the characteristic polynomial. The characteristic polynomial obtained turns out to be of degree 4 in t=tan(φ 1 /2) and can be written as follows:

a 4 t 4 + a 3 t 3 + a 2 t 2 + a 1 t + a 0 = 0 (6)
where :

a 4 = (b + 1) 2 (b 2 y 2 + x 4 + 2x 2 y 2 + y 4 + 4x 3 + 4xy 2 + 5x 2 + y 2 + 2x) (7)
a 3 = 4y(b + 1)(2b 2 x + b 2 -2x 2 -2y 2 -4x -1) (8)
a 2 = 2(b 4 y 2 + b 2 x 4 + 2b 2 x 2 y 2 + b 2 y 4 + b 2 x 2 -10b 2 y 2 + x 4 + 2x 2 y 2 + y 4 -3x 2 + 9y 2 ) (9) a 1 = 4y(b -1)(2b 2 x -b 2 + 2x 2 + 2y 2 -4x + 1) (10)
a 0 = (b -1) 2 (b 2 y 2 + x 4 + 2x 2 y 2 + y 4 -4x 3 -4xy 2 + 5x 2 + y 2 -2x) (11)
Note that L was set equal to 1 to simplify the calculations without loss of generality. For each solution φ 1 , α 1 is then solved from the first equation in ( 5) (disregarding the solution α 1 = π corresponding to the parallelogram assembly):

tan(α 1 /2) = -L cos(φ 1 ) + b L sin(φ 1 ) (12)
Finally, ψ 2 and α 2 are solved from system (4), which gives two solutions, one of which never satisfies the closed-loop equations [START_REF] Arsenault | Kinematic, static and dynamic analysis of a planar 2-dof tensegrity mechanism[END_REF]. In total, thus, one comes up with a maximum number of four solutions. The inverse kinematics is solved for a manipulator defined by L = 1 and b = 2/3 at x = 0.03 and y = 1.6. Four solutions are found (see figure 3), showing that the characteristic polynomial ( 6) is of minimal degree.

Singularity analysis and aspects

The Jacobian matrix of the manipulator is derived from system (3) and its determinant is calculated and can be put in the following form:

Det(J) = (sin(θ 1 ) 2 b 2 cos(θ 1 ) -(L 2 -b 2 cos(θ 1 ) 2 )(cos(θ 1 ) + 2 cos(θ 2 + 2θ 1 ))) (cos(θ 2 + 2θ 1 )b 2 cos(θ 2 ) sin(θ 2 ) -(L 2 -b 2 cos(θ 2 ) 2 ) sin(θ 2 + 2θ 1 )) -(-sin(θ 2 + 2θ 1 )b 2 cos(θ 2 ) sin(θ 2 ) -(L 2 -b 2 cos(θ 2 ) 2 ) cos(θ 2 + 2θ 1 )) (cos(θ 1 ) 2 b 2 sin(θ 1 ) -(L 2 -b 2 cos(θ 1 ) 2 )(sin(θ 1 ) + 2 sin(θ 2 + 2θ 1 ))) (13)
Solving Det(J) = 0 for θ 1 yields two solution sets. The first one is θ 1 = -θ 2 ± π and means that the two modules are coincident. It is equivalent to the fully-folded back configuration of a planar 2R serial manipulator. When the manipulator is in this singularity, the reference point is at the origin x = 0 and y = 0, whatever θ 1 . The second solution is given by:

θ 1 = atan2(-sin(θ 2 )(4b 4 cos(θ 2 ) 4 -4L 2 b 2 cos(θ 2 ) 2 -L 2 b 2 + L 4 ), cos(θ 2 )(4b 4 cos(θ 2 ) 4 -4b 4 cos(θ 2 ) 2 -4L 2 b 2 cos(θ 2 ) 2 + 3L 2 b 2 + L 4 )) ( 14
)
This solution is associated with the configuration where the two instantaneous centers of rotation (ICR) are aligned with the end-effector control point, like in the fully outstretched configuration of a planar 2R serial manipulator. In this singularity, the manipulator cannot produce an instantaneous motion along a line passing through the two mechanism ICR and the end-effector control point. The above singularities divide the joint space into singularity-free domains called aspects [START_REF] Borrel | A study of manipulator inverse geometric solutions with application to trajectory planning and workspace determination[END_REF]. The singularity θ 1 = -θ 2 ±π produces two lines while the singularity defined by equation ( 14) gives a curve, as shown in figure 4. In the absence of joint limits, the opposite sides of the square are in fact and the singularity curves divide the joint space into only two aspects. Since the manipulator admits up to four solutions, there are two solutions in each aspect, which means that the manipulators can move from one inverse kinematic solution to another without meeting a singularity, namely, it is cuspidal [START_REF] Wenger | Cuspidal and noncuspidal robot manipulators[END_REF]. To the authors' knowledge, it is the first time that a planar positioning manipulator is shown to be cuspidal.

Workspace analysis

When plotted into the workspace, the singularity curves define its boundaries. The equation of these curves can be obtained by deriving the discriminant of the characteristic polynomial [START_REF] Crane | Kinematic analysis of a planar tensegrity mechanism with presstressed springs[END_REF]. By doing so, a polynomial of degree 16 in x and y is obtained. Its expression is quite large and shall not be reported here for lack of space. Figure 5 shows the plot of these curves for three examples. In the second and third figures, they divide the workspace into three regions. In the largest one, the manipulator admits two inverse kinematic solutions. In the two smaller regions, there are four solutions. Clearly, the 4-solution regions get larger when b is increased. The boundaries of these two 4-solution regions have three singular points: a node and two cusps. The existence of cusps confirms the fact that the manipulator is cuspidal. A non-singular solution changing motion can be defined by encircling one of the cusps [START_REF] Omri | How to recognize simply a non-singular posture changing manipulator[END_REF], [START_REF] Wenger | Cuspidal and noncuspidal robot manipulators[END_REF]. Note that the two aspects map onto the whole workspace, namely, figure 5 also shows the image of any of the two aspects.

Conclusion

The complete kinematic analysis of a planar manipulator made of two X-four bar mechanisms in series has been carried out. The manipulator was shown to have two or four solutions depending on its geometric parameter values. When it has four solutions, the manipulator turns out to be cuspidal, a result that was not expected. The influence of the geometric parameters on the shape and size of the workspace was analyzed. The influence of joint limits was not reported for lack of space and shall be studied in a near future as they also play an important role. Internal collisions were not considered since a suitable design by assembling the rods in different planes allows avoiding any physical interference. Future work will be conducted on the actuation strategy.

Fig. 1 :

 1 Fig. 1: Snelson's X-shape mechanism (left) and a series assembly of several such mechanisms, mimicking a bird neck (right)

1 Fig. 2 :

 12 Fig. 2: Manipulator description

Fig. 3 :

 3 Fig. 3: The four inverse solutions at x = 0.03 and y = 1.6 (L = 1 and b = 2/3)

2 Fig. 4 :

 24 Fig. 4: Singularity curves in the joint space and aspects when L = 1 and b = 2/3

Fig. 5 :

 5 Fig. 5: Workspace boundaries when L = 1 and b = 2/5 (left), b = 2/3 (center), b = 9/10 (right)

Acknowledgement This work was conducted with the support of the French National Research Agency (AVINECK Project ANR-16-CE33-0025).


Romain Tisserand

Thomas Robert

Pascal Chabaud

Marc Bonnefoy

Elderly fallers enhance dynamic stability through anticipatory postural adjustments during a choice stepping reaction time

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

Elderly fallers enhance dynamic stability through anticipatory postural adjustments during a choice stepping reaction time

Tisserand R. 1 , Robert T. 1 , Chabaud P. 2 , Bonnefoy M. 3 , Chèze L.

RESULTS:

Total stepping time was longer in F compared to NF, independently of the target direction. This elongation is due to a significant increase of the APA phase duration. Both APA subphases ("loading" and "unloading" mechanisms) were significantly elongated. Elderly F did not make more APA errors than NF but used two distinct balance strategies during APA, depending on the axis (Fig B). In the AP direction, F had a smaller backward movement and slower peak velocity of the CoP. In the ML direction, the CoP movement was similar in amplitude and peak velocity between groups but lasted longer. The biomechanical consequence was an increased MoS at FO in both directions.

CONCLUSIONS:

The results about the step timings are concurring with previous studies in the literature. The presence of APA error seems however not to be an evident reason for APA elongation in F. Analysis of APA tend to indicate that F voluntarily elongate their APA during a CSRT. This strategy allows increasing the condition for dynamic stability at the end of the APA, i.e. when the base of support is reduced to only one foot. By delaying the FO, elderly F chose to prioritize the stability to the detriment of the objective of the task (a quick step), probably because a higher fear of falling. It is concluded that elderly F used a "safer" balance strategy than NF during the CSRT. If delaying the FO is possible during a voluntary step initiation task, it may however become a severe balance issue in a more demanding context such as protective steps. An elongated APA duration during the CSRT could thus be understood as an indicator of the risk of fall in community-dwelling elderly. ACKNOWLEDGEMENTS AND FUNDING: Romain Tisserand held a doctoral fellowship from La Région Rhône-Alpes.

As APA are not invariant but adapted to external and internal constraints, we hypothesized that elongation of the APA in elderly F is the result of a strategy to increase their conditions for dynamic stability.

Trajectories of 39 reflective markers and ground reaction forces from 4 forceplates were collected.


Beya Bachtobji Bouachir

Ali Khorchani

Frédéric Guibal

Mohamed Hedi

El Aouni

Abdelhamid Khaldi

Beya Bachtobji Bouachir

Mohamed Hédi

Dendroecological study of Pinus halepensis and Pinus pinea in northeast coastal dunes in Tunisia according to distance from the shoreline and dieback intensity

Keywords:

come

Introduction

Forests, which cover about 30 percent of the world's land surface (FAO, 2006), are being rapidly and directly impacted in many areas by human expansion. Less evident are the pervasive effects of ongoing climatic changes on the condition and status of forests around the world. Increased greenhouse gas emissions are now widely acknowledged by the scientific community as a major cause of the recent rise in global mean temperature and changes in the world's hydrological cycle (IPCC, 2007). Understanding and predicting the consequences of these climatic changes on ecosystems is one of the greatest challenges for scientists, and forecasting the impacts on forests is particularly important [START_REF] Boisvenue | Impacts of climate change on natural forestproductivityevidence since the middle of the 20th century[END_REF][START_REF] Bonan | Forests and climate change: forcings, feedbacks, and the climatebenefits of forests[END_REF]. Of these impacts on forests, tree dieback is a serious phenomenon on a local, regional and national level [START_REF] Allen | A global overview of drought and heat-induced tree mortality reveals emerging climate change risks for forests[END_REF]. This phenomenon alters the external appearance of the tree due to organ mortality, reducing the quality and quantity of foliage, and may cause stunted growth or even death in some trees [START_REF] Delatour | Dépérissement des chênes et pathogènes Revue Forestière Français XLII 2[END_REF]. This dieback phenomenon is either caused by pathogens, parasites or conditions like acid rain and drought [START_REF] Allen | Climate-induced Forest Dieback: an Escalating Global Phenomenon[END_REF].

Examples of drought and heat-related forest stress and dieback are being documented by all forested continents, making it possible to begin to see global phenomena. Two of the nine tipping points for major climate changes forecast for the next century are directly related to forest dieback (IPCC, 2014). Examples of dieback have been particularly well documented in southern Europe [START_REF] Breda | Temperate forest trees and standsunder severe drought: a review of ecophysiological responses, adaptation processes and long-term consequences[END_REF][START_REF] Bigler | Drought as aninciting mortality factor in Scots pine stands of the Valais, Switzerland[END_REF] and in the temperate and boreal forests of western North America, where background mortality rates have increased rapidly in the past few decades [START_REF] Van Mantgem | Widespreadincrease of tree mortality rates in the western United States[END_REF]. The common causal factor in these examples is elevated temperatures and/or water stress. Recent extreme drought in North Africa [START_REF] Touchan | Long term context for recent drought in northwestern Africa[END_REF] has been linked to severe mortality of Atlas cedar (Cedrus atlantica) from Morocco to Algeria [START_REF] El Abidine | Forest decline in Morocco: causes and control strategy[END_REF][START_REF] Bentouati | La situation du cèdre de l'Atlas en Algérie[END_REF]. Disturbing events have occurred in some forest areas in Tunisia over the last few years [START_REF] Jamaa | Le dépérissement du chêne liège dans la subéraie de Bellif (Tunisie): caractérisation et évaluation de son impact sur l'accroissement du Liège[END_REF], 2006a), raising a number of issues including the effects of drought and global warming (Ben Jamaa et al., 2006b;[START_REF] Khorchani | Evolution des relations croissance-climat de trois espèces de pin en Kroumirie (Nord-Ouest de la Tunisie)[END_REF]. Sudden and widespread mortality struck cork oak forests in Tunisia in the late 80s and early 90s after three years of consecutive drought, affecting more than 20% of the trees in different cork oak forests [START_REF] Hasnaoui | Contribution à l'étude du dépérissement du chêne-liège dans la forêt de Tabarka: relations possibles entre nutriments et état sanitaire de l'arbre[END_REF]. The same dieback phenomenon was observed between 2000 and 2003 in Aleppo pine forests in a semiarid bioclimate [START_REF] Aloui | Changements climatiques en Tunisie: impacts sur les forêts[END_REF].

One of the most vulnerable areas to climate change is the coast where about 30% of the entire coastline is affected by erosion (EEA, 2006). According to commonly accepted models of coastal dynamics [START_REF] Bruun | The Bruun Rule of erosion by sea level rise: a discussion on large-scale two-and three-dimensional usages[END_REF][START_REF] Davidson-Arnott | Conceptual model of the effects of sea level rise on sandy coast[END_REF], an increase in coastal erosion should therefore be expected in the future together with an increase in vulnerability of the coastal ecosystem (IPCC, 2007). Tree dieback along the coast is due to a combination of factors unfavorable to tree growth such as drought, storms, and nutritional problems which lead to overall weakening of the tree and possible death [START_REF] Garrec | Les dépérissements littoraux d'arbres forestiers[END_REF]. Moreover, pollution from urbanization, industry, maritime traffic and agriculture converge in coastal waters. This ecosystem degradation directly affects vegetation under stress and demonstrates a rupture in forest management. Since the 80s, considerable damage has been observed in the maritime pine forests of northern Tunisia, in Bizerte (forests of M'hibes and Rimel) and Tabarka (Sidi Badr forests), following the attack of the insect Tomicus piniperda L. (Ben Jamaa et al., 2000). According to a dendroclimatological study of the impact of drought on the growth of three pine species in Tunisia (maritime pine, stone pine and Aleppo pine), the number of dry years and observably narrower rings increased from 1978 to 2001 compared to the period between 1954and 1977[START_REF] El Khorchani | Approche Dendrochronologique De l'influence Des Changements Climatiques Sur La Productivité Des Forêts De Pin d'Alep (Pinus Halepensis Mill[END_REF]. Deterioration of coastal vegetation in the north of Tunisia is also related to the presence of several pollutants in the marine spray such as anionic surfactants and hydrocarbons [START_REF] El Ayeb | Effets des embruns marins pollués sur les feuilles d'Acacia cyanophylla Lindl. et d'Eucalyptus gomphocephala Dc. du littoral tunisien[END_REF]. The acceleration of climate change, extreme climatic events (storms, heat, frost, drought and excess water), anthropogenic changes in land use, and pollution are all factors which can contribute directly or indirectly, alone or in interaction, to the explosion of cases of ecosystem dysfunction. Sustainable and responsible management therefore requires new adaptation strategies. These may include assessing forest vulnerability to climate change, reviewing forest use expectations, identifying research and education needs, developing forest policies to facilitate and anticipate adaptation and maintaining the health and vitality of forest ecosystems [START_REF] Spittlehouse | [END_REF]. Knowing the extent of the territory affected by dieback will contribute greatly to the decision-making process concerning this crisis. For example, some species currently widely used for reforestation of coastal dunes in Tunisia may be restricted in the future and replaced by other species more resistant to drought, or their silvicultural management techniques may be revised in order to adapt to climate and territorial changes.

Two of the main species used to reforest Mediterranean dune ecosystems are Pinus pinea and Pinus halepensis. These pine species have to survive in hard environmental conditions with constant exposure to threatening marine spray and water stress conditions [START_REF] Garrec | Les dépérissements littoraux d'arbres forestiers[END_REF]. In southeast Spain, [START_REF] Raventós | Growth of Pinus pinea and Pinus halepensis as affected by dryness, marine spray and land use changes in a Mediterranean semiarid ecosystem[END_REF] showed that Pinus pinea and Pinus halepensis are highly sensitive to exposure both to sea spray and to surfactants in marine aerosols, as reported in other parts of the Mediterranean coast of Spain (Diamentapolos et al., 2000). Reduced growth of Pinus pinea along the Tyrrhenian coast of Tuscany (Italy) is due to the crown's greater exposure to salty winds and surfactants and the increased salinity of the water table [START_REF] Raddi | The impact of sea erosion on coastal Pinus pinea stands: a diachronic analysis combining tree-rings and ecological markers[END_REF].

Remel and Menel Belgacem forests, pine coastal woodlands of Tunisia, are good examples for studying the vulnerability of these two species under severe conditions of marine spray and drought. In these areas located along the northern coastal dunes, Pinus pinea and Pinus halepensis have clearly suffered canopy damage due to the proximity of the shoreline. In this study, a dendroecological approach was used to estimate the effect of distance from the shoreline on two pine species, Pinus pinea and Pinus halepensis, growing in two forest sites in northeast Tunisia.

Material and methods

Study area

The study was carried out in northeastern Tunisia (Fig. 1) on two coastal Pinus pinea and Pinus halepensis planted forests. The first site is located in Remel Forest (37°11′N, 10°04′E) in the region of Bizerte, and the second is in Menzel Belgacem Forest (36°57′N, 10°57′E) in Kelibia in the Cap Bon region. These forest sites have a sandy dune ecosystem and belong to the Mediterranean subhumid bioclimate. The mean annual rainfall in the Bizerte site is 656 mm, concentrated mainly in the winter. In Kelibia, the mean annual rainfall is 550 mm, mostly falling in autumn. Annual temperature varies from 7 °C (January) to 32 °C (August) in Bizerte and from 8 °C (January) to 31 °C (August) in Kelibia. Northwestern winds with a mean annual speed of 3.8 m s -1 and 3 m s -1 are most dominant in Bizerte and Kelibia, respectively.

The sand dunes of both sites have been afforested with species such as Acacia sp, Eucalyptus sp., Pinus halepensis, Pinus pinea and Pinus pinaster since the 1900s to fix the dunes along the coastal area.

Collection of data and measurements

Structural data

From 2010-2015, 12 plantation stands of Pinus pinea and Pinus halepensis with different environmental conditions were studied in the Remel and Menzel Belgacem forests. For each species in each forest site, three stands were sampled along a latitudinal transect according to distance from the shoreline. Characterization of the stands was analyzed by spiral plots according to the protocol used by the Department of Forest Health in France for determining tree dieback classes (Office National des Forêts, 2010). The center of the plot being fixed by the sampling plan, the first tree is the closest to the center of the plot and the following trees are chosen step by step in a spiral to reach the defined number (recommended: 20 trees within a maximum radius of 40 m). The method consists of estimating leaf deficit at the crown level of each sampled tree. Leaf deficit includes the absence of needles due to drop; twig drop, dried by wind or insects; reduced growth resulting in fewer branches or a smaller individual leaf area due to the phenomenon of microphylla or the impact of insects or foliar diseases. To assess leaf deficit, the appearance of the crown is described by assigning a dieback index. The dieback index in% is calculated by observing the foliar deficit in the crown. In order to limit the interference between phytosanitary problems and symptoms related to competition or lack of light, only dominant or codominant trees were sampled. For each plot, tree density (trees ha -1 ), diameter at breast height (DBH, cm), height (m), height and crown diameter (m) for 20 trees were measured. Table 1 shows the characteristics of each stand based on the distance from the shoreline and dieback intensity.

Dendroecological data

For the dendroecological study, 15-30 trees were selected in each plot and 2-3 cores were extracted from each tree. Cores were collected at breast height (1.30 m) with a Swedish increment borer 5 mm in diameter. All cores were placed on wooden supports and specimen details such as site name, sample number and collection date were recorded for identification purposes. These cores were polished using a sanding machine with progressively finer sandpaper (×100 × 180) to obtain a smooth surface.

Cores were visually crossdated with each other by using regional pointer years (missing rings, false rings and narrow rings). The computer program COFECHA [START_REF] Holmes | Computer-assisted quality control in tree ring dating and measurement[END_REF]) was run to examine the statistical quality of the association between the aligned series. COFECHA standardizes raw ring width series and subsequently evaluates the statistical significance of the correlation of each whole series and series segment with a master series by using the Pearson coefficient (r-value) [START_REF] Grissino-Mayer | Evaluating crossdating accuracy: a manual and tutorial for the computer program COFECHA[END_REF]. The segment length of 20 years lagged by 10 years was used for this study. Placement of missing rings was checked by direct comparison of the wood samples under a microscope and by COFECHA. The frequency of missing rings in each series was calculated for each calendar year. Ring widths were measured from bark to pith with a precision of 0.01 mm using the TSAP Win software and LINTAB ® [START_REF] Rinn | TSAP-Win: Time Series Analysis and Presentation for Dendrochronology and Related Applications[END_REF] measuring table in the National Institute for Research in Rural Engineering, Water and Forests of Tunis and in the Mediterranean Institute of Biodiversity and Ecology of Marseille. Ring width series were standardized using the ARSTAN program [START_REF] Cook | The smoothing spline: a new approach to standardizing forest interior tree-ring width series for dendroclimatic studies[END_REF][START_REF] Holmes | Treeing Chronologies of Western North America: California, Eastern Oregon and Northern Great Basin, with Procedures Used in the Chronology Development Work, Including User Manuals for Computer Programs COFECHA and ARSTAN[END_REF]. The long-term trend was removed from each raw chronology by fitting a curve and calculating an index defined as measured ring width for each year divided by the curve-fit value. This made it possible to remove age trend, non-climatic factors and the effects of stand dynamics [START_REF] Cook | Methods of Dendrochronology[END_REF]. Thus, we applied a double detrending with a negative exponential curve or linear regression equation in the first step and a cubic smoothing spline in the second step.

Finally, for each stand, a non-autoregressive chronology was computed by a technique known as "bi-weight robust estimate of the mean" designed to reduce the influence of isolated outlier values [START_REF] Cook | A Time Series Analysis Approach to Tree Ring Standardization[END_REF].

The ARSTAN program produces three chronologies: standard, residual and arstan. Residual chronology uses autoregressive modeling to remove any signal in the data that carries over from year to year and that may cause an unrealistic increase in the correlation with climate variables [START_REF] Henderson | Climate -tree growth relationships of longleaf pine (Pinus palustris mill.) in the southeastern Coastal Plain, USA[END_REF]. Because residual chronology showed the strongest and most interpretable relationship, we selected it to model the tree growth to climate relationship [START_REF] Parker | The growth response of slash pine (Pinus elliottii) to climate in the Georgia Coastal Plain[END_REF].

Dendrochronological variables calculated by ARSTAN and expressing the climate signal such as mean sensitivity, standard deviation and autocorrelation are good indicators for tree-ring to climate relationship studies [START_REF] Fritts | Tree Rings and Climate[END_REF]) (Table 2).

Climate data

To establish tree growth relationships to climate, reliable climate data close to the sampling sites are crucial. Available climate data from several meteorological stations around the sampling sites were scrutinized for their quality assessments especially in terms of homogeneity, duration and continuity of data. The monthly average of maximal and minimal temperature and monthly total precipitation data from 1950 to 2014 were collected from the two nearest meteorological stations: Bizerte (37°15′N, 09°48′E) and Kelibia (36°51′N, 11°05′E).

Tree-growth relationship to climate

A response function analysis [START_REF] Fritts | Tree Rings and Climate[END_REF] and bootstrap technique [START_REF] Guiot | The boostrapped response function[END_REF] were used to determine the relationship between tree rings and climate with 3PBase software [START_REF] Guiot | PPPBASE, software for statistica analysis of paleoecology data[END_REF]. A response function analysis is a multiple regression technique after extracting the main components of monthly climatic data (predictors) to determine the significant climatic variable influencing ring width (predictants). With the bootstrap technique, 200 iterations were done to obtain 200 sets of regression coefficients, 200 multiple calibrations and 200 verification correlation coefficients. In tree-ring analysis, this method offers the advantage of avoiding errors caused by colinearity among variables and provides a more realistic estimate of tree response to climate. Whether for regression coefficients or for the overall significance of the response function, the different calculated ratios (mean multiple correlation coefficient or mean regression coefficient divided by standard deviation) give an approximate value of the t student coefficient [START_REF] Gadbin-Henry | Etude Dendroécologique De Pinus Pinea L. Aspects méthodologiques[END_REF].

Analyses were done between residual tree ring chronologies and monthly climatic variables (precipitation, maximal and minimal temperature). These climatic variables were organized according to the biological calendar for the Mediterranean region from October of the previous year to September of the current year [START_REF] Serre | Contribution à l'étude Dendroclimatologique Du Pin d'Alep (Pinus Halepensis Mill[END_REF].

Data analysis

Mean and standard deviation were calculated for each stand parameter (Mean Height, DBH, Height of the crown, Diameter of the crown and Mean ring width). For missing rings, frequency was calculated for each stand in ten-year increments from 1955 until 2014. An analysis of variance (ANOVA) under SAS software (9.1) was applied with distance from the shoreline as a source of variation to five stand parameters. To test the effect of distance from the shoreline and the effect of time period on missing rings, a two-way analysis of variance (ANOVA) was used. The main effects of distance from the shoreline and time periods were tested for statistical differences using the Newman-Keuls test.

Results

Crossdating

From the available samples, 627 ring width series from 12 stands were crossdated successfully. Statistics for each site are listed in Table 3. Using regional pointer years (missing rings, false rings and narrow ring) made crossdating much easier. Mean series inter-correlations were reasonable and gave high values for all sites (0.709-0.980). The number of 20-ring segments with possible problems was low. Comparing site composite skeleton plots and each series with the master series showed clear similarities in growth patterns. However, correlation coefficients for some segments were not statistically significant at 99% but no negative correlation was detected in any segment. The critical correlation coefficient at the 99% confidence level associated with the selected segment length (20 years) was 0.515. For each species at each site, localizing missing rings (Table 3) and false rings (Fig. 2) in all measurement series certified good crossdating.

Stand characteristics

The results of the analysis of variance (ANOVA) revealed a significant difference in the structural measurement between stands of each species in each forest site. Along the transect, the closer the distance to the shoreline, the greater the decrease in diameter at breast height, tree height, height and crown diameter (Table 4). When comparing the three stands in each transect for each species, trees growing near the shoreline were more sensitive and smaller in radial growth on average. Stands with the highest percentage of dieback index always showed the lowest growth and structural values regardless of stand age. BZPP2 is the oldest stand (90 years) for Pinus pinea transect in Bizerte; however, the lowest growth and structural values were recorded in BZPP3 (70 years). KBPH1 is the oldest stand (69 years) for Pinus halepensis transect in Kelibia; however, the lowest growth and structural values were recorded in KBPH3 (64 years).

Missing and false rings

Table 3 shows that missing rings were observed only in stands near the shoreline for both species in both sites. The number of missing rings was higher for Pinus pinea than Pinus halepensis. The number of series with missing rings could indicate the years when there were more affected trees with missing rings. The results of crossdating showed that in the years 1988-1989 and 2002, there were more affected trees with missing rings.

Fig. 2 shows the distribution of false rings by species and site. At each site, there was a similarity in the distribution of false rings. However, this distribution was often lacking in some stands.

Table 5 shows the statistics on missing rings for the period Segments with possible problems is the number of 20-ring segments where the correlation with the master series is less than 0.515.

Fig. 2. shows the distribution of false rings by species and site. At each site, there was a similarity in the distribution of false rings. However, this distribution was often lacking in some stands.

B. Bachtobji Bouachir et al. Dendrochronologia 45 (2017) 62-72

1955-2014 using a variance analysis. For both species, no missing rings were observed for the first period (1955)(1956)(1957)(1958)(1959)(1960)(1961)(1962)(1963)(1964), but the appearance of missing rings was observed from the period 1965-1974 until 2005-2014 but only for trees growing near the shoreline. In contrast, no missing rings were recorded in trees growing far from the shoreline. Their frequency was only statistically significant for Pinus pinea in the two forest sites for the periods 1985-1994 and 1995-2004.

Climatic influence on tree-growth

The tree-growth to climate relationship of the two pine species in both studied forest sites was illustrated using a response function analysis (Figs. 3 and4). Only response functions for the combination of maximal temperature and precipitation were significant. The calculated ratios (mean multiple correlation coefficient divided by standard deviation) show that all response functions were significant. The climategrowth relationship showed two types of correlation: positive correlation with precipitation and negative correlation with maximal temperature. The monthly variables significantly correlated with growth varied between stands depending on the species and the distance from the shoreline. Most stands showed positive correlation with precipitation between October of the previous year and April of the current year. Concerning the negative correlation with maximal temperature, difference occurred along the transect, between stands for both species in both forest sites. In the Kelibia forest site, both species growing in stands far from the shoreline showed negative correlation only with the March maximal temperature, while in stands near the shoreline both species showed negative correlations with both March and April maximal temperatures. In the Bizerte forest site, negative correlation with maximal temperature was observed in May for both species growing in stands far from the shoreline, in February and March for the Pinus halepensis stand and in March and May for the Pinus pinea stand near the shoreline.

Fig. 5 shows a mixed graph of missing rings, residual site chronologies, temperature and precipitation based on the results of response function. With this graph, the reason for the increased number of missing rings in the coastal sites can be understood clearly. Dry conditions from October to April and high temperatures from March to April in Kelibia and from February to May in Bizerte seem to be the main climatic elements causing missing rings in both species. In Bizerte, the rainfall deficit was 55% and 50% for 1988 and 2002, respectively, and the increase in maximum temperature was 1.3 °C and 1.4 °C, respectively. In Kelibia, this deficit was 39% and 45% and the increase in maximum temperature was 0.67 °C and 1.67 °C, respectively. Comparisons of site chronologies for each species and site showed clear similarities in growth patterns. Missing rings occurred where rings were usually narrow. This is explained by the fact that, for a year, when a tree shows a missing ring, another tree shows a narrow ring. Missing rings are also included when computing the master series.

Discussion

Pines are important components of coastline landscapes in North Africa. Pine forests can be found along a coastal strip of this region. Of the Mediterranean pines which occur at low altitude close to the coast, Pinus halepensis, P. pinaster and P. pinea are the most frequent. P. pinea's natural range is difficult to define because it has been planted widely for so long [START_REF] Barbero | Pines of the mediterranean basin[END_REF]. Pine forests play a crucial role in this region in preventing water erosion by decreasing the effect of erosive forces and keeping the soil in place (Mazzoleni et al., 2004). The extensive root system and large crown make the species resistant to strong winds, which is why P. pinea has been used to consolidate sand dunes and protect soil from erosion in exposed coastal areas. Moreover, the multi-layered root system makes it possible for mature trees to obtain water from different levels of the soil and from various sources of water: precipitation, soil water and the water table [START_REF] Frattegiani | Quantitative analysis of Stone pine (Pinus pinea L.) root system morphology and its relationship with water table and soil characters[END_REF].

Table 4 Analysis of variance (ANOVA) with distance from the shoreline as source of variation to 5 stand parameters.

Chronology name

Common Intervals DBH (cm) H (m) Height of the crown (m) Diameter of the crown (m) Mean radial growth (1/100 mm)

Table 5 Mean number of missing rings in Pinus pinea and Pinus halepensis per decade for various types of canopy damage. 1955-1964 1965-1974 1975-1984 1985-1994 1995-2004 2005-2014

Site

BZPH1 0ab 0ab 0ab 0ab 0ab 0ab BZPH2 0ab 0ab 0ab 0ab 0ab 0ab BZPH3 0b 0b 0b 0.40b 1.89b 0b BZPP1 0b 0b 0b 0b 0b 0b BZPP2 0b 0b 0b 0b 0b 0b BZPP3 0b 1.88b 1.11b 12.44a 8a 2b KBPH1 0ab 0ab 0ab 0ab 0ab 0ab KBPH2 0ab 0ab 0ab 0ab 0ab 0ab KBPH3 0ab 0ab 0.33ab 2.66ab 5.66ab 1ab KBPP1 0ab 0ab 0ab 0ab 0ab 0ab KBPP2 0ab 0ab 0ab 0ab 0ab 0ab KBPP3 0ab 2.10ab 1.97ab 15.39a 16.71a 0.92ab
In the same line the followed figures by the same letter indicate the absence of significant differences to the threshold of 5%.

B. Bachtobji Bouachir et al. Dendrochronologia 45 (2017) 62-72 In this study, the differences in Pinus halepensis and Pinus pinea stand structure and growth reflect the effect of the distance from shoreline. Stands with the highest percentage of dieback index always showed the lowest growth and structural values regardless of stand age. Tree age should also be taken into consideration as one of the reasons for differences in growth between stands but its effect is masked by that of dieback intensity and remains minor. However, the effect of age could be important during the first years of life, when even-aged stands of Pinus spp. have high radial growth rates. These high rates are only induced by competition from shrubs and understory vegetation because of the light-demanding behavior typical of the pioneer species. However, competition in planted pinewoods is not as strong as in naturally regenerating pines. [START_REF] Mazza | Growt rate and climate responses of Pinus pinea L. in Italien oastal stands over the last centry[END_REF].

The distance from the shoreline influences environmental variables such as mean temperature, water availability, growth period extension, land use, erosion and exposition to sea spray. This characteristic was observed for these species on the Mediterranean coast in Italy [START_REF] Gellini | Colloque International d'Ecologie littorale méditerranéenne[END_REF][START_REF] Bussotti | The role of marine salt and surfactants in the decline of Tyrrhenian coastal vegetation in Italy[END_REF][START_REF] Nicolotti | Morphological and physiological damage by surfactant-polluted sea spray on Pinus pinea and Pinus halepensis[END_REF][START_REF] Raddi | The impact of sea erosion on coastal Pinus pinea stands: a diachronic analysis combining tree-rings and ecological markers[END_REF] and in Spain [START_REF] Diamantopouolos | Meccanismi di resistenza/sensibilità nei confronti dell aerosol marino inquinato nella vegetazione costiera della zona di Barcelona[END_REF]Raventos et al., 2001). According to [START_REF] Raddi | The impact of sea erosion on coastal Pinus pinea stands: a diachronic analysis combining tree-rings and ecological markers[END_REF], growth decline in Pinus pinea on the eroded coastal dunes in Italy may be attributed to greater exposure of the crowns to salty winds and surfactants and increased salinity of the water table. On the other hand, Pinus pinea is highly sensitive to exposure both to sea spray and to surfactants in marine aerosols (Raventos et al., 2001;[START_REF] Rettori | Ecophysiological responses of Mediterranean pines to simulated sea aerosol polluted with an anionic surfactant: prospects for biomonitoring[END_REF]. In the Mezel Belgacem forest, El Ayeb et al. ( 2004) noted increased Na + concentration in leaf tissue when trees were closer to the shoreline (Na + increased from 0.54 to 0.62% in Acacia and from 0.39 to 2.3% in Eucalyptus). A synergistic effect due to seawater contamination with anionic surfactants was recognized to be the cause of growth decline on the north coast of Tunisia [START_REF] El Ayeb | Effets des embruns marins pollués sur les feuilles d'Acacia cyanophylla Lindl. et d'Eucalyptus gomphocephala Dc. du littoral tunisien[END_REF]. Sea-spray containing the surfactants and hydrocarbons had a significant impact on leaves with particular deterioration of the cuticle. Indeed, deleterious effects of Na + and Cl -ions and the pollutants present in the spray on leaf tissue occurred when these substances crossed the cuticular barrier [START_REF] Bussotti | Cytological and structural changes in Pinus pinea L. needles following the application of an anionic surfactant[END_REF]. This phenomenon could explain the reduction of photosynthetic activity in decaying trees and thereafter the growth reduction in trees growing near the shoreline.

The results of this study show that the frequency of missing rings was surprisingly high in Pinus pinea compared to Pinus halepensis. However, in a semiarid Mediterranean site in Spain, Pinus. halepensis was shown to be more sensitive to drought than Pinus pinea [START_REF] Novak | Frequency and variability of missing tree rings along the stems of Pinus halepensis and Pinus pinea from a semiarid site in SE Spain[END_REF]. These results confirm that, with increasingly severe environmental conditions, production of wood may be partly or totally absent. This might be the case for the trees in our study, where the frequency of missing rings has increased since the 1980s and there have been more and more missing rings in those stands nearest the shoreline. Several factors may be the cause of this increase in missing ring frequency. It may be associated with an increase in water stress, which could be of climatic origin (dry years) or edaphic origin (sandy soils of the littoral dunes). However, dry conditions from October to April and high temperature in late winter and spring were found to be the main climatic factors causing missing rings in both species. In a study on the impact of drought on the growth of three tree species in Tunisia (Pinus halepensis Mill., Pinus pinea L., and Pinus pinaster Sol.), El [START_REF] Khorchani | L'impact de la sécheresse sur la croissance de trois espèces forestières en Tunisie (Pinus halepensis Mill., Pinus pinea L. et Pinus pinaster Sol.)[END_REF] found a correlation between increased drought and the et al., 2001;[START_REF] Novak | Frequency and variability of missing tree rings along the stems of Pinus halepensis and Pinus pinea from a semiarid site in SE Spain[END_REF].

In addition to climate, other stressors might contribute to the frequency of missing rings. The coastal vegetation in north Tunisia is harmfully affected by chemical components contained in marine spray [START_REF] El Ayeb | Effets des embruns marins pollués sur les feuilles d'Acacia cyanophylla Lindl. et d'Eucalyptus gomphocephala Dc. du littoral tunisien[END_REF]. As reported in other parts of the Mediterranean coast in Spain (Raventos et al., 2001) and in Italy [START_REF] Bussotti | The role of marine salt and surfactants in the decline of Tyrrhenian coastal vegetation in Italy[END_REF], the appearance of missing rings might accompany alteration or death of needles, decreased photosynthetic production and reduced availability of assimilates for wood production [START_REF] Raddi | The impact of sea erosion on coastal Pinus pinea stands: a diachronic analysis combining tree-rings and ecological markers[END_REF].

Trees on coastal sites also often suffer from underground problems (e.g. root damage). This problem has become more common on sandy coasts throughout Mediterranean regions due to the filtering factor which limits the water retention capacity of the sandy soils of the coastal dunes where the availability of water could decrease abruptly during dry years. Accordingly, underground problems are associated with disturbed water and mineral uptake, leading to reduced wood production [START_REF] Mc Laughlin | Dendroecological alications in air pollution and environmental chemistry: research needs[END_REF].

A missing ring in a particular year should not endanger tree survival [START_REF] Cherubini | Tree-life history prior to death: two fungal root pathogens affect tree-rings differently[END_REF][START_REF] Sass_Klassen | Juniper from Ethiopia contains a large-scale precipitation signal[END_REF]. However, long-term disturbances of cambial activity often lead to physiological problems associated with disturbed water transport and reduced storage capacity of reserve assimilates [START_REF] Bigler | Growth patterns as indicators of impending tree death in silver fir[END_REF][START_REF] Wunder | Predicting tree mortality from growth data: how virtual ecologists can help real ecologists[END_REF].

The relationship between tree-ring index and precipitation from October to April shows that water availability is the main factor influencing tree growth of both pine species in Tunisia. These results are consistent with several previous dendroecological studies in Mediterranean regions (Raventos et al., 2001;[START_REF] El Khorchani | Approche Dendrochronologique De l'influence Des Changements Climatiques Sur La Productivité Des Forêts De Pin d'Alep (Pinus Halepensis Mill[END_REF][START_REF] Campelo | Climatic significance of tree-ring width and intra-annual density fluctuations in Pinus pinea from a dry Mediterranean area in Portugal[END_REF][START_REF] Raddi | The impact of sea erosion on coastal Pinus pinea stands: a diachronic analysis combining tree-rings and ecological markers[END_REF][START_REF] Mazza | Growt rate and climate responses of Pinus pinea L. in Italien oastal stands over the last centry[END_REF] and showed that a large part of a tree ring is formed during the late winter and spring. These findings reflect the behavior of evergreen conifers that grow in regions with mild winters and are characterized by a photosynthetic activity in winter that produces carbohydrates for early wood formation in the following year (Kozlowski et al., 1991). The number of months that precipitation is positively correlated with growth is always less for trees growing near the shoreline than for those growing in distant stands. Thus, low amounts of precipitation over the past months in Tunisia, which is the only available freshwater, has reduced soil water content above the water table. The degree to which trees regulate water loss during drought may explain patterns of carbohydrate production and subsequent susceptibility to drought or biotic attack [START_REF] Mcdowell | Mechanisms of plant survival and mortality during drought: why do some plants survive while others succumb to drought? Tansley review[END_REF]. Stomatal responses to drought vary from drought avoidance, in which stomata close at a threshold water potential to minimize further transpiration, to drought tolerance, in which stomatal closure is less severe and transpiration continues at relatively high rates [START_REF] Mcdowell | Mechanisms of plant survival and mortality during drought: why do some plants survive while others succumb to drought? Tansley review[END_REF].

The negative relationships with maximal temperatures show that the number of months that maximal temperature is correlated negatively with growth is always higher for trees growing near the shoreline (2 months) than for trees growing in distant stands (1 month). This observation could be interpreted by the possibility of intense water stress for trees growing near the shoreline. However, precipitation remains the primary explanatory variable of tree growth decline. High temperatures might also affect water shortage because of increased evapotranspiration and soil water evaporation (Campelo et al., 2006). Furthermore, recent research indicates that warmer temperatures alone can increase forest water stress, independent of the amount of precipitation [START_REF] Barber | Reduced growth of Alaskan white spruce in the twentieth century from temperature-induced drought stress[END_REF]. Moreover, on coastal sand dunes where water availability could suddenly decrease in dry years, the negative effect of maximal temperature on radial growth in the spring could accentuate the phenomenon of water stress [START_REF] Aussenac | La sécheresse de 1976: influence de déficit hydrique sur la croissance des arbres forestiers[END_REF][START_REF] Khorchani | L'impact de la sécheresse sur la croissance de trois espèces forestières en Tunisie (Pinus halepensis Mill., Pinus pinea L. et Pinus pinaster Sol.)[END_REF]. Water availability still remains a factor more limiting than temperature. Thus, it might be the case that Pinus pinea, like other Mediterranean species (e.g. Pinus halepensis), responds by becoming increasingly dependent on moisture from deeper soil horizons that have not been exhausted in previous years [START_REF] Sarris | Recent decline in precipitation and tree growth in the eastern Mediterranean[END_REF]. In a study on the process based model shedding light on climate sensitivity of Mediterranean tree-ring width, conducted on Pinus halepensis from semi-arid areas in Tunisia, [START_REF] Touchan | Process based model sheds light on climate sensitivity of Mediterranean tree-ring width[END_REF] showed that the average duration of the growing season is 191 days, and that on average soil moisture limits tree-ring growth for 128 days and temperature for 63 days in that 191-day window. Due to water deficiency and subsequent stomata closure, dryness causes a decrease in photosynthesis and consequently fewer carbohydrates are available for radial growth [START_REF] Sitte | Strasburg-Lehrbuch Der Botanik[END_REF]. The combination of response function, dendrochronological measurements and structural characteristics of Pinus halepensis and Pinus pinea stands has shown that distance from the shoreline influences environmental conditions. The closer the trees are to the shoreline, the more they are put under stress conditions. Among these stress conditions, drought can operate as a trigger ("inciting factor") that may ultimately lead to mortality or dieback in trees that are already under stress (by "predisposing factors" such as old age, poor site conditions and air pollution) and succumb to subsequent stem and root damage by biotic agents ("contributing factors" such as wood-boring insects and fungal pathogens) [START_REF] Manion | Forest Decline Concepts[END_REF].

Dieback of pine forests involves multiple interacting factors, ranging from particular sequences of climate stress and stand life histories to insect pests and diseases [START_REF] Manion | Decline diseases of complex biotic and abiotic origin[END_REF][START_REF] Miao | Vegetation responses to extreme hydrological events: sequence matters[END_REF]. Of the many factors of climate stress, drought is a concern affecting forests in coastal zones. This type of damage causes long-term degradation that may predispose trees to attack by pathogens and pests. Forests may become ecologically unstable, reducing their efficiency to protect coastal dunes and increasing forest fire risks due to the abundance of dry canopies.

Conclusion

The northeastern pine coastal woodland of Tunisia has great ecological and economic importance in protecting the soil from erosion and controlling water shortage. The major threats to the coastal landscapes of Tunisia, are common along the Mediterranean coastal zone, and related to uncontrolled development, urbanization, increasing national and international tourism and unplanned or over-exploitation of natural resources. Depletion and degradation of resources, including freshwater, forests and coastal areas, is a growing concern, as is pollution within the region itself [START_REF] Vogiatzakos | Coastal Landscape of Tunisia-A Proposed Landscape Character Assesment[END_REF]. Our study indicates that Pinus pinea and Pinus halepensis on subhumid Mediterranean coastlines are growing under stress conditions when trees are close to the shoreline. These stress conditions are manifested by a negative effect on radial growth and structural characteristics of the trees, namely the development of the crown and the needles which can lead to conditions of dieback. Several climate models indicate that drought frequency and intensity are expected to increase in the Mediterranean basin [START_REF] Giorgi | Climate change projections for the mediterranean region[END_REF][START_REF] Lelieveld | Climate change and impacts in the eastern Mediterranean and the Middle East[END_REF][START_REF] Ozturk | Projections of climate change in the Mediterranean Basin by using downscaled global climate model outputs[END_REF]. In sensitive areas such as the coastal dunes of Rimel and Menzel Belgacem, maximum temperature is significantly negative in late winter and spring, high precipitation from autumn of the previous year to mid-April of the current year has a significantly positive influence on tree-ring width. Long-term low precipitation and high temperature during these months in Tunisia influence the availability of water resources and cause drought stress. The higher water stress induced by an increase in air temperature and a decrease in rainfall for successive years could seriously reduce growth accompanied by an increased number of missing rings of the Mediterranean pine species, despite their drought-tolerant behavior. Such conditions trigger physiological disturbances in trees and predispose them to be attacked by pathogens and pests and thus accelerate their decline. The Pinus pinaster decline observed in recent years on northern coastal dunes of Tunisia could be the consequence of this imbalance caused by such factors (drought, sea spray…). Our results suggest that stands growing in stressed conditions need different management approaches in order to achieve sustainability.

Fig. 1 .

 1 Fig. 1. Location of studied stands of Pinus halepensis and Pinus pinea, Northeast Tunisia.

Fig. 3 .

 3 Fig. 3. Response functions for Pinus halepensis and Pinus pinea in Kelibia with monthly average precipitation and maximal temperature.

Fig. 4 .

 4 Fig. 4. Response functions for Pinus halepensis and Pinus pinea in Bizerte with monthly average precipitation and maximal temperature.

Fig. 5 .

 5 Fig. 5. Mixed graph for missing rings, temperature and precipitation based on the result of response function.

Table 1

 1 Characteristics of each stand based on the distance from the shoreline and dieback intensity.

	Region	Site name	Stand name	Distance from the shoreline (km)	Dieback index in% (dieback intensity)	Tree density (trees ha -1 )	Age (year)
	BIZERTE	RIMEL	BZPH1	3.154	12.66 (Low)	668	54
	BIZERTE	RIMEL	BZPH2	1.155	26.27 (moderate)	684	52
	BIZERTE	RIMEL	BZPH3	0.105	76.26 (very strong)	660	64
	BIZERTE	RIMEL	BZPP1	3.187	13.25 (Low)	557	64
	BIZERTE	RIMEL	BZPP2	1.263	26.31 (moderate)	716	90
	BIZERTE	RIMEL	BZPP3	0.123	76.25 (very strong)	724	70
	KELIBIA	MEZEL BELGACEM	KBPH1	7.464	14.65 (Low)	636	69
	KELIBIA	MEZEL BELGACEM	KBPH2	1.725	24.93 (moderate)	652	52
	KELIBIA	MEZEL BELGACEM	KBPH3	0.727	76.27 (very strong)	684	64
	KELIBIA	MEZEL BELGACEM	KBPP1	7.619	9.53 (Low)	605	62
	KELIBIA	MEZEL BELGACEM	KBPP2	1.635	27.67 (moderate)	668	50
	KELIBIA	MEZEL BELGACEM	KBPP3	0.680	76.43 (very strong)	700	65

Acknowledgements

Special thanks are due to Mr. Abdelaziz Kebaier Heah of the National Institute of Meteorology that supplied the climatic data used in this paper. Thanks are also addressed to Mr. Adel Sakouhi, Head of Afforestation and Soil Protection at the Regional Office of Agricultural Development (CRDA of Bizerte) that allowed sampling from their own areas. We also thank Mrs. Salima Bahri for her help in Statistical Analysis.


Laila El Bouzidi

Widad Ben Bakrim

Valérie Mahiou

Nadine Azas

Mustapha Larhsini

Mohamed Markouk

Evelyne Ollivier

Khalid Bekkouche

In vitro antiplasmodial activity of Withania frutescens-Solanaceae

Keywords: Antiplasmodial activity, Withania frutescens, Plasmodium falciparum, Cytotoxicity, Selectivity index

Introduction: Drugs resistant Plasmodium falciparum is a recurring issue that threatens public health. New anti-plasmodial drugs are needed to overcome this problem. The aim of this study was to characterize in vitro an-tiplasmodial activity of Withania frutescens (Solanaceae) against chloroquine-resistant Plasmodium falciparum strain. Methods: The in vitro antiplasmodial activity of leaves and roots extracts from Withania frutescens was performed in 96 well plates and preliminary phytochemical analysis was performed for the active fractions. The toxicity of the plant extract against CHO (Chinese Hamster Ovary) cells was assessed using the tetrazolium salt MTT col-orimetric assay. The selectivity index (SI) was calculated as the ratio between the cytotoxic and antiparasitic activities. Results: The methanol extract of W. frutescens leaves showed a good antiplasmodial activity (CC 50 18.1 μg/ml). Furthermore, ethyl acetate and butanol fractions showed promising in vitro antiplasmodial activity with a se-lectivity index of 3.1 and 2.4, respectively. The roots of W. frutescens were found to be inactive with a CC 50 value > 80 μg/ml.

Conclusion:

The antiplasmodial activity of W. frutescens may in part be attributed to the presence of polyphenolic and flavonoid compounds. Based on our results, ethyl acetate and butanol leaves fractions could be considered as a promising source for the development of putative antiplasmodial drugs.

Introduction

Malaria caused by Plasmodium parasites is amongst many prevalent public health concerns in several tropical regions of the world. Amongst the five existing species of Plasmodium causing malaria in humans, Plasmodium falciparum is the most widely studied species since it causes a high number of deaths [START_REF] Who | World Malaria Report 2012[END_REF]. In spite of considerable control efforts, malaria remains a major cause of global morbidity and mortality in most tropical countries, especially those in sub-Saharan Africa [START_REF] Alonso | A research agenda to underpin malaria eradication[END_REF][START_REF] Müller | Malaria in Africa. Challenges for Control and Elimination in the 21st Century[END_REF]. Resistance to the majority of available antimalarial drugs has been reported in a growing number of countries worldwide and such resistance threatens future progress in malaria control [START_REF] Who | World Malaria Report 2012[END_REF]. Medicinal plants have been a reliable source of therapeutic agents and nowadays still represent an inexhaustible pool for the discovery of novel drug leads.

Withania frutescens is a perennial plant mainly distributed in Morocco, Algeria, Spain and Canary Islands [START_REF] Hepper | Old World Withania (Solanaceae): A taxonomic review and key to the species[END_REF]. Previous investigation on the pharmacology of this plant showed that its crude extracts displayed an interesting bioactivity profile, possessing various bioactivities including a protective and curative action against carbon tetrachloride (CCl4)-induced hepatotoxicity, neuroprotective properties, antioxidant and cytotoxic effects [START_REF] Montilla | The protective and curative action of Withania frutescens leaf extract against CCl4-induced hepatotoxicity[END_REF][START_REF] Bouzidi | Antioxidant and antimicrobial activities of Withania frutescens[END_REF][START_REF] Bouzidi | Cytotoxic withanolides from the leaves of Moroccan Withania frutescens[END_REF][START_REF] Fewell | An assessment of the in vitro neuroprotective properties of selected Algerian and South African medicinal plant extracts[END_REF]. The major chemical constituents reported from W. frutescens are called withanolides. These compounds are structurally diverse steroidal compounds with an ergosterol skeleton in which C-22 and C-26 are oxidized to form a δlactone. Many of these withanolides are reported to be responsible for the wide array of pharmacological activities, including antitumor, cytotoxic, cancer preventive, antifeedant, anti-inflammatory, leishmanicidal and immune-modulating effects [START_REF] Bouzidi | Cytotoxic withanolides from the leaves of Moroccan Withania frutescens[END_REF][START_REF] Chen | Natural withanolides: an overview[END_REF][START_REF] Glotter | Withanolides and related ergostane-type steroids[END_REF][START_REF] Ray | Withasteroids, a growing group of naturally occurring steroidal lactones[END_REF][START_REF] Veleiro | Chemistry and bioactivity of withanolides from South American Solanaceae[END_REF][START_REF] Anjaneyulu | Withanolides, biologically active natural steroidal lactones: A review[END_REF]. In our ongoing research on W. frutescens, we aim to explore its antiplasmodial potential in view to discover new drugs. This paper deals, for the first time, with the in vitro evaluation of the antiplasmodial activity of W. frutescens against a chloroquine-resistant strain of Plasmodium falciparum.

⁎ Corresponding author. E-mail address: laila_elbouzidi@yahoo.fr (L. El Bouzidi).

Extracts and fractions preparation

The leaves and roots were separately air-dried in the shade at room temperature (28 °C) until no further changes in their weight was observed (10 days), at Cadi Ayyad University of Marrakech (Morocco), in the laboratory of biotechnology, protection and valorization of plant resources, phytochemistry and pharmacology of aromatic and medicinal unit, and then reduced to fine powder with a grinder. Thus, the powdered parts of plants (100 g each) were exhaustively extracted using a Soxhlet apparatus. The leaves were first defatted with n-hexane (400 ml). The roots and defatted leaves were Soxhlet extracted with MeOH for 72 h (400 ml). After removing the solvent under vacuum at 40 °C, the residues obtained from the leaves (25.7 g) and roots (26.3 g) were dissolved in distilled water, filtered and extracted successively with CH 2 Cl 2 , EtOAc and n-BuOH, then concentrated under vacuum to yield 2.7 g, 0.6 g and 3.4 g of the CH 2 Cl 2 , EtOAc and n-BuOH leaf fractions, respectively and 0.9 g, 0.15 g and 3.1 g of the CH 2 Cl 2 , EtOAc and n-BuOH root fractions, respectively.

Phytochemical analysis of the plant extracts using TLC

The major secondary metabolite classes such as tannins, alkaloids, flavonoids and steroidal lactones were analyzed in W. frutescens by thin layer chromatography (TLC), on silica gel 60 F254 Merck. The solvent system used was: hexane/CH 2 Cl 2 /MeOH (18/3/2, v/v/v) for hexane extract, CH 2 Cl 2 /MeOH (9/1, v/v) for dichloromethane fraction, EtOAc/ MeOH/H 2 O (100/20/10, v/v/v) for ethyl acetate fraction and CH 2 Cl 2 / MeOH/H 2 O (65/35/5, v/v/v) for methanol extract and butanol fraction. Visualization of the TLC plates was achieved under UV at 254 and 365 nm and by spraying with NP/PEG reagent for flavonoids, ferric trichloride for tannins and Dragendorff's reagent for alkaloids [START_REF] Wagner | Plant Drug Analysis: A Thin Layer Chromatography Atlas[END_REF]. The presence of steroidal lactones was revealed with 4-4 nitrobenzylpyridine (NBP) followed by ethylenediamine (EDA) [START_REF] Hammock | Detection and analysis of epoxides with 4-(p-nitrobenzyl)-pyridine[END_REF].

Determination of total phenolic compounds

The total phenolic content (TPC) of leaf extracts and fractions was determined using the Folin-Ciocalteu assay [START_REF] Kähkönen | Antioxidant activity of plant extracts containing phenolic compounds[END_REF]. Aliquots of 300 μl containing 100 μg of each sample, dissolved in MeOH, were introduced into test tubes, followed by 1.5 ml of a Folin-Ciocalteu's reagent (diluted 10 times) and 1.2 ml of sodium carbonate (7.5% w/v). The contents of the tubes were mixed and placed in dark for 30 min before the absorbance was measured at 765 nm. The analyses were performed in triplicate and the results were expressed, as p-coumaric acid equivalent (CAE), in mg per g of dry extract or fraction (y = 0.065 x + 0.057; r2 = 0.984).

Determination of total flavonoid content

The total flavonoid content of leaf extracts and fractions was determined by the aluminium chloride colorimetric method [START_REF] Zhishen | The determination of flavonoid contents in mulberry and their scavenging effects on superoxide radicals[END_REF]. In brief, 200 μl of each concentration (5 and 2.5 mg/ml) of the samples in methanol were mixed with 800 μl of distilled water and 60 μl of aqueous solution of NaNO 2 (5%). The mixture was allowed to stand for 5 min before the addition of 40 μl of the solution of AlCl 3 , and the mixture was allowed to stand for 5 min. Then, 400 μl of 1 M of NaCO 2 and 500 μl of distilled water were added. The absorbance of all samples was measured at 510 nm. The total flavonoid content was calculated from a calibartion curve, and the result was expressed as mg catechin equivalent per g dry weight.

In vitro antiplasmodial assay

In this study, P. falciparum strain, resistant to chloroquine was used in the in vitro culture. Cultures were maintained in fresh A+ human erythrocytes at 2.5% haematocrit in complete medium (RPMI 1640 with 25 mM HEPES, 25 mM NaHCO 3 , 10% of A+ human serum) at 37 °C under reduced O 2 atmosphere (gas mixture 5% O 2 , 5% CO 2 , and 90% N 2 ). Parasitaemia was maintained daily between 1% and 6%.

The P. falciparum drug susceptibility test was carried out by comparing quantities of DNA in treated and control cultures of parasite in human erythrocytes according to a SYBR Green I fluorescence-based method using a 96-well fluorescence plate reader. Parasite culture was synchronized at ring stage with 5% sorbitol. Compounds were incubated in a total assay volume of 200 μl (RPMI, 2% haematocrit and 1% parasitaemia) for 72 h in a humidified atmosphere (5% O 2 and 5% CO 2 ) at 37 °C, in 96-well flat bottom plates.

Triplicate assays were performed for each sample. After incubation, the supernatant was discarded and cells were washed with 150 μl of PBS. 15 μl re-suspended cells were transferred to 96-well flat bottom non sterile black plates (Greiner Bio-one) already containing 15 μl of the SYBR Green lysis buffer (2X SYBR Green, 20 mM Tris base pH 7.5, 20 mM EDTA, 0.008% w/v saponin, 0.08% w/v Triton X-100). Negative control, treated by solvents (DMSO) and positive control (chloroquine) was added to each set of experiments. Plates were incubated for 15 min at 37 °C and then read on a TECAN Infinite F-200 spectrophotometer with excitation and emission wavelengths at 497 and 520 nm, respectively. The concentrations of compounds required to induce a 50% decrease of parasite growth (CC 50 ) were calculated from three independent experiments.

In vitro cytotoxicity evaluation on CHO cell line

The evaluation of the tested extracts cytotoxicity was conducted on the Chinese Hamster Ovary (CHO) cells according to the method described by Mosmann [START_REF] Mosmann | Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays[END_REF] with slight modifications. Briefly, cells in 100 μl of complete medium, [RPMI supplemented with 10% foetal bovine serum, 1% L-glutamine (200 mM) and 1% penicillin (100 U/ ml)/streptomycin (100 μg/ml)], were inoculated into each well of 96well plates and incubated at 37 °C in a humidified 6% CO 2 , 14% O 2 and 80% N 2 atmosphere. After 24 h incubation, 100 μl of medium with various product concentrations was added and the plates were incubated for 72 h. At the end of the treatment and incubation, each plate-well was microscope-examined for detecting possible precipitate formation before the medium was aspirated from the wells. 10 μl of MTT solution (5 mg MTT/ml in PBS) were then added to each well with 100 μl of medium without foetal calf serum. Cells were incubated for 2 h at 37 °C to allow MTT oxidation by mitochondrial dehydrogenase in the viable cells. After this time, the MTT solution was removed and DMSO (100 μl) was added to dissolve the resulting blue formazan crystals. Plates were shaken vigorously (300 rpm) for 5 min. The absorbance was measured at 570 nm with a microplate spectrophotometer (ELX 808, IU, BIOTEK). DMSO was used as blank and doxorubicine as positive control. Cell viability was calculated as percentage of control (cells incubated without compound). The 50% cytotoxic concentration was determined from the dose-response curve.

Selectivity index (SI)

The selectivity index (SI), which corresponds to the ratio between the cytotoxic and antiparasitic activities of each tested samples, was calculated to assess the selectivity of samples to P. falciparum.

Results

The antiplasmodial activity of the methanol extracts obtained from W. frutescens leaves and roots was tested against chloroquine resistant (W2) strains of Plasmodium falciparum. The in vitro antiplasmodial assay revealed that the methanol leaves extract exhibited an interesting activity against the W2-R Plasmodium falciparum strain with a CC 50 of 18.1 μg/ml (Table 1). Whereas, the methanol roots extract was shown to be inactive with a CC 50 > 80 μg/ml.

To further characterize the chemical class associated with the observed antiplasmodial activity, different organic fractions we prepared from the methanol leaves extract using solvents with different polarity. Each fraction was then tested against W2-R Plasmodium falciparum strains.

The most potent antiplasmodial activity was observed in the CH 2 Cl 2 (CC 50 9.4 μg/ml) followed by butanol (CC 50 12.4 μg/ml) and ethyl acetate (CC 50 22.3 μg/ml) fractions. However, the selectivity indexes were proportionally inversed to the determined inhibitory concentration (Table 2).

The result of the phytochemical screening showed the presence of flavonoids in ethyl acetate and butanol fractions, steroidal lactones were detected in dichloromethane and butanol fractions. Based on the polarity of the used solvents, dichloromethane and butanol fractions probably contain free withanolides and glycowithanolides, respectively [START_REF] Jadhav | Flux and retention analysis during micellar enhanced ultrafiltration for the removal of phenol and aniline[END_REF], while none of the tested extracts and fractions contained tannins or alkaloids.

Phenolic compounds including flavonoid are one of the most important classes of antiplasmodial compounds [START_REF] Azebaze | Antiplasmodial activity of some phenolic compounds from Cameroonians Allanblackia[END_REF]. The ethyl acetate fraction possess high phenolic and flavonoid content (254.8 ± 2.9 mg CAE/g dry fraction and 122 ± 0.77 mg CE/g dry fraction, respectively) which may be responsible for its antiplasmodial activity. The concentration of flavonoid in butanol fraction (186.7 ± 0.12 mg CE/g dry fraction) was more significant. Therefore, these compounds might have improved the antiplasmodial activity of this fraction (Table 3).

Discussion

According to Jonville et al. [START_REF] Jonville | Screening of medicinal plants from Reunion Island for antimalarial and cytotoxic activity[END_REF] antiplasmodial activity has been classified as follows: high (CC 50 < 5 μg/ml), promising (5 < C-C 50 < 15 μg/ml), moderate (15 < CC 50 < 50 μg/ml) and inactive (CC 50 > 50 μg/ml). This is the first report showing antiplasmodial activity of W. frutescens. From the obtained data, it appears that methanol extract of W. frutescens leaves exhibited good activity in vitro against chloroquine resistant strains (W2-R) of Plasmodium falciparum, whereas methanol roots extract was inactive (Table 1).

The obtained fractions from leaves methanol extracts of W. frutescens displayed promising activity on the tested strain (CC 50 9.4-22.3 μg/ml). The dichloromethane fraction exhibited the highest antiplasmodial activity with a CC 50 of 9.4 μg/ml, nonetheless it exhibited significant cytotoxic activity against CHO cell lines. Our previous study has shown that the dichloromethane fraction of W. frutescens leaves exhibited cytotoxic activity against cancer cell lines (Hep G2), which can be ascribed to the phytochemical constituents; the withanolides [START_REF] Bouzidi | Cytotoxic withanolides from the leaves of Moroccan Withania frutescens[END_REF]. Earlier studies reported the antiplasmodial activity of some other sesquiterpenoid lactones, but in both reports high cytotoxicity was also associated with the compounds [START_REF] Ramanandraibe | Pseudoguanolide sesquiterpene lactones from Vernoniopsis c audata and their in vitro antiplasmodial activities[END_REF][START_REF] Pillay | Antiplasmodial hirsutinolides from Vernonia staehelinoides and their utilization towards a simplified pharmacophore[END_REF].

The ethyl acetate and butanol fractions enriched in polyphenols and flavonoids proved to be active against malaria with a promising to moderate activity (specificity index of 3.1 and 2.4, respectively). Numerous studies have been reported wide varieties of pharmacological activities of polyphenols and flavonoids, including antiplasmodial activity [START_REF] Joseph | Larvicidal and mosquitocidal extracts, a coumarin, isoflavonoids and pterocarpans from Neorautanenia mitis[END_REF][START_REF] Kaur | Antimalarials from nature[END_REF]. Antiplasmodial activity has been reported for some biflavonoids primarily (I-30, II-8)-biflavones, in which a high number of methoxy substituents were associated with high activity [START_REF] Dhooghe | Antiplasmodial activity of (I-3, II-3)-biflavonoids and other constituents from Ormocarpum kirkii[END_REF]. This group enhances the lipophilicity of the substance, improving its incorporation into cells [START_REF] Monks | In vitro cytotoxicity of extracts from Brazilian asteraceae[END_REF]. It is concluded from of the present study that the ethyl acetate and butanol fractions of W. frutescens leaves possess a promising antiplasmodial activity. This activity may be ascribable to high level of polyphenols and flavonoids in the fractions. Pure drugs that are industrially produced or isolated from plants against a humans disease have not always the same degree of activity as the unrefined extract or fraction at comparable concentraction of the active component, and in many cases there is evidence of synergistic or multifactorial effects between compounds present in the extracts [START_REF] Wagner | Synergy research: approaching a new generation of phytopharmaceuticals[END_REF][START_REF] Wright | Ancient Chinese methods are remarkably effective for the preparation of artemisinin-rich extracts of Qing Hao with potent antimalarial activity[END_REF]. In a recent study, it was found that the antiplasmodial IC 50 values of Artemisia annua extracts were 6 to 18 fold lower than was expected in terms of their artemisinin content, suggesting that the activity of the extracts could not be accounted entirely by their artemisinin content [START_REF] Wright | Ancient Chinese methods are remarkably effective for the preparation of artemisinin-rich extracts of Qing Hao with potent antimalarial activity[END_REF]. In another study, the flavone casticin enhanced the in vitro activity of artemisinin by 3-5 fold [START_REF] Elford | Potentiation of the antimalarial activity of qinghaosu by methoxylated flavones[END_REF]. This result demonstrates that the mode of action of artemisinin depends on the formation of free radicals as a consequence of the interaction of haem iron in malaria infected red bood cells with the endoperoxide moiety of artemisinin [START_REF] Wright | Mode of action of artemisinin and its derivatives[END_REF]. Thus, alternative antiplasmodials must be needed to overcome any Chloroquine was used as positive control for antiplasmodial activity and Doxorubicine was used as positive control for cytotoxicity activity. deficiencies in present therapy. In this context, further study should be conducted for the purification of bioactive fractions of Withania frutesence in regard to isolate active antiplasmodial compounds and thereafter the investigation of the synergistic interaction between the isolated compounds and also between them and the commercial antimalarial drugs.

MARK 2 .

 2 Materials and methods 2.1. Plant material Withania frutescens (L.) Pauquy roots and leaves were collected in El Hassania (20 km from Marrakech), Morocco, in March 2009. The material was authenticated by Prof. A. Abbad, a plant taxonomist, in the Department of Biology, Faculty of Sciences Semlalia, Cadi Ayyad University, Morocco. A voucher specimen (No. mhnm.bk.kh.1) has been deposited at the herbarium of Natural History Museum of Marrakech, Morocco.

Table 1

 1 In vitro antiplasmodial and cytotoxic activities of Withania frutescens methanol extracts on W2 (CQ-R) strains of Plasmodium falciparum.

		CC 50 CHO (μg/ml)	CC 50 Plasmodial (μg/ml)	SI
	Leaves	14	18.1	0.8
	Roots	> 100	86.8	> 1.2
	Chloroquine	-	0.13	-
	Doxorubicine	0.98	-	-
	Chloroquine was used as positive control for antiplasmodial activity and Doxorubicine
	was used as positive control for cytotoxicity activity.

Table 2

 2 In vitro antiplasmodial activity of different fractions of methanol leaves extract on W2 (CQ-R) strains of Plasmodium falciparum.

			CC 50 CHO (μg/ml)	CC 50 Plasmodial (μg/ml)	SI
	Leaves	CH 2 Cl 2 F.	7	9.4	0.7
		EtOAc F.	70	22.3	3.1
		BuOH F.	30	12.4	2.4
	Chloroquine	-	0.13	-
	Doxorubicine	0.98	-	-
	CH

2 Cl 2 F.: dichloromethane fraction; EtOAc F.: ethyl acetate fraction; BuOH F.: n-butanol fraction.

Table 3

 3 Total phenolic content (TPC) and total flavonoid content (TFC) of leaves extracts and fractions of W. frutescens. EtOAc F.: ethyl acetate fraction; BuOH F.: butanol fraction. ACE: p-coumaric acid equivalent; CE: catechin equivalent. All assays were determined in triplicates ± standard deviation. For each assay, values followed by the same letter are not significantly different at P < 0.05, as measured by the SNK test.

	Samples	TPC (mg CAE/g dry extract or	TFC (mg CE/g dry extract or
		fraction)	fraction)
	Hexane E.	26.61 ± 1.4e	9.87 ± 0.43e
	MeOH E.	144.15 ± 3.73c	133 ± 0.13b
	CH 2 Cl 2 F.	32 ± 1.07d	19 ± 0.3d
	EtOAc F.	254.77 ± 2.88a	122 ± 0.77c
	BuOH F.	200.61 ± 0.96b	186.7 ± 0.12a
	Hexane E.: hexane extract; MeOH E.: methanol extract; CH2Cl2F.: dichloromethane
	fraction;

Acknowledgments

The authors express their gratitude to the intergovernmental program of France-Morocco FSP-ARESM (Priority Solidarity Fund on the reform of higher Moroccan education) for the mobility grant to the first author and the National Center for Scientific and Technical Research (CNRST) of Morocco for providing a research grant n°: E3/006.

Funding source

This work was supported in part by the intergovernmental program of France-Morocco FSP-ARESM (Priority Solidarity Fund on the reform of higher Moroccan education) and the National Center for Scientific and Technical Research (CNRST) of Morocco.