New Water Soluble Polysaccharide From Periploca Laevigata: Functional Characterization and Evaluation of Multi-biological Activities.

The water-soluble polysaccharide was isolated from Periploca laevigata root barks and puried. The structurale, surface, functional properties, and biological activities of P. laevigata polysaccharide (PLP1) were investigated. Scanning electron microscopy (SEM), X-ray diffraction crystallography (XRD), were used for structurale characterization of PLP1. SEM analysis suggested that the polysaccharide had irregular particle size with a large number of cavities, mostly seen as aggregates, and brous in nature. The samples had peaks at approximately 22°, and 38° 2θ degrees in the XRD pattern, which indicated both crystalline and amorphous structure. Functional properties of PLP1 were investigated based on water and oil holding capacity, solubility, emulsication activity, and foaming ability. Interestingly, PLP1 exhibited, a noticeable ability to emulsify corn and olive oil. Experimental analysis indicated that PLP1 able to decrease surface tension of water of 67.5 mN/m to 43.7 mN/m. On other trend, bioactivities evidenced that PLP1 is a potent protease and lipase inhibitor. The cell viability proved the cytoprotective effect of PLP1 on cell treated with hydrogen peroxide H 2 O 2 , suppressing apoptosis and ameliorating oxidative lesions. The experimental data and multi functionality provides enough evidence and open many new possibilities for biomedicine and food applications.


Introduction
Polysaccharides are biopolymer constituted by repeating subunits linked together by glycosidic bonds.
These macromolecules, are frequently existed in plants and algae, play a prominent role in the development of green products including pharmaceuticals, packaging materials and foods 1,2 . Recently, the water-soluble polysaccharides (WSP) have developped a great deal of interest cause of their bioactivities such as antibacterial, hypoglycemic, and hypolipidemic potential 3,4 . In fact, the functions of this macromolecules are strongly linked to their chemical composition, tertiary structure, linkage type, branch degree, and molecular weight.
Oxidation of polyunsaturated fatty acids, resulting from storage conditions and/or during processing, is among the major factors that lead to the allteration of food quality and the aproduction of toxic compounds 5 . likewise, it has been demonstrated that antioxidants are important in the pre-theurapie of cancer, biological effect in living tissues, cardiovascular and neurodegenerative diseases, where the Reactive Oxygen Species (ROS) are the main effectors 6 . Among the synthetic antioxidants the most most commonly implicated in stabilization of foods, are butylated hydroxyanisole (BHA) and butylated hydroxytoluene (BHT) which are very effective in the prevention of oxidative damages 7 . However, the use of these chemical compounds that undoubtedly induced genotoxicity and DNA damages, has become restricted 8 . From the health point of view, governmental authorities and consumers are concerned to checked food additives for their potential harmful effects 9 . Therefore, it is extremely important to develop green antioxidant to overcome the limits of synthetics compounds. Due to its high stability and biosafety plant-derived polysaccharides has often served as an effective natural antioxidants in pharmaceutical and food elds 10 .
On the other hand, microbial contamination associated with food borne diseases is the major challenge of the third world and developing countries, and even in developed nations 11 . The consumption of foods contaminated with some microorganisms is cosely related to the appearance of serious humans health risk. Thereby, The subsistence and multiplication of microorganisms in foods may lead to fobtaining food products with high toxicity and low quality 12 . In this approach, several researchers were challenged to discover green molecules caracerized by number of advantages in comparison to other food additives and preservatives including the high quality and the low cost, which are important issues faced by the food industry. P. laevigata that belongs to the Asclepiadaceae family, is very abundant specie in North Africa. In Tunisia, this plant is widely grow in the steppes of desert, arid and semi-arid areas. It has been extremly used as medicinal plant in both modern and traditional medicine. Further, It is reported that P. laevigata polysaccharides have high antioxidant and antibacterial activities 3 . However, there is no systematic study on the functional properties and anti-lipase and anti-proteases activities of P. laevigata polysaccharides. homogenized using a vortex to incorporate the air. The whipped sample was immediately transferred into a graduated cylinder, and the total volume was immediately measured after whipping at 0 min, and 60 min.

Materials And
Foam capacity was expressed as volume increase percentage after homogenization at 0 min, which was calculated according to the following equation: Foam stability was calculated as the foam remaining volume of after 60 min.
Where VT is the total volume after whipping (ml); V0 is the volume before whipping; Vt is the total volume after leaving at room temperature.

Surface tension measurements
The surface tension of PLP1 (1 to 5 g/100 mL) was evaluated according to the method of Wilhelmy plate using a manual digital surface tensiometer (GIBERTINI Elettronica™ TSD Digital Tensiometer).

X-ray diffraction (XRD)
X-ray powder diffraction patterns of PLP1 were recorded at room temperature on an X-ray diffractometer (D8advance, Bruker, Germany). Samples were scanned between diffraction angles of 5 to 80° 2θ with a step size of 0.05• and acounting time of 5 s/step.

Scanning electron microscopy
The morphological features of the PLP1 were visualized using a scanning electron microscope (Cambridge Scan-360 microscope). Bre y, in order to make the dried PLP1 conductive, the sample was mounted on a metal stub and sputtered with gold. The images of sample were taken at an accelerating voltage of 3.0 kV.

Lipase inhibition
The lipase activity was measured titrimetrically at pH 7.2 and 37°C with a pH-Stat (Metrohm, Switzerland) using olive oil emulsion. One lipase unit corresponds to 1 µmol of fatty acid released per minute.
A preincubation of lipase with inhibitor was use in order to determine the lipase inhibitory activity of PLP1. This method aimed to test, in an aqueous medium and in the absence of the substrate, the possible reactions between the lipase and the inhibitor. Lipase was pre-incubated at room temperature for 1 h with different concentrations of the inhibitor dissolved distilled water. The reaction medium contained the correspondent inhibitor concentration and 100 µL of lipase. A negative control (distillated water) was incubated in the same medium than the test of inhibition without the inhibitor. The lipase inhibition (% Inhibition) was calculated as compared to the initial activity measured in the absence of inhibitor.

Protease inhibition
The trypsin inhibitor activity was determined using casein (1%) as substrate 17 . Enzyme solution (40 μg of trypsin) was pre-incubated with different concentration of PLP1 in a total volume of 2 ml at 37 °C for 10 min in 0.01 M Tris-HCl buffer, pH 8.0. Absorbance of the mixture was measured at 280 nm and the residual activity was determined. Trypsin inhibitory unit is de ned as the number of trypsin units inhibited under the assay conditions. The inhibiton of pepsin activity was performed according to Anson method 18 , using haemoglobin as a substrate at pH 3.0 and 37 °C for 15 min. To 100 µl substrate was added 50 µl of pepsin in 0.1 M glycine-HCl buffer, and 350 µl of PLP1 in water at different concentration. Pepsin inhibitory unit is de ned as the amount of enzyme units inhibited in the absorbance at 280 nm per minute under the assay conditions.
The activity of the enzymes (trypsin and pepsin) assayed without PLP1 was taken as control (100% activity). Embryonic rat cardiomyocyte derived cell line H9c2 was obtained from America Tissue Type Collection (CRL-1446). Cells were cultured in DMEM medium supplemented with 10% fetal bovine serum, 100 U/ml penicillin, and 100 lg/ml streptomycin in 75 cm 2 tissue culture asks at 37 °C in a humidi ed atmosphere of 5% CO 2 . They were fed every 2-3 days, and sub-cultured when reaching 70-80% con uence in order to prevent the loss of the differentiation potential. Cells were then differentiated into cardiomyocytes by methods described by Ménard et al 19 .

Cell viability
Cell viability was determined using an MTT assay. H9c2 cells were plated in 96-well plates at 200 H9c2 cells/well. After cell differentiation, cells were treated with water-soluble polysaccharide (PLP1) at various concentrations (0.5, 1, and 2 mg/ml) or with DMSO used to dilute the PLP1. To evaluate the cytoprotective assay, cells were exposed to H 2 O 2 (0.1 mM) as well as PLP1 at the same time for 12 h at 37 °C. At 12 h after the treatment, the medium in the wells was replaced with 5 mg/ml MTT solution, and the cells were incubated for 2 h at 37 °C. Following incubation, the MTT solution was discarded, and 100 µl DMSO was added to dissolve the precipitate completely at room temperature. The optical density was then measured at 540 nm. The cell viability was expressed as relative viable cells (%) to control H9c2 cells.

Statistical analysis
Analysis was performed using SPSS (Version 17.0 for windows, SPSS Inc., Chicago, IL, USA). DATA were subjected to analysis of variance (ANOVA) and mean comparisons were carried out by Ducan's multiple range test (p< 0.05). All tests and measurements were carried out in triplicate As it can be seen, for the WHC the value determined for was of ∼7.92 g water/g PLP1, while OHC value was of ∼3.57 g oil/g PLP1. The WHC value was signi cantly lower than those of polysaccharides from Nostoc agelliforme (27.82 g/g) 20 , Gigartina pistillata (10.22 g/g) 21 , higher than that from watermelon rinds (2 g/g) 22 . Similar value of WHC was observed with polysaccharide from potato (8 g/g) 23 . A pioneering study exploring the functional properties of polysacchrides, published by Elleuch et al. 24 highlighted that the high WHC of polysaccharide present a key parameter to avoid syneresis and modify the viscosity and texture of some formulated foods. From table 1, PLP1 also showed the highest OHC with the value of ∼23.57 g/g, which was much higher than that polysaccharide from black cumin seeds (2.86 and 1.26 g/g at the temperature 25°C and 75°C) 25 .
As for OHC, it is partially correlated to the chemical composition, but it is more closely linked to and polysaccharide source to the porosity of the ber structure than to the a nity of the ber molecule to oil 26 . Solubility de ned by Song et al. 27 as a typical representative of product behavior in aqueous phase and the common standard that determine the quality of powder reconstitution. From the result presented in table 1, we found that the solubility value is arround 78.34%, wich is slightly lower than that of polysaccharide from Vigna radiate L.(~ 88.8%) 27 . Researches conducted by Ahmadi et al. 28 showed that the interaction between hydrocolloids and water molecules is in uenced by the hydrogen bonding, and particle size.

FC and FS of PLP1
The foaming property is a surface property de ned by its size and stability. High-performance foaming agents are widely required in the food industry 29 . Polysaccharides as an important food macromolecule, play a crucial role in the stabilization of foams 30 . The foaming properties were investigated in terms of FC and FS shown in Fig. 1.
It can be observed, PLP1 accounted a FC betwen 27.5% and∼49.7% whereas the FS varied from ∼25.5% to ∼41.25% (p < 0.05), depending on the concentration. Our results were in line with earlier reports of polysaccharides extracted from Trigonella foenum-graecum 31 . According to Shen et al. 20 , the foam capacity and stability of N. agelliforme is related to the molecular weigh, to the concentration and source of polysaccharides. The important FC and FS of PLP1 may be associated with their ability to increase the viscosity of the aqueous phase and to create a network that stabilizes the interfacial lm. The avor, texture, and shelf life of food can be greatly enhanced by using the polysaccharides as gelling or thickening agents for stabilizing dispersions.

EC and ES of PLP1
Knowledge of the emulsients properties of polysaccharide is an important parameter in determining its suitability in food formulations. Figure 2 illustrates the emulsion properties that include the emulsion capacity (EC) and emulsion stability (ES) of PLP1.
Interestingly, PLP1 exhibited, after 1 h, a noticeable ability to emulsify corn oil and olive oil, whose index value (E1) was 96.13% and 75.63%, respectively. Tan and Gan 32 reported that emulsiant activity of polysaccharides from Momordica charantia, where the emulsion prepared using palm oil at the same concentration, was 66.4%. However, at a concentration of 0.5% the EC of polysaccharide extracted from Trigonella foenum-graecum seeds, reported by Ktari et al. 31 , was 79.08% and 62.66%. Consistent with the previous reported observations 33 , the emulsifying ability of polysaccharides depends on the amount of protein linked to their structure. From previous study of Hajji et al. 3 , we found that the protein content for PLP1 is less thant 0.1%, which could explain the low EC compared to that of exopolysaccharide from Lactobacillus sp. Ca6 34 , which is arround 100%. In addition, it should be highlighted that higher concentration would result in a higher EC because of the presence of high amounts of polysaccharide that can completely saturate the oil-water interface, thus resulting in higher surface coverage. The ES of PLP1 decreased after 168 h and reached about 62.47% and 52.13% for corn aoil and olive oil, respectively. Ben Slima et al 35 stated that emulsion stability had a positive effect on its physical stability, such as occulation, gravitational separation and coalescence. ES was closely related to the size of the droplets in an emulsion based on food product.
The size of the droplets is therefore useful to accurately specify droplets size which are present within an emulsion. Thus, microscopic evaluation was performed on PLP1 corn oil and olive oil emulsions (Fig. 2B). As shown by emulsion's light micrographs, smaller and more densy droplets were observed in the emulsions prepared with corn oil, resulting in more stable emulsions, as reported by Maalej et al. 36 . Among the characteristic required for a polysaccharide to be most effective in stabilizing emulsion droplets, is the high-molecular weight of biopolymer 37 . According to our previous study 3 , minor proteinaceous fraction combined with its high-molecular weight, contribute to the ability of PLP1 to act as emulsifying and emulsion.

SEM analysis of PLP1
SEM analysis was used to illustrate the surface morphology of extracted polysaccharides. As presented in Fig. 3, and their various derivatives at 55 and 160 times magni cation to evaluated the microstructures of PLP1. SEM showed that PLP1 existed in closely aligned particles with a smooth surface, which con rmed that PLP1 is amorphous under vacuum freeze-drying process. The shape and structure, or the surface topology of the macro-biomolecules including polysaccharide may be affected by the method of extraction and puri cation or preparation of the product 38 .

Surface tension of PLP1
The evaluation of the potential of PLP1 to reduce surface tension at the air-water interface, was made by determining their surface activity at different concentrations. Figure 4A illustrates that PLP1 surfaceactive macromolecule, since it is able to reduce interfacial tension relative to water (67.5 mN/m). In the scope of the testing concentration (1-5 g/100mL), the surface tension of PLP1 was concentrationdependent, which is corresponded with the results of polysaccharides from black cumin seeds 25 .
From the results of surface tension, PLP1 had the greatest surface activity (43.25 mN/m) at the highest concentration (5 g/100 mL) which revealed a rapid diffusion into the air-water interface. It was likely that the surface tension of polysaccharide was strogly related with the amphiphilic character, means that polysaccharide can be adsorbed and oriented at liquid-liquid interfaces to reduce e ciently the interfacial tension.

XRD analysis of PLP1
XRD is an effective analytical tool employed to evaluate the crystalline characteristics and unraveling the structure of the polysaccharides, which can predict their various properties like solubility, swelling, exibility, and other physical properties 39 .
By studying the data obtained from the Fig. 4B, PLP1 showed a peaks at approximately 22°, and 38° 2θ degrees.
Consequently, it can be stated that the polysaccharide puri ed from P. laevigata (PLP1) exhibits both crystalline and amorphous portions. This nding is congruent with the crystalline/amorphous contrast obtained by XRD of puri ed polysaccharides from Musa sapientum L. and Vigna radiate L. 27,40 , but it is far different from the pattern of okra polysaccharide 41 .

Effect of PLP1 on cell viability
The cytoprotective effects of PLP1 against hydrogen peroxide induced cardiomyocyte toxicity was evaluated (Fig. 5).
The viability of control cells (no PLP1 or H 2 O 2 added) was considered to be 100%. It was observed that at concentration of 0.5 mM H 2 O 2 signi cantly reduced the cell viability and the total viable cells were 9.11%.
The PLP1 solution at a concentration of 2 mg/mL, was found non-toxic to H9c2 cells, and the viability of cells was 97.12%. It was found that PLP1 exhibited signi cant (p < 0.05) cytoprotective potential in a dose-dependent manner. In MTT assay, PLP1 increased the viability of H 2 O 2 -treated cells, thus attenuated the cytotoxic effect of H 2 O 2 and provided protection against oxidative stress-induced cytotoxicity 3.5.2. Anti-protease activity of PLP1 As proteases are important in tumor growth and spread, the effect of PLP1 was tested on two selected proteases (Trypsin and Pepsin). The effect of PLP1 on acidic and alcalin proteolytic activity is shown in Fig. 6B.
The results suggested that PLP1 had a strong inhibitory effect on proteolytic activity of pepsin and trypsin. The acidic proteolytic enzyme retained about 21.5 % of their initial activities, after incubation with 2 mg of PLP1. At the same concentration trypsin was lost the totality of its activity. Furthermore, enzyme activity was decreased in concentration depending-manner. The obtained results were in agreement with several works reported in the literature with regards to the anti-protease effects of polysaccharides 42,43 .
In a recent study, Wang et al. 43 reported that there may be multiple ways in which polysaccharides affect protease activity and protein digestibility. The decrease in proteolytic activity may have been due to the ability of polysaccharide to reduce protein digestibility to some extent, possibly because of their monosaccharide compositions.
It was reported that there was a relationship between polysaccharide viscosity, protease activity 43 and reduction of protein digestibility 44 . Therefore, there may be multiple mechanisms by which polysaccharides affect protease activity.

Anti-lipase activity of PLP1
The plant kingdom have large scope to be used as an effective oral antiobesity or weight loss agents that have insigni ants side effects and that are costeffective alternative to synthetic drugs 45 . The inhibitory activity towards pancreatic lipase displayed by the PLP1 is illustrated in Fig. 6B. The analysis of pancreatic lipase activity after addition of polysaccharide, highlights that PLP1 signi cantly (p < 0.5) decreased lipase activity, with a dose-response manner. A total inhibition of lipase was obtained with concentration of 3 mg/ml (PLP1). Interestingly, PLP1 exhibited strong lipase inhibitory effects with IC 50 values of 0.59 mg/ml. This result suggest that the new puri ed polysaccharide from P. laevigata root barks could be potential candidate speci cally for the extraction of antiobesity green molecules. Our nding are in high concordance with results reported by Tsujita et al. 46 who indicated that a basic polysaccharide is able to bloked dietary fat absorption from the small intestine by inhibiting pancreatic lipase activity to get the purpose of losing weight. Hu and co-authors 47,48 suggest that based on their structure, including glycosidic bonds, monosaccharide composition, and anionic groups, polysaccharide can inhibit pancreatic lipase activity. Therefore it is ensured that the molar ratio of 1,4-linked-β-D-Glcmanp which in turn affect the activity of pancreatic lipase, as an increasing percentage of mannose decreased pancreatic lipase activity. Tsujita et al. 49 suggest the paramount importance of extent of polymerization as a key factor in the lipase inhibition.

Conclusion
In this study, a structural characteristics, functional properties and bioactivities of a novel puri ed polysaccharide from P. laevigata root barks (PLP1) were investigated. The spectroscopic analyses by SEM and XRD, showed that PLP1 corresponded to biopolymer with crystalline and amorphous structure. Furthermore, PLP1 exhibited good functional properties, particularly for emulsion forming and stabilizing capacity, under certain conditions. Intestinal functions were characterized, and the results indicated that PLP1 could signi cantly improve intestinal functions, including signi cantly lowering protease and pancreatic lipase activities. Furthermore, the bioactivity studies demonstrated that PLP1 could slightly  Anti-protease and anti-lipase activity of water-soluble polysaccharide (PLP1) as a function of concentration