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Full Research Article
Effect of Five Mediterranean Shrubs Extracts on Larval Exsheathment of Haemonchus contortus
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First Online 05-10-2021|
Background: Mediterranean shrub species cover more than 70% of the total area in Tunisia and in summer when the herbaceous species have wilted, they constitute feeding resource for livestock. The use of tanniniferous shrubs seems to be a good alternative to control gastrointestinal nematodes infections in small ruminants. This study evaluated the in vitro anthelmintic (AH) effect of Ceratonia siliqua (C. siliqua), Periploca angustifolia Labill. (P. angustifolia) and Medicago arborea (M. arborea) against Haemonchus contortus third stage larvae (L3).
Methods: The larval exsheathment assay (LEA) was used to determine the proportions (%) of exsheathment of five acetonic extracts at different concentrations (1200, 600, 300, 150 μg/ml). To confirm the role of tannins in the AH effects of extracts, polyvinyl polypyrolidone (PVPP) was used as deactivating chemical tannins.
Result: The highest % L3 exsheathed was recorded for M. arborea (55.01%) and the lowest value was founded for C. siliqua and P. angustifolia leaves (16.26%). Our results were concentration-dependent (P<0.001). The % of exsheathment increased as the time of incubation increased (P<0.001). P. angustifolia pods recorded the lowest EC50 value (P<0.05). After PVPP addition, all the acetonic extracts showed a restoration of L3 exsheathment values similar to control values (P<0.001).
Some studies have focused on the AH properties of legume forage. The aims of the current study were to verify (i) the in vitro AH effect of three Mediterranean woody shrubs against Haemonchus contortus third stage larvae using LEA and (ii) to verify tannins AH activity, a tannins inhibitor, the polyvinyl polyrrolidone (PVPP) was used.
MATERIALS AND METHODS
Five samples from three different plants were collected in spring-summer using the diagonal Methods (Theau et al., 2010): C. siliqua (leaves and pods), P.angustifolia (leaves and pods) and M. arborea: (pods). They were collected from two regions in central Tunisia:
• El Nathour (latitude 36°07' 12.91" N, longitude 10°05' 40.25" E, altitude 114 m) for C. siliqua .
• Saouaf (latitude 36°13' 53.07" N, longitude 10°10' 25.21" E, altitude 158 m) for P. angustifolia and M. arborea.
Preparation of plant extracts
After being freeze-dried and grounded at 1 mm screen, 10 g of each shrub sample was shaking in 70:30 acetone: water (v/v) solution for 1 hour in a water bath (32-35°C). The acetone was removed under low pressure at a temperature below 35°C and the aqueous solution was washed three times with 100 ml of dichloromethane to remove chlorophyll and lipids. The remaining fraction was frozen then freeze-dried for 24 h and kept at 4°C in air-tight containers until used in the in vitro biological assay.
Representative samples of shrubs were dried at 60°C for 72 h and then grounded at 1 mm screen for chemical analysis composition. Ash, crude protein (CP) and crude fiber (CF) contents were quantified according to AOAC (2000) and acid detergent fiber (ADF), neutral detergent fibre (NDF) and acid detergent lignin (ADL) were analyzed according to Van Soest et al. (1991).
Polyphenolic compounds and biological activity
The Folin-Ciocalteu method described by Makkar (2003) was used to quantify the concentrations of total polyphenols (TP) and total tannins (TT) in the shrub samples. For each sample, we measured polyphenols without and with addition of PVPP (Sigma Aldrich Ltd), then we determined TT by difference between TP measured without PVPP and non TT measured with PVPP. The quantification of TP and TT was done in three replicates, made at 725 nm using of a spectrophotometer (UV-visible Spectronic Unicum, Genesys 8). A tannic acid standard curve was performed and total phenols and total tannins were expressed as g-equivalent tannic acid/100 g DM (g-equiTA).
The condensed tannins (CT) of each plant samples were determined by the butanol-HCl method (Makkar, 2003). In test tubes, we deposited 0.05 ml of tannin extract, 0.45 ml of 70:30 acetone:water (v/v) solution, 3 ml of butanol-HCl and 0.1 ml of ferric reagent. After covering their open sides, the tubes were boiled for 60 min. A blank containing the reagents without extract was used as a control. They were then cooled and absorbances at 550 nm were measured on a spectrophotometer (UV-visible SpectronicUnicum, Genesys 8). Concentrations of CT were expressed as g - equivalent of leucocyanidin/100 g of DM.
The biological activity (BA) of tannins was quantified using the Radial Diffusion method (Hagerman and Bulter, 1978). This technique is based on the property of tannins to form insoluble complexes with protein. We used bovine serum albumin BSA (Sigma Aldrich Ltd) and tannic acid (Sigma Aldrich Ltd) as standards. The activity was expressed as g-equiTA.
Larval exsheathment assay (LEA)
The larval exsheathment assay was artificially performed (Bahuaud et al., 2006) on the infective stage larvae (L3) of H. contortus (INRA goat strain, France) with extracts of each shrub at different doses (1200, 600, 300, 150 µg/ml). One thousand ensheathed L3 were incubated for 3 h at 20°C. After incubation, the larvae were washed and centrifuged (1000 rpm at 20°C during 3 mn) three times in phosphate buffer saline solution (PBS: 0.1M phosphate, 0.05M NaCl, pH 7.2). Then, the larvae were subjected to an artificial exsheathment process by contact with a sodium hypochloride solution (2%, w/v) and sodium chloride solution (16.5%, w/v) diluted 1-400 in PBS. The kinetics of larvae exsheathment was measured at 20 min intervals for 60 min under microscopic observation at a magnification of ×100. PBS was used as a negative control. 4 replicates were considered for each shrub extract. In order to check the role of tannins in the anthelmintic effects of extracts an inhibitor of tannins, PVPP was used.
Data were subjected to analysis of variance using SPSS Statistics 20. The model included shrub type, dose of extract, time of incubation and all their interactions. The Duncan test was used to detect differences between treatments and values biochemical analysis and % of LEA are reported means with corresponding standard deviation. The effective concentration for 50% inhibition (EC50) ratios for each plant extract for the LEA was calculated with the PoloPLUS 2002-2003 (Probit and Logit Analysis). EC50 was obtained by non-linear regression analysis of 4 replicates for each of 5 dilutions (PBS, 150, 300, 600, 1200 µg/ml).
RESULTS AND DISCUSSION
Carob leaves exhibited the highest concentrations of CP and ADF (P<0.05) than pods (Table 1). For carob leaves, Silanikove et al., (1996) reported higher DM, ash and NDF contents; however, the CP level was similar (9%). Concerning carob fruits, the CP, NDF and ADF contents were slightly lower than those founded by Obeidat et al., (2010).
The contents of chemical constituents (ash, CP, ADL) of P. angustifolia leaves were higher than pods (P<0.05).
For M. arborea, Ventura et al., (1999) founded similar ash value, lower NDF content and higher ADF rate. This chemical composition variation’s can be due to several parameters such as: soil and air moisture, species and plant organ harvested (Jarrige et al., 1995).
Polyphenolic compounds and biological activity
C. siliqua leaves showed the highest polyphenolic compounds and BA (P<0.05) than C. siliqua pods (Table 2). In comparison to values reported by Manolaraki et al., (2010), TP and TT values of carob were higher for leaves and lower for pods, CT rates of carob leaves and pods were lower and the carob leaves BA was similar.
P. angustifolia pods presented highest TP and TT contents than P. angustifolia leaves (P<0.05). For CT and BA values, the tendency was reversed (P<0.05). This difference may be due to assay methods that do not measure all the same entities (Aufrere et al., 2012). It should be noted that the highest values in CT and BA were recorded for leaves. Indeed, Aufrere et al., (2012) reported that the majority of tannins were accumulated in young leaves and the secondary compounds rates varied according pedoclimatic conditions (water stress, light intensity), species, locality and the plant exploited organ’s of the plant.
Shrub’s effect on larval exsheathement assay
The effect of shrub was demonstrated without PVPP (P<0.001) and with PVPP, a tannin inhibitor (P=0.004) (Table 3). The highest % L3 exsheathed was recorded for M. arborea (55.01%) and the lowest value was founded for C. siliqua and P. angustifolia leaves (16.26%). The % L3 exsheathed values for carob pods were higher than carob leaves. The leaves and pods of P. angustifolia showed close values for %L3 exsheathed and also inhibition.
The % L3 exsheathed values founded for carob pods were higher than for leaves. P. angustifolia leaves and pods showed close values for the % L3 exsheathed. The recorded value was equivalent to 39.20% for M. arborea.
The highest inhibition was recorded for C. siliqua leaves and P. angustifolia leaves and pods. This strong inhibition could be due to their high content of secondary compounds and mainly to their high BA content. Indeed, C. siliqua leaves and P. angustifolia leaves and pods showed an inhibition higher than 50% so it may be considered as a bioactive plant with an interesting anthelmintic power.
Dose extract effect on larval exsheathement assay
Our results were concentration-dependent (P<0.001). As the doses increased, the % of exsheathment decreased to 29.74, 24.28, 18.80 and 16.39 for the doses 150, 300, 600 and 1200 µg/ml, respectively (Table 4). After addition of PVPP, the exsheathment trend reversed and was closer to that of PBS. Previous studies showed the same trend (Alonzo-Diaz et al., 2008; Manolaraki et al., 2010; Oliveira et al., 2011).
The restoration of L3 exsheathment to values similar to PBS, after PVPP addition, indicates that tannins of these shrubs are involved in the AH effects against H.contortus.
Incubation time effect on larval exsheathement assay
The result of incubation time effect on the % of exsheathment without and with PVPP was reported in Table 5. The % of exsheathment increased as the time of incubation increased (P<0.001) as reported by Aïssaet_al(2015).
Table 6 summarized data from the LEA. The interaction (shrub×time×dose) was significant (P<0.001). For PBS, the %L3 exsheathed of H.contortus ranged from 91.51% for C.siliqua leaves to 96.43% for P. angustifolia pods at 60 min. At the highest concentration, the % L3 exsheathed at 60min ranged from 0.00% to 93.75% for C. siliqua leaves and M. arborea, respectively. Indeed, AH was important for C.silique and P. angustifolia (leaves and pods). These results are in agreement with other studies that reported shrub’s AH activity (Alonso-Diaz et al., 2008; Oliveira et al., 2011). The variability of the AH effect is mainly due to variability in their phenolic compounds at the quantitative and qualitative level (Manolaraki et al., 2010).
After PVPP addition, all the extracts of the studied shrubs showed a restoration of the L3 exsheathment values similar to the control values (P<0.001) (Table 7). Alonso-Diaz et al., (2008) and Oliveira et al., (2011) also noted that the % of exsheathment was completely reversed. This suggests a major role of tannins, especially CT. Molan et al., (2003) mentioned that CT have the property of forming complexes with macromolecules including proteins and in our case the proteins of parasites (Manolaraki et al., 2010).
Effective concentration for 50% inhibition
EC50 was different (p<0.05) for plant extract (Table 8). C. siliqua leaves showed a lower EC50 than pods. However, the EC50 of P. angustifolia pods and leaves were similar. M. arborea had an EC50 around 954.08 μg/ml. More EC50 was lowest and the shrub sample’s was more effective against the exsheathment of L3 larvae, helping to prevent animal infestation.
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