In the current study, we synthesized eugenol (EU) based Pickering emulsion (PE) stabilized by food grade ingredients such as chitosan (CS) and tripolyphosphate (TPP) not only to enhance water miscibility of EU but also to decrease stress and damage to the immune system of fish due to anesthetic procedures.
Carbohydrate Polymers 265 (2021) 118096 Contents lists available at ScienceDirect Carbohydrate Polymers journal homepage: www.elsevier.com/locate/carbpol Opening a new gateway towards the applications of chitosan nanoparticles stabilized Pickering emulsion in the realm of aquaculture ´k, Josef Velíˇsek, Jan Mra ´z Bakht Ramin Shah *, Petr Dvoˇra ˇ e Budˇejovice, Faculty of Fisheries and Protection of Waters, South Bohemian Research Center of Aquaculture and Biodiversity of University of South Bohemia in Cesk´ Hydrocenoses, Czech Republic A R T I C L E I N F O A B S T R A C T Keywords: Pickering emulsions Eugenol Characterization Fish anesthesia Common carp In the current study, we synthesized eugenol (EU) based Pickering emulsion (PE) stabilized by food grade in gredients such as chitosan (CS) and tripolyphosphate (TPP) not only to enhance water miscibility of EU but also to decrease stress and damage to the immune system of fish due to anesthetic procedures The formulated EUPEs were characterized in terms of droplet size, size distribution and the effects of environmental conditions e.g pH and temperature on the behavior of the EUPEs The results showed that EU PEs with 5% EU had smaller size with uniform distribution and were stable in the range of pH 5–7.5 and temperature 30–80 ◦ C The anesthetic effect of the EUPE was investigated by taking Common carp as a sample species Interestingly, it was found that the induction time to anesthesia and recovery for the fish that received the PE was significantly shorter than that received EU at the same eugenol concentration (50 ppm) Most importantly, the improved hematological and bio-chemical parameters in the PE group further confirmed the immuno-protective and stress control efficacy of the PE The results of this study propose a novel useful and potential application of PE in fishery where sedation is needed Introduction Emulsions are regarded as thermodynamically unstable systems of two immiscible or partly miscible phases e.g oil and water (BertonCarabin & Schroăen, 2015) The interfaces between these phases are required to be stabilized by some type of amphiphilic or surface active compounds (Matos et al., 2018) The surfactants used to stabilize conventual emulsions such as nano or micro (McClements, 2012), can exert adverse effects on health as well as environment (Perrin et al., 2020) On the other hand, Pickering emulsion (PE) is an emulsion which is stabilized by solid particles instead of surfactants (Pickering, 1907; Ramsden, 1904) The particles are partly wetted by both the phases and are irreversibly adsorbed at their interfaces thereby conferring these emulsions with stability for months or even years In contrast with conventional emulsions (stabilized by small molecular surfactants), PEs have garnered tremendous attention in scientific community due to its non-toxic safe and long-term stable nature and therefore, are known as much more advantageous and efficient option for the encapsulation and delivery of a wide range of bioactive compounds (Shah & Mraz, 2020) So far a wide range of particles have been implied to stabilize PEs including inorganic or synthetic silica, latex, clay and so on (Yang et al., 2017) or natural food grade particles derived from protein such as lac toferrin, soy protein isolate (SPI), pea protein isolate (PPI) etc (Shao & Tang, 2016); from lipids in the form of fat crystal which have been documented as potential stabilizers for water in oil (W/O) emulsions (Rousseau, 2013) However, carbohydrates-based particles in the form of modified starches comprise a large group of emulsifiers for PEs This predominantly includes cellulose, starch and chitosan (CS) etc (Chen et al., 2020; Soltani & Madadlou, 2016) CS is generally obtained by the alkaline deacetylation of chitin (the second most abundant polymer in nature after cellulose) present in the exoskeletons of crustaceans as well as the cell walls of some algae and fungi (Mohammed et al., 2013) Being inexpensive, commercially available, biocompatible, biodegradable due to the free amino and hydroxyl groups along its backbone, CS has a wide range of useful applications in biomedicine, pharmaceutics and food industries (Mwangi, Ho, Ooi, et al., 2016) During the last few decades there has been a considerable research interest in using CS nanoparticles (NPs) as drug delivery carriers for sustained and controlled release of the encapsulated drugs and as stabilizers for stabilizing the oil water in terfaces of PEs To synthesize these NPs, different methods have been * Corresponding author at: University of South Bohemia in Ceske Budejovice, Faculty of Fisheries and Protection of Waters, South Bohemian Research Centre of ˇ e Budˇ Aquaculture and Biodiversity of Hydrocenoses, Institute of Aquaculture and Protection of Waters, Na S´ adk´ ach 1780, 370 05 Cesk´ ejovice, Czech Republic E-mail address: bshah@frov.jcu.cz (B.R Shah) https://doi.org/10.1016/j.carbpol.2021.118096 Received November 2020; Received in revised form March 2021; Accepted 22 April 2021 Available online 24 April 2021 0144-8617/© 2021 The Author(s) Published by Elsevier Ltd This is an (http://creativecommons.org/licenses/by-nc-nd/4.0/) open access article under the CC BY-NC-ND license B.R Shah et al Carbohydrate Polymers 265 (2021) 118096 implied so far including pH induce CSNPs (Liu et al., 2012) or by cross linking through ionotropic gelation method using different compounds such as glutaraldehyde (GLA), glyoxal (GO), epichlorohydrin (ECH), ethylene glycol diglycidyl ether (EGDE) and tripolyphosphate (TPP) (Babakhani & Sartaj, 2020; Shah, Li, et al., 2016) However, TPP is regarded as low cost and environmentally safe in comparison to the rest of these cross-linking agents which are known to be toxic and exert adverse effects on the environment (Vakili et al., 2014) In our previous studies we successfully synthesized CS cross linked TPP NPs which proved to be efficient stabilizers for a long-term stable PEs (Shah, Li, et al., 2016; Shah, Zhang, et al., 2016) Available literature supports the claim that till date PEs have a broad spectrum of useful applications in food, cosmetic and pharmaceutical industries including delivery of bioactive compounds and in the synthesis of porous scaffold, environment-responsive material and thermo-responsive material so on (Jiang et al., 2020; Yang et al., 2017) In addition, most recently, a research group fabricated zein particle stabilized clove oil (CO) PE and found that the addition of the synthesized COPE boosted up the anti microbial efficacy of the chitosan-based edible films (Xu et al., 2020) Eugenol (EU) an essential oil present in CO (70–90% by weight) is obtained from the buds, leaves, flowers and stem of the clove tree (Syzygium aromaticum) (Soltanian et al., 2018) It contains active com pounds isoeugenol and methyleugenol and is considered a potential anesthetic in fish (Hur et al., 2019) However, due to the issue of its poor dissolution in water, researchers employ alcohols or detergents as dis solving agents which can exert unfavorable effects on the fish and other living organisms (Gholipourkanani et al., 2015) Fish anesthesia is often required during different experimental procedures or during their transportation, grading or vaccination to make them sedated for extended period of time And applying higher doses of anesthetics can put a huge amount of stress on fish, that can result in anomalous metabolic rates, oxygen utilization, blood pressure, and blood physio logical responses, which can even last for several hours after recovery from anesthesia and can ultimately affect the nutritional quality of fish for human consumption (Herrera et al., 2019; Park et al., 2009) Now in order to avoid these unfavorable conditions, it is desired to reduce the stress to the fish up to the maximum level during anesthetic procedures And hence ecofriendly, non-toxic and cost-effective formulations such as PEs stabilized by food grade ingredients will be the best choice to serve the role So far no such study has ever been conducted to use EUPEs based formulations as anesthetizing agents in fishery Therefore, the main aim of the current study was to fabricate and characterize EUPEs stabilized by CSTPP NPs and evaluate their anesthetizing role in com mon carp as a sample specie room temperature (≈25 ◦ C) After overnight stirring the solution was filtered to remove the large dust or undissolved particles A solution of TPP at the concentration of 1.0 mg/ml (0.1 wt%) was prepared by dis solving TPP powder in ultrapure water and stirred to ensure its complete dissolution The NPs were then prepared by dropwise addition of TTP solution onto the CS solution at a ratio of 1:1 (w/w) under continuous stirring 2.3 Preparation of the required EUPEs Using the synthesized CSTPP NPs as stabilizer, the desired PEs were prepared with different EU contents (5, 10, 20, 30, 50 and 70 wt%) To so, aqueous phase containing NPs (95, 90, 80, 70, 50 and 30 v% respectively) was taken in a glass vial and was mixed with the oil phase (EU) at the specified amounts This mixture was then vigorously ho mogenized using UltraTurrax® T25 device equipped with a S25N-18G shaft (IKA, Germany) rotating at a speed of 10,000 rpm for The emulsions so prepared were immediately transferred to tightly covered glass bottles and stored at room temperature (≈25 ◦ C) for further analysis 2.4 Emulsions characterizations 2.4.1 Mean droplet size & droplet size distribution Particle size (mean diameter) and size distributions of the PEs sam ples were determined using a Malvern Mastersizer 3000 instrument (Malvern Instruments Ltd., Worcestershire, UK) The samples were dispersed in deionized water (≈1000 ml) and stirred at a rate of 2200 rpm to avoid multiple scattering effects The refractive indices of 1.53 and 1.33 were used for clove oil droplets and water (the dispersant), respectively 2.4.2 Effect of pH The effect of pH was assessed by adjusting pH of the samples to different values i.e 5, 6, and using either 0.1 M NaOH or 0.1 M HCl solution Thereafter, mean particle diameter and size distributions of these samples were measured in the same way as described above in Section 2.4.1 2.4.3 Effect of temperature In order to evaluate the effect of temperature on the behavior of the emulsions, emulsion samples were incubated in a water bath at 30, 50, 60, 80 and 90 ◦ C for 30 The visual changes of the emulsions were observed Then the mean particle diameter and size distributions of the samples were determined after the samples cooled down to room tem perature following the same procedure mentioned in Section 2.4.1 Experimental section 2.1 Materials and methods 2.4.4 Optical images Optical micrographs of the ZNCSPs based PEs were taken by an Optical microscope OLYMPUS (U-TVO.63XC, Tokyo, Japan) fitted with a digital camera (Olympus, DP 50) Emulsions samples were placed directly onto a microscope slide and softly covered with a cover slip The images were recorded at 25 ◦ C under the magnification from 10 to 40× CS (3 × 105–7 × 105 Da), ≥99.5% acetic acid (AA), TPP, NaOH were purchased from Sigma-Aldrich, Sokolovska, Prague, Czech Republic EU (100%) was purchased from Mach chemical spol s.r.o Ostrava, Czech Republic NaCl and 36–38% HCl were purchased from PENTA phar maceuticals Radiova, Prague, Czech Republic All of the materials guaranteed analytical grade and were used without further purification Water used in the preparations of all solutions was purified by deion ization and filtration with a IWA 30 iol, WATEK apparatus (Czech Re public) to a resistivity higher than 18.0 MΩ⋅cm 2.5 Fish trials 2.5.1 Experimental fish A total of 28 common carps with average body weight 206 ± 41, total body length 223 ± 21 and specific length of 182 ± 12 were selected from the already acclimatized common carp in the aquarium of the Institute of Aquaculture and Protection of Waters, Faculty of Fishery and Pro ˇ ´e Budˇejovice The tection of Waters, University of South Bohemia in Cesk fish were individually stocked in plastic tanks (10 L) equipped with aeration Parameters such as water dissolved oxygen, pH and tempera ture of the water were measured to be 90%, 7.3 and 19 ◦ C respectively In order to test the anesthetizing efficacy of the designed EUPEs, the 2.2 Preparation of chitosan tripolyphosphate nanoparticles (CSTPP NPs) CSTPP NPs were synthesized using the same method as described in our previous work (Shah, Li, et al., 2016) Briefly, 0.5 wt% CS solution was prepared by dissolving CS powder in AA solution maintaining CS to AA ratio of 2:3 and was diluted with deionized water to obtain the required volume The resultant mixture was then stirred for overnight at B.R Shah et al Carbohydrate Polymers 265 (2021) 118096 selected fish were divided into seven groups (six experimental and one control with no EU) with four fish in each group EU at three different concentrations i.e 12.5, 25 and 50 ppm either in the form of PE (0.25 ml, 0.5 ml & ml of 5% PE/l respectively) or free (original form) were applied to the respective tanks with the individual fish Statistics All experiments were done at least in triplicates The data were presented as mean ± SD Statistical evaluation of the anesthesia in duction and recovery times as well as hematological and biochemical parameters were carried out by analysis of variance (ANOVA) procedure using the Statistix 8.1 software Least significant difference (LSD) at 5% level was used for multiple comparison tests among the treatments 2.5.2 Onset and recovery from anesthesia The observations of the stages & 6-anesthesia onset and recovery (according to Table 1) were made after using either EUPE or EU under completely similar experimental conditions The duration to achieve stages & anesthesia (A5 & A6) for each replicate were noted using a stopwatch Once an individual fish would reach the onset of stage anesthesia, a dip net was used to immediately remove it from the experimental tank The fish was then washed off in clean water tank and transferred to another 10 L, well-oxygenated tank termed as ‘Recovery’ tank (i.e., no anesthesia present) maintained at 19 ◦ C and observed until it fully recovered During this recovery period, the fish behavior was observed and times to observe stages & recovery (R5 & R6) were noted using a stopwatch Results and discussion 4.1 Fabrication and characterization of EUPEs 4.1.1 Optimization and droplet size of the EUPEs In order to prepare stable PE systems, the oil to water ratio should be optimized as it will affect particles concentration which is the vital factor in controlling the droplet size of PEs With this consciousness, initially we prepared the required EUPEs with different oil (EU) frac tions of 5, 10, 20, 30, 50 and 70 wt% shown in Fig 1a As can be seen, the formulations with higher oil contents of 30, 50 and 70 wt% didn’t turn into well homogenized emulsions systems and the oil fractions were clearly seen separated soon after the homogenization On the other hand, formulations with lower oil contents i.e 5, 10 and 20 wt% gave well homogenized and stable emulsions systems at the same homoge nizing speed and time The reason for this emulsification failure at higher oil contents could be due to the poor coverage of the oil water interface by the stabilizing CS-TPP NPs Based on these outcomes, we therefore, didn’t further characterize the formulations with higher oil contents but rather proceeded with the ones having lower oil contents Optical images in Fig 1b, c and d for emulsions with oil contents 5, 10 and 20 wt% respectively, exhibited an increase in size with increasing oil contents This trend in increase with increasing oil contents was further confirmed by measuring mean droplet diameter through laser diffraction instrument (Malvern Mastersizer 3000, UK) It is evident from Fig that although the three emulsions had uni form particle distributions, the droplet sizes in terms of the Sauter mean diameter (d32) and volumetric diameter (d43) increased significantly with increasing the oil contents from to 20 wt% These results are in agreement with the previous study where increase in sizes with increase in oil contents of olive oil based PE was observed at all tested pH values (i.e 2, and 7) (Jutakridsada et al., 2020) As a matter of fact, this can be attributed to the alteration in the oil to particles ratio caused by increasing the oil contents in the system This phenomenon has been well explained in previous work by Arditty and colleagues who described the phenomenon of droplet size variation with the amount of particles by a so-called limited coalescence The emulsion droplets tend to coalesce as a result of the decrease in the total particles concentration required to completely cover the oil-water interfaces (Arditty et al., 2003) Similar results of the link between particles concentration and droplet size were also presented by Tzoumaki et al., who obtained emulsions with smaller droplets, enhanced stability and distinct elastic responses by increasing the concentrations of chitin nanocrystals (ChNs) used to stabilize oil-in-water emulsion (Tzoumaki et al., 2011) 2.5.3 Blood sample collection, hematological and biochemical assay Blood samples were collected from all the anesthetized fish included in six experimental groups and one control group (without EUPE and EU administration) The samples were drawn from vena caudalis using a syringe with heparin as anticoagulant (Heparin inj., Leciva, Czech Re public) at a concentration of 5000 IU heparin sodium salt in ml Plasma samples obtained by centrifugation of blood at 3000g for 15 were stored at − 80 ◦ C until analysis The hematological parameters were measured that included red blood cell count (RBC), haemoglobin concentration (Hb), hematocrit (PCV), white blood cell count (WBC) mean erythrocyte volume (MCV), mean corpuscular haemoglobin con centration (MCHC), mean corpuscular haemoglobin (MCH), differential % WBCs (lymphocyte, monocyte, neutrophile granulocytes segment, neutrophile granulocytes bands, developmental phases – myeloid sequence) The indices used to evaluate hematological profile were based on unified methods for hematological examination of fish (Svo bodova et al., 1991) The bichemical parameters were measured that included glucose (GLU), total protein (TP), albumin (ALB), globulin (GLOB), triacylglycerols (TAG), aspartate aminotransferase (AST), lactate (LACT), inorganic phosphate (PHOS), cholesterol (CHOL) Globulin content was calculated by subtracting albumin values from serum total protein Measurement of biochemical plasma parameters was conducted using the Catalys One analyser (IDEXX Laboratories Inc., Maine, USA) Table Detailed description of different stages of anesthesia induction and recovery in EU based tests performed in common carp (Park et al., 2009; Yousefi et al., 2018) Stage Characteristic behavior Anesthesia A1 Normal swimming; opercular movement and normal general movement A2 Swimming speed slowed; rolling from side to side A3 Partial loss of equilibrium; swimming erratic A4 Complete loss of equilibrium; swimming perfectly inside out; pectoral fin, pelvic fin and dorsal fin movement stop A5 Little sedation; anal fin and tail fin movement stop A6 Perfect sedation; only opercular movement Recovery R1 R2 R3 R4 R5 R6 4.1.2 Effects of temperature and pH on the behavior of the EUPEs Droplet size of an emulsion has a crucial role in its stability The smaller the emulsion droplets, the more stable the emulsions are and vice versa This higher stability of an emulsion with smaller droplets can be due to the strong resistance of smaller droplets against different destabilization phenomena like aggregation, coalescence, and floccula tion because of the Brownian motion that overcomes gravitational forces (Hidajat et al., 2020) Thus a stable emulsion is the one capable of resistance to the instability mechanisms such as Ostwald ripening (development of the larger droplets by the expense of the smaller ones) or very much similar to that coalescence as well as the flocculation, where the droplets collide and bunch together to make flocs (larger Resume opercular movement Preferential movement of pectoral fin and tail fin Dorsal fin, pelvic fin and anal fin movement Swimming perfectly inside out Swimming erratic; redress the balance Normal swimming; responsiveness to visual stimuli B.R Shah et al Carbohydrate Polymers 265 (2021) 118096 Fig Digital photographs of the PEs prepared with different EU contents (wt%) a) and optical micrographs of the ones prepared with b) 10 c) and 20 d) wt% of EU Fig Mean droplet sizes and size distributions of the EUPEs prepared with 5, 10 and 20 wt% EU droplets) which then finally lead to sedimentation or creaming of the emulsion due to gravitational separation (Low et al., 2020) Based on these facts, the emulsion prepared with wt% EU was selected to evaluate the effects of temperature and pH on its stability in terms of visual observations and droplet sizes, as it had smaller sizes compared to the ones prepared with 10 and 20 wt% (Fig 2) In the first instance, the effect of temperature was evaluated by incubating emulsion samples (original temperature 20 ◦ C) in a water bath at 30, 50, 60, 80 and 90 ◦ C for 30 Visual observations of the samples showed that there was no cream formation or even sedimen tation of the particles on the bottom of the sample tubes till 80 ◦ C And hence, although the mean droplet diameter slightly increased at tem perature 30 ◦ C, no significant changes were observed in the sizes thereafter till 80 ◦ C (Fig 3a) Also, all the samples showed uniform size distributions (Fig 3b) which further confirmed the capability of the PE to withstand the influence of temperature However, when the tem perature was raised to 90 ◦ C, the emulsion apparently turned into cream and therefore at this temperature no mean droplet size of the emulsion was measured This observation can be attributed to the enhanced en counters among the particles due to the rise in temperature and ulti mately their average kinetic energy, thereby leading to enhancing their motion and rate of collision finally making them to agglomerate (Mwangi, Ho, Tey, & Chan, 2016) Similarly, the effect of pH on the emulsion stability was monitored by adjusting pH of the samples (original pH 4.3) to 5, 6, 7, 7.5 and As no destabilization occurred during changing pH from to 7.5, it can be stated that emulsion was highly stable against droplet coalescence and creaming till pH 7.5 The droplet sized increased linearly with increasing pH values (Fig 3c) but B.R Shah et al Carbohydrate Polymers 265 (2021) 118096 Fig Influence of temperature on the droplet size a) size distribution b) and influence of pH on the droplet size c) and size distribution d) of the EUPE prepared with EU contents of wt% exhibited uniform distribution (Fig 3d) This finding can be well explained by the fact of relatively low net droplet charge at high pH with lowered electrostatic repulsion between the droplets (Shah, Li, et al., 2016) which could even be reason for the complete destabilization of the emulsion at pH (shown in the inset of Fig 3c) These results can be regarded to present an ideal scenario of releasing the encapsulated EU as main anesthetizing agent into the water (with pH ≈ 7.3) in which the experimental fish are stocked Furthermore, to assess the influence of storage time on its stability, the emulsion was stored at room temperature for three months and was observed visually with time It was found that no visual change appeared in the behavior of the emulsion during this time which clearly indicated its long-term stable nature PE groups, whereas no change in the behavior of fish was observed even after 30 of applying EU in their respective water tanks This dif ference was also reflected in the analysis of different blood parameters of both the groups For example, it can be seen in Table 2, that hematocrit (PCV) of 0.3925 in the PE group with 25 ppm of EU was significantly higher (P < 0.05) than EU group (0.3425) These results comply with the previous studies where significant increase in PCV levels were observed after administrating EU as an anesthetic in O tshawytscha (Cho & Heath, 2000) and olive flounder (Paralichthys olivaceus) (Hur et al., 2019) Interestingly, when the EU at a concentration of 50 ppm in the form of EUPE or EU was administrated, the induction and recovery times from anesthesia were significantly lower (P < 0.05) for the EUPE group than EU As shown in Fig 4a, the induction time of stage and anesthesia (A5 and A6) with ≈3.3 min, was significantly lower (P < 0.05) than the EU group with A5 and A6 of 12.4 and 14.2 respectively Similarly, the recovery time for stage and recovery (R5 and R6) of 11.9 and 15 respectively for EUPE group was significantly lower (P < 0.05) than R5 and R6 of 15 and 19.9 respectively for the EU group (Fig 4b) These findings of EUPE are closely coincided with the claims of previous studies mentioning optimum anesthetic concentrations to induce anes thesia around and recovery around 10 (Park et al., 2009) This promising capability of EUPE was further hallmarked by different hematological and biochemical plasma parameters as given in Tables and respectively It is evident from Table that neutrophil granulocytes segment (%) of 1.8000 ± 1.1106 for EUPE group was significantly higher (P < 0.05) than EU group with 0.5000 ± 0.7572, but was non-significant with the control group (1.4750 ± 0.4717) where no EU (neither EUPE nor EU) was administered Similarly, another important indicator developmental phases – myeloid sequence (%) with 1.8000 ± 1.2410 for EUPE group was significantly higher (P < 0.05) 4.2 Evaluating anesthetizing efficacy of the synthesized EUPE in fish model (common carp) With the aim to introduce EU based PE an unprecedented non-toxic formulation composed of biocompatible and biodegradable materials as anesthetizing agent in fish, we evaluated the anesthetizing role of the prepared EUPE by choosing common carp as a sample species The selected fish were divided into different groups, and three different concentrations of EU i.e 12.5, 25 and 50 ppm in the form of EUPE or free EU were tested to evaluate their anesthetizing efficacy It was found that the EUPE ensured complete dissolution of the loaded EU as no oil droplets were visualized on the water surface after the administration Conversely, the oil droplets were seen on the water surface even after powerful agitation when free EU was applied And therefore, it could be the reason that even at lower concentration of EU i.e 12.5 and 25 ppm, anesthesia up-to stage (A3 & A4 shown in Table 1) was achieved in the B.R Shah et al Carbohydrate Polymers 265 (2021) 118096 Table Hematological parameters of common carp following treatment with different concentrations of EU in the form of EUPE or free EU Hematological parameters Control PCV 0.3525 0.0150 79.048 2.3276 1.9875 0.0981 10.525 1.3672 39.859 2.5597 177.80 13.556 224.37 4.6701 ± bc ± a ± a ± a ± a ± a ± a 96.000 1.2083 0.8250 0.3403 1.4750 0.4717 0.1000 0.2000 1.6000 0.8446 ± bc ± ab ± ab ± a ± ab Hb (g/l) RBC (T/l) WBC (G/l) MCH (pg) MCV (fl) MCHC (g/l) Differential WBC count Lymphocyte (%) Monocytes (%) Neutrophil granulocytes segment (%) Neutrophil granulocytes bands (%) Developmental phases – myeloid sequence (%) Group I (12.5 ppm EU) Group II (25 ppm EU) Group III (50 ppm EU) PE EU PE EU PE EU 0.3238 ± 0.0075 c 80.481 ± 5.4021 a 1.7975 ± 0.3715 a 13.275 ± 4.9466 a 45.896 ± 7.8457 a 185.37 ± 33.917 a 248.68 ± 17.346 a 0.3250 ± 0.0220 c 78.153 ± 11.806 a 1.7525 ± 0.2323 a 7.8750 ± 2.4281 a 45.272 ± 10.132 a 187.05 ± 19.772 a 240.10 ± 28.368 a 0.3925 ± 0.0419 a 86.658 ± 15.477 a 1.8900 ± 0.3153 a 6.7500 ± 3.3789 a 47.721 ± 16.919 a 210.04 ± 22.324 a 223.40 ± 53.929 a 0.3425 ± 0.0328 bc 88.627 ± 6.5280a 1.9725 ± 0.2478 a 11.525 ± 4.5353 a 45.780 ± 9.1281 a 176.46 ± 32.849 a 260.24 ± 27.369 a 0.3725 ± 0.0260 ab 88.806 ± 6.7754 a 2.0050 ± 0.2478 a 9.7500 ± 2.5331 a 44.837 ± 5.7419 a 187.97 ± 23.121 a 239.60 ± 28.651 a 0.3650 ± 0.0297 ab 85.315 ± 4.6171 a 1.8325 ± 0.1047 a 9.5000 ± 3.9370 a 46.753 ± 4.9958 a 200.01 ± 23.743 a 235.19 ± 25.914 a 97.950 ± 0.8583 ab 0.6000 ± 0.6928 b 1.0250 ± 0.2754 abc 0.0000 ± 0.0000 a 0.4250 ± 0.6131 cd 98.975 ± 0.6076 a 0.3250 ± 0.3948 b 0.5750 ± 0.4272 c 0.1250 ± 0.2500 a 0.0000 ± 0.0000 d 95.350 ± 2.2368 c 2.4500 ± 1.7823 a 0.8250 ± 0.2217 bc 0.2375 ± 0.2750 a 1.1375 ± 0.5764 abc 98.100 ± 1.3928 ab 0.9500 ± 1.2557 ab 0.5000 ± 0.4082 c 0.0000 ± 0.0000 a 0.4500 ± 0.5196 cd 94.425 ± 2.2232 c 1.7250 ± 1.0468 ab 1.8000 ± 1.1106 a 0.2500 ± 0.5000 a 1.8000 ± 1.2410 a 96.625 ± 2.2750 abc 1.8500 ± 1.8448 ab 0.5000 ± 0.7572 c 0.3250 ± 0.2217 a 0.7000 ± 0.5944 bcd Similarly, significant difference (P < 0.05) in lymphocyte (%) i.e 95.350 ± 2.2368 and 98.100 ± 1.3928 for EUPE and EU group respectively was found between the two groups As lymphocytes have fundamental importance in body’s immune system, these findings highlight significant influence of anesthesia on the immunity of the fish Fig Comparison between the times elapsed during the stages of anesthesia induction and recovery in the EUPE and EU groups of the experimental common carp than 0.7000 ± 0.5944 for the EU group Furthermore, as given in Table 3, we also analyzed different biochemical plasma parameters It is important to mention that being one of the most common stress indicators in fish, glucose cannot be ignored while speaking about stress Because under stress conditions, the release of stress hormones is altered which cause changes in the blood as well as tissue chemistry including increasing blood glucose levels, in an attempt to prepare the fish for emergency situations (Makaras et al., 2020) Referring to the fascinating results of EUPE group for the glucose levels in Table 3, it can be seen that the glucose values of 4.7950 ± 0.7735 mmol/l for EUPE was significantly lower (P < 0.05) than for the EU with 7.1800 ± 0.7963 mmol/l What’s more, the glucose levels of the EUPE group (4.7950 ± 0.7735 mmol/l) was almost similar with the control group (4.0775 ± 0.9859 mmol/l) having no significant difference (P < 0.05) Looking at these findings it can postulated that the EUPE reduced the stress level of fish by ≈50% than free EU These re sults clearly favor the immuno-protective as well as stress reducing potentials of the synthesized EUPE The fast induction of anesthesia and recovery of fish in the EUPE group due to short time of exposure would favor less stress in these fish compare to the ones in the EU group exposed for longer time This can be the key immuno-protective factor as there is a well-established link between stress and immune system modulation of fish (Tort, 2011) B.R Shah et al Carbohydrate Polymers 265 (2021) 118096 Table Biochemical plasma parameters of common carp following treatment with different concentrations of EU in the form of EUPE or free EU Biochemical plasma parameters Control ALB (g/l) 11.750 a 24.500 a 176.00 a 1.3125 a 36.000 ab 4.0775 c 2.2850 ab 1.3725 c 3.0875 abc GLOB (g/l) AST (U/l) PHOS (mmol/l) TP (g/l) GLU (mmol/l) TAG (mmol/l) LAC (mmol/l) CHOL (mmol/l) Group I (12.5 ppm EU) PE ± 0.9574 ± 2.3805 ± 118.74 ± 0.1204 ± 2.7080 ± 0.9859 ± 0.2694 ± 0.1335 ± 0.2708 10.750 b 23.250 ab 141.25 a 1.3650 a 33.500 bc 5.4650 bc 2.2325 ab 4.5675 a 2.8025 c Group II (25 ppm EU) EU ± 0.5000 ± 1.5000 ± 23.796 ± 0.0311 ± 1.7321 ± 1.3986 ± 0.1056 ± 2.8222 ± 0.2702 11.750 ab 21.750 bc 176.25 a 1.2800 a 33.750 bc 6.9825 a 1.9500 cd 5.4675 a 2.8300 bc PE ± 0.5000 ± 1.5000 ± 44.192 ± 0.0455 ± 0.9574 ± 0.6547 ± 0.1013 ± 0.9652 ± 0.0648 10.750 b 19.250 d 102.50 a 1.1400 b 30.000 d 6.1050 ab 2.0250 bcd 2.3625 bc 2.4700 d Group III (50 ppm EU) EU ± 0.9574 ± 0.9574 ± 16.862 ± 0.0906 ± 0.8165 ± 0.9425 ± 0.1318 ± 0.8982 ± 0.1564 10.750 b 20.000 cd 122.00 a 1.2950 a 30.750 d 4.6825 bc 1.9200 d 4.0575 ab 2.3050 d PE ± 0.9574 ± 1.4142 ± 33.872 ± 0.1480 ± 0.9574 ± 1.1598 ± 0.1454 ± 0.9251 ± 0.1666 10.500 b 21.250 bcd 165.50 a 1.3250 a 31.750 cd 4.7950 bc 2.2900 a 5.4725 a 3.1250 ab EU ± 1.2910 ± 0.5000 ± 42.587 ± 0.1439 ± 1.7078 ± 0.7735 ± 0.1525 ± 0.5861 ± 0.1028 12.250 b 25.000 a 149.00 a 1.4250 a 37.750 a 7.1800 a 2.1900 abc 4.7850 a 3.1550 a ± 0.5000 ± 1.1547 ± 49.214 ± 0.0954 ± 2.3629 ± 0.7963 ± 0.2652 ± 1.4121 ± 0.2908 Glucose (GLU), total protein (TP), albumin (ALB), globulin (GLOB), triacylglycerols (TAG), aspartate aminotransferase (AST), lactate (LACT), inorganic phosphate (PHOS), cholesterol (CHOL) The values are expressed as mean ± SD Measurements were performed in triplicate (n = 4/group) Means in the rows with different letter are significantly different (LSD test, P < 0.05) Conclusion concentrations of EUPE based on EU in common carp Although no full sedation was achieved at lower concentrations (i.e 12.5 and 25 ppm EU), however with higher concentration (50 ppm), the times for anes thesia induction and recovery was significantly lower for EUPE group than EU groups We also analyzed different hematological and biochemical plasma parameters which showed that compared to EU, EUPE significantly control the stress with no attenuating effects on the immunity of the fish Overall outcomes of the current study categorically demonstrated EUPE to be a potential anesthetizing formulation in fish ery with immuno-protective and stress control capabilities In the current study we for the first time introduced EU based PE as an anesthetizing formulation in fish As drawn schematically in Fig 5, the EUPEs were successfully fabricated using CS-TPP NPs for stabilizing the oil water interfaces The preparation of the required EUPE was initially optimized by its preparation with different oil contents Droplet size and size distribution of all the samples were measured and the one with the smallest size was chosen for further analysis Effects of influ encing environmental conditions such as temperature and pH on the behavior of the EUPE were evaluated The EUPE was found to be stable against different tested temperature ranges up-to 80 ◦ C as no significant change in droplet size was observed On the other hand, the droplet size was found to increase with increasing pH until pH 7.5 as at pH the emulsion was completely destabilized Thereafter, the anesthetizing efficacy of the formulated EUPE was evaluated by testing three different CRediT authorship contribution statement Dr Bakht Ramin Shah: Conceptualization, Methodology, Formal analysis, Data curation, Writing - original draft, Visualization, Ac tivities administration Fig Schematic representation of the NPs and EUPEs synthesis and the potential application of the EUPE as an anesthetizing formulation in comparison with free EU in common carp B.R Shah et al Carbohydrate Polymers 265 (2021) 118096 ´k: Methodology, 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