Abstract
Oxidative stability of fish oil supplements poses a considerable health risk which can be prevented by novel delivery systems. A newly developed formulation of microencapsulated sardine oil showed excellent oxidative stability in vitro. The present study’s objective is to evaluate the new formulation in vivo as a potential new supplement which may improve antioxidant, haematological, and lipid profile. The optimisation of the sardine oil loaded microparticles (SO-M) and the characterisation have been presented briefly. The SO-M formulation was fed to male albino rats for two months. Following the feeding experiment, haemoglobin content, platelet and RBC count were assessed in the control and treated group. Similarly, levels of serum cholesterol, HDL, LDL, triglycerides, and metabolic enzyme biomarkers, namely catalase, SOD, GST, AST, ALT, ACP and ALP, were compared. The blood analysis showed a significant increase in haemoglobin, platelets and RBC count in the treated group. Lipid profiling showed that both triglycerides and LDL levels were decreased in the sample treated group. This study also showed significant modulation of antioxidant enzymes such as catalase, SOD and GST. The new formulation of PUFA rich sardine oil significantly improved the in vivo antioxidant, haematological and lipid profile, suggesting potential use as a dietary supplement.
Supplementary Information
The online version contains supplementary material available at 10.1007/s13197-021-05329-5.
Keywords: Microencapsulation, Vanillic acid grafted chitosan, Sardine oil, Antioxidant enzymes, Lipid profiling, Haematological profile
Introduction
The health benefits of long-chain polyunsaturated fatty acids (LC-PUFA), especially docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) containing marine fish oils are well known (Vishnu et al. 2017). An informal estimate pegs the market size at USD 2.5 billion. PUFA rich supplements help in overcoming adverse inflammatory effects, prevents excessive accumulation of subcutaneous fat, and maintains cardiac health (Luo et al. 2013; Iizuka et al. 2016; Chu et al. 2021). On the contrary, unsaturated fatty acids are highly susceptible to peroxidation. Oxidised oil consumption leads to increased lipid peroxidation in the liver and impairs gut barrier function (Tan et al. 2019). Such peroxidation products can adversely affect their nutritional quality. Hence, fish oil needs to be carefully protected from oxidising factors, such as oxygen, light, free radicals and pro-oxidants (Jeyakumari et al. 2018).
Microencapsulation is one of the potential methods that protect unsaturated oil containing ingredients during the processing and manufacture of food products (Intarasirisawat et al. 2015). The choice of versatile wall material is a crucial factor (Linke et al. 2020). Chitosan is an amino polysaccharides with excellent emulsifying property. It has often been used as a wall material, as well as an emulsifier for microencapsulation of highly unsaturated oils. Chitosan is generally soluble in dilute acid solutions, whereas some modified chitosan exhibit improved solubility and emulsifying properties (Jayakumar et al. 2005). Modification of chitosan with phenolic acids has been reported previously (Chatterjee et al. 2015). The derivatives have promising applications as food additives or functional food ingredients due to improved antioxidant, antimicrobial and emulsifying properties (Liu et al. 2017). Microencapsulation of fish oil with such chitosan derivatives will open up new avenues in the food industry as novel functional ingredients.
Our previous work on encapsulation of PUFA rich sardine oil in vanillic acid grafted chitosan microparticles (SO-M)showed excellent oxidative stability, antioxidant property, and cardioprotective activity (Vishnu et al. 2017, 2018). However, it is vital to evaluate novel dietary supplements in an experimental animal model to further evaluate and gain insights on applicability as dietary supplement. The present study investigated the effect of dietary supplementation of SO-M on in vivo antioxidant status, haematological and lipid profile in albino rats.
Methods and materials
Materials
Sardine oil (Sardinella longiceps) was obtained from Arbee agencies Kottayam, Kerala, India and was stored in dark amber-coloured glass bottles at −20 °C. Chitosan was prepared in Central Institute of Fisheries Technology (Molecular weight: 100 KDa, Degree of deacetylation: 80–85%), Cochin, Kerala, India. Vanillic acid, Tween 20 and all other chemicals used in the enzyme assay were obtained from Sigma-Aldrich (USA).
Synthesis of vanillic acid grafted chitosan, preparation of SO-M, and characterisation
Vanillic acid grafted chitosan was synthesised and characterised as previously described in our earlier report. FTIR and NMR experiments confirmed successful synthesis. Spray-dried SO-M were prepared as described earlier. Encapsulation efficiency, particle size, zeta potential, and oxidative storage stability of the SO-M were established as described in our earlier report (Vishnu et al. 2017).
Experimental animals and design of the study
The experiment used male albino rats of the “Wister” strain (120–200 g), which were bred, reared and housed hygienically at room temperature (28 ± 2 °C) in separate polyurethane cages in the ICAR-CIFT animal house facility. The activities in the animal house are approved by the “Committee for the Purpose of Control and Supervision of Experiments on Animals” (CPCSEA, New Delhi, India). The animals received a standard diet and water and the experiments followed all CPCSEA guidelines. The study received prior approval from the “Institutional Animal Ethics Committee” (IAEC) (Approval code: CIFT/B&N/IAEC/16(4)/2016). Each of the Control (C), Treatment 1 (T1) and Treatment 2 (T2) groups contained six animals. The animals in “C” received a standard diet, while animals in T1 and T2 received a diet supplemented with 1% SO-M and 3% SO-M, respectively. The feed which the animals did not consume was weighed before disposal, and consumption data were recorded. At the end of the experiment (60 days), animals were anaesthetised with intraperitoneal administration of ketamine (80 mg/kg) and xylazine (10 mg/kg) and blood was collected. After blood collection, liver, kidney, muscle and heart were removed. The tissue parts were immediately homogenised in in chilled sucrose solution (0.25 M), the aliquots were flash-frozen and stored at −20 °C.
Growth study
The animals were weighed at the start and every seven days’ intervals until the experiment’s termination on the 60th day. The growth performance was calculated in terms of weight gain (%) and specific growth rate (SGR) as described in Eqs. 1 and 2:
| 1 |
| 2 |
where Wf is final weight, Wi is initial weight, AvgWf is average final weight, and AvgWi is the average initial weight.
Lipid and blood profile
An automated lipid analyser (Abacus-250) measured the total cholesterol, high-density lipoprotein (HDL), low-density lipoprotein (LDL) and triglycerides (TG). Blood profile, including haemoglobin, platelets and RBC count, was measured in an automated clinical analyser (Minbray-3200 BC). Blood glucose was estimated using a method reported earlier (Somogyi 1945).
Enzyme biomarker assay
The aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALP), acid phosphatase (ACP), superoxide dismutase (SOD), catalase, and glutathione-s-transferase (GST) activities in liver, kidney, muscle, and heart tissues were determined following literature reported methods (Harper 1965; Garen and Levinthal 1960; Misra and Fridovich 1972; Claiborne 1985; Habig et al. 1974). Tissue protein content was determined using the Folin phenol reagent method of Lowry et al. (1951).
Statistical analysis
A one-way analysis of variance was carried out to compare the direct and interaction effect of different treatments. Tukey’s test was performed to compare the marginal means, and a t-test was used to compare the interaction means at 5% level of significance. All the statistical analysis was done using SAS 9.3 software.
Results and discussion
In this study, a novel biofunctional biopolymer Va-g-Ch was used to develop SO-M that maintains the oil’s oxidative stability for a longer duration. Our previous reports suggest excellent antioxidant activity of Va-g-Ch (Chatterjee et al. 2015). Also, vitamin loaded Va-g-Ch microparticles enhanced growth, immune, and metabolic performance in an animal model (Tejpal et al. 2017).
The feed of the animal was supplemented with 1% and 3% level. Initially trial feeds were prepared with different percentage of supplementation. At doses higher than 3% the animals showed less consumption of feed. Since, all the material used in the formulation is food grade and oxidative stability already been established in our previous published study, we did not perform routine toxicological study. The study results presented in subsequent sections do not indicate any adverse effect of the sardine oil microencapsulated formulation.
Physicochemical characterisation of Va-g-Ch and SO-M
The successful synthesis of Va-g-Ch was confirmed as previously published in our work (Vishnu et al. 2017). The details of characterisation, characteristic FTIR bands, and NMR spectra assignments are presented as the Supplementary Material to the manuscript. Similarly, physicochemical characterisation of the SO-M demonstrated successful microencapsulation of sardine oil in Va-g-Ch. The detailed descriptions are in the Supplementary Material to the document.
Effect of SO-M on growth performance and haematological parameters
Table 1 presents the weight gain percentage (%) and specific growth rate (SGR) of the experimental rats. Animals in group T1 and T2 showed significant improvement in weight gain percentage and SGR when compared to control animals. The blood glucose level, haemoglobin content, RBC count, and Platelet count of the animals in treated and control groups were estimated at the end of the experiment. Figure 1a shows a dose-dependent decrease in the blood glucose levels in the treatment groups when compared to control. The blood glucose level of the control group was 118 mg/dL, whereas dietary supplementation with SO-M at 1 and 3% levels resulted in a blood glucose level of 100 and 82 mg/dL. When compared to control, there was a significant increase in haemoglobin concentration in the treatment groups (Fig. 1b).
Table 1.
Impact of dietary supplementation of SO-M on weight gain% and SGR
| Parameter | Control | T1 (1% SO-M) | T2 (3% SO-M) |
|---|---|---|---|
| Weight gain% | 300.8 ± 5.28a | 324.6 ± 6.49b | 320 ± 7.21b |
| SGR | 2.98 ± 0.02a | 3.19 ± 0.07b | 3.12 ± 0.02b |
Values are presented as the mean ± SD. Different small letters as superscript indicate the significant difference (p < 0.05)
Fig. 1.
Effect of dietary supplementation of SO-M on a Blood glucose level, b Hemoglobin concentration, c RBC count, and d Platelet count. Different superscript (a, b and c) in the graph indicate a significant difference (p < 0.05) between the control and treatment groups
Similarly, a significant increase in the RBC and Platelet counts were observed in the treated groups when compared to control (Fig. 1c, d). However, there was no dose-dependent increase of haemoglobin concentration, RBC and Platelet counts in the treated groups. A higher dose did not result in any significant increase or decrease. Nutritional benefits of the consumed products can be better monitored by analysing hematological parameters since any biochemical change ultimately reflects in clinical parameters. Better Hb concentration, RBC and platelet counts play a critical role in host defense and in many physiological functions. Supplementation with SO-M markedly improved in the platelet counts. However, a higher dose of SO-M did not significantly change the hematological parameters further indicating no adverse effect at higher dose.
Effect of SO-M on lipid profile
A significant decrease in the total serum cholesterol and total triglyceride levels were observed in animals supplemented with 1% SO-M (T1). With a 3% SO-M (T2) supplemented diet, a decrease in the total serum cholesterol and total serum triglyceride levels were observed, even though such changes were not statistically significant. Serum-LDL levels in animals belonging to both T1 and T2 groups were significantly less than the control group. However, dose-dependent reduction in serum-LDL was not observed when supplemented with 3% SO-M. Similarly, the serum-HDL level was considerably higher in both the treated groups than the control group. Here too, no significant increase in the serum HDL level was observed when supplemented with 3% SO-M (Fig. 2). Supplementation at higher doses did not cause any adverse effect on the overall lipid profile but failed to cause a dose dependent improvement. This could be due to higher content of sardine oil at higher concentration, but need further confirmation. Plateauing of beneficial effect at higher doses is normal and emphasizes the need for further future trials at even lower doses. A recent study suggests that even the younger population may have mildly abnormal serum cholesterol levels, triglyceride, LDL-cholesterol and VLDL-cholesterol. They are at risk of myocardial infarction (Park et al.2020). Our previous study demonstrated that SO-M attenuates myocardial oxidative stress and apoptosis in cardio myoblast cell lines (H9c2) (Vishnu et al. 2018). Further, the present in vivo experiment established that dietary supplementation with SO-M reduces serum cholesterol, triglyceride, and LDL while increasing the serum HDL.
Fig. 2.
Effect of dietary supplementation of SO-M on lipid profile in Wister rats. Different superscript (a, b, c) in the graph indicate a significant difference (p < 0.05) between the control and treatment groups
Effect of SO-M on organ biomarker enzymes and antioxidant enzymes
The mean AST and ALT activities in four tissue types decreased in a dose-dependent manner with dietary supplementation of 1% and 3% SO-M (Table 2). The reduction in AST and ALT activities were most pronounced in the liver, kidney, and heart tissues (~ 40%). The muscle tissue, too, had a significant reduction of AST and ALT activities in the treatment groups than in control.
Table 2.
Effect of SO-M dietary supplementation on AST and ALT activity in muscle, liver, kidney and heart tissues of albino rats
| Response | Tissue | Treatments | ||
|---|---|---|---|---|
| Control | T1 | T2 | ||
| AST | Liver | 18.04 ± 00.21a | 11.26 ± 0.31b | 10.03 ± 0.28b |
| Kidney | 10.26 ± 0.20a | 7.43 ± 0.18b | 5.52 ± 0.17c | |
| Muscle | 61.26 ± 3.47a | 48 ± 2.89b | 40.25 ± 3.12c | |
| Heart | 14.25 ± 0.34a | 10.38 ± 0.37b | 7.95 ± 0.41c | |
| Mean | 26.48A | 19.94B | 16.50C | |
| ALT | Liver | 14.21 ± 0.28a | 11.35 ± 0.34b | 9.64 ± 0.22c |
| Kidney | 6.34 ± 0.31a | 5.21 ± 0.27b | 3.56 ± 0.22c | |
| Muscle | 54.26 ± 3.10a | 50.12 ± 3.24b | 46.4 ± 2.01c | |
| Heart | 9.12 ± 0.41a | 7.68 ± 0.35b | 5.32 ± 0.31c | |
| Mean | 21.88 A | 19.42 B | 16.87 C | |
Different superscript (a, b, c) in the row indicate a significant difference (p < 0.05) between the control and treatment groups. Marginal means are written in bold (A, B, C, D). Values are expressed as mean ± SE (n = 6). Units: nanomoles oxaloacetate released/ mg protein/ minute at 37 °C (AST); nanomoles of sodium pyruvate formed/mg protein/minute at 37 °C (ALT)
The mean ALP and ACP activities of all the tissue types decreased significantly in T2 and T1 in a dose-dependent manner (Table 3). However, the ACP activities in liver and kidney tissues were not significantly different in the treatment groups. Similarly, ALP activity in liver, muscle and heart tissue does not significantly differ between the two treatment groups. Reduction in ACP activity was most pronounced in heart and muscle tissue, whereas ALP activity reduced prominently in kidney and liver tissue.
Table 3.
Effect of SO-M dietary supplementation on ACP and ALP activity in muscle, liver, kidney and heart tissues of albino rats
| Response | Tissue | Treatments | ||
|---|---|---|---|---|
| Control | T1 | T2 | ||
| ACP | Liver | 9.02 ± 0.52a | 7.83 ± 0.64b | 8.14 ± 0.32b |
| Kidney | 12.43 ± 0.31a | 10.25 ± 0.28b | 10.94 ± 0.32b | |
| Muscle | 48.5 ± 0.28a | 36.54 ± 0.29b | 30.21 ± 0.31c | |
| Heart | 18.61 ± 0.51a | 11.26 ± 0.58b | 9.32 ± 0.49c | |
| Mean | 22.03 A | 16.91 B | 14.88 C | |
| ALP | Liver | 8.42 ± 0.18a | 6.12 ± 0.22b | 5.92 ± 0.24b |
| Kidney | 29.3 ± 0.29a | 26.40 ± 0.31b | 22.5 ± 0.33c | |
| Muscle | 4.27 ± 0.21a | 3.46 ± 0.22b | 3.05 ± 0.21b | |
| Heart | 9.25 ± 0.19a | 8.34 ± 0.18b | 8.01 ± 0.11b | |
| Mean | 12.58A | 11.05B | 10.01C | |
Different superscript (a, b, c) in the row indicate a significant difference (p < 0.05) between the control and treatment groups. Marginal means are written in bold (A, B, C, D). Values are expressed as mean ± SE (n = 6). Units: Mole of PNP (p-Nitrophenol) released min-1 mg-1 protein (ACP), and Mole of PNP released min−1 mg−1protein (ALP)
SOD, catalase, and GST activities in liver, kidney, muscle, and heart tissues are presented in Table 4. Dietary supplementation with SO-M at 1% and 3% significantly reduced (p < 0.05) the SOD and Catalase activities. A statistically significant decrease in SOD activity with increased doses of SO-M was only observed in liver tissue. Similarly, a dose dependent decrease in Catalase activity was significant (p < 0.05) in liver, muscle and heart tissue. GST activity in liver tissue reduced significantly in both the treatment groups. There was no significant reduction in GST activity in kidney and muscle tissue. A significant decrease in GST activity in heart tissue was observed only when the diet was supplemented with 3% SO-M.
Table 4.
Effect of SO-M dietary supplementation on SOD, Catalase and GST activity in muscle, liver, kidney and heart tissues of albino rats
| Response | Tissue | Treatments | ||
|---|---|---|---|---|
| Control | T1 | T2 | ||
| SOD | Liver | 138.47 ± 0.44a | 129.04 ± 0.58b | 120.58 ± 0.49c |
| Kidney | 86.41 ± 0.38a | 80.18 ± 0.52b | 78.57 ± 0.54b | |
| Muscle | 49.21 ± 0.77a | 44.27 ± 0.61b | 41.51 ± 0.68b | |
| Heart | 32.85 ± 0.08a | 30.05 ± 1.30b | 28.06 ± 0.94b | |
| Catalase | Liver | 30.01 ± 0.82a | 26.82 ± 0.59b | 23.51 ± 0.71c |
| Kidney | 60.24 ± 3.21a | 58.54 ± 4.2b | 56.30 ± 3.3b | |
| Muscle | 19.01 ± 0.21a | 14.26 ± 0.27b | 10.04 ± 0.22c | |
| Heart | 22.89 ± 1.21a | 20.04 ± 1.30b | 18.95 ± 1.14c | |
| GST | Liver | 1.89 ± 0.02a | 1.34 ± 0.07b | 1.21 ± 0.02b |
| Kidney | 0.28 ± 0.01a | 0.21 ± 0.03a | 0.17 ± 0.02a | |
| Muscle | 0.31 ± 0.07a | 0.28 ± 0.05a | 0.21 ± 0.02a | |
| Heart | 0.24 ± 0.07a | 0.22 ± 0.08a | 0.16 ± 0.04b | |
Different superscript (a, b, c) in the row indicate significant difference (p < 0.05) between the control and treatment groups. Values are expressed as mean ± SE (n = 6). Units: millimoles H2O2 decomposed min−1 mg−1 protein at 37 ºC (Catalase); 50% inhibition of epinephrine auto oxidation mg−1 protein min−1 (SOD) and μ moles of DNCB (2,4-Dinitrochlorobenzene)-GSH conjugate formed min−1 mg−1 protein (GST)
Previous studies reported that increased activities of AST and ALT accelerate the metabolism of amino acids to form TCA (Tricarboxylic acid) intermediates. These amino acids intermediates enter into gluconeogenesis and gradually decrease the bioavailability for growth and development (Tejpal et al. 2017; Taha et al. 2014). Both ACP and ALP are associated with different diseased condition.
Increased level of ACP activity is mainly associated with diseased conditions like carcinoma, and some skin disorders (Hillmann 1971). Decreased level of organ biomarkers including AST, ALT, ACP and ALP is highly desirable to maintain better health as an elevated level indicates disease or toxic effect of xenobiotics. Ability of fish oil to lower these biomarkers are reported (Kamat and Roy 2015). Similarly, in our study feeding of SO-M significantly maintained level of these biomarkers to around normal range without any abnormal increase. This indicates the efficacy and safety of SO-M as an effective health supplementation.
Detoxification of many chemical compounds occurs in liver and that may be the reason for high SOD activity in liver. The catalase activity in liver, kidney, muscle and heart tissue showed significant (p < 0.05) decrease in the treated groups. Higher catalase activity was observed in kidney. In the biological system, both SOD and catalase protect the cell from free radical damage generated during oxidation metabolism. Previous reports suggested a decreased level of SOD activity following dietary supplementation with catechin-rich oil palm leaf extract in an animal model due to the less oxidative stress (Jaffri et al. 2011). Similarly, SOD and catalase levels were not elevated in rats fed with thiamine and pyridoxine-loaded vanillic acid-grafted chitosan microspheres (Tejpal et al. 2017). The antioxidant nature of Va-g-Ch might have helped to maintain healthy antioxidant status. Similar trend was observed in GST expression in different organ tissues.
Conclusion
Vanillic acid grafted chitosan is a novel encapsulating material for PUFA rich fish oil. The SO-M formulation was stable against oxidation during storage and can be used for development of functional foods. The in vivo experiment further confirmed the suitability of SO-M as a functional food formulation. Supplementation with SO-M significantly improved Weight gain percentage, SGR, haemoglobin concentration, RBC and Platelet counts. There was a reduction in the blood glucose level too. The new formulation increased the serum HDL level while reducing the harmful serum cholesterol, triglycerides, and LDL. All the enzyme markers of stress were lower in the treated groups. At higher doses, no adverse change in the enzyme markers were observed. In some cases the beneficial effects were dose-dependent. Building on our previous reports, the study demonstrated that dietary supplementation with SO-M clearly improved growth performance, reduced blood glucose level, and enhanced haemoglobin concentration, RBC & Platelet counts. Hence, the new SO-M formulation is a potential functional food ingredient.
Declaration
We declare that (i) the work described has not been published before (except in the form of an abstract, a published lecture or academic thesis), (ii) it is not under consideration for publication elsewhere, (iii) its submission to JFST publication has been approved by all authors as well as the responsible authorities –tacitly or explicitly—at the institute where the work has been carried out, (iv) if accepted, it will not be published elsewhere in the same form, in English or in any other language, including electronically without the written consent of the copyright holder, and (v) JFST will not be held legally responsible should there be any claims for compensation or dispute on authorship.
Supplementary Information
Below is the link to the electronic supplementary material.
Acknowledgements
We acknowledge the analytical instrumentation support of the Sophisticated Test and Instrumentation Centre (STIC) in the Cochin University of Science and Technology, Cochin, India.
Author contribution
K. V. Vishnu, K. K. Ajeeshkumar, R. G. K. Lekshmi and B. Ganesan carried out the in vivo experiment and other bench work. N. S. Chatterjee conceptualized the work, synthesized the vanillic acid grafted chitosan, optimized the encapsulation process, and drafted the manuscript. R Anandan, Suseela Mathew, and C. N. Ravishankar received project funding, guided the team and edited the manuscript.
Funding
We sincerely acknowledge the funding support (Grant no. MOES/10/MLR/01/2012) received from Centre for Marine Living Resources and Ecology (CMLRE), Ministry of Earth Sciences, India.
Declarations
Conflict of interest
The authors declare that there is no conflict of interests.
Footnotes
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