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Table of Contents
ORIGINAL ARTICLE
Year : 2019  |  Volume : 5  |  Issue : 4  |  Page : 214-219

Effects of sialic acid from edible bird nest on cell viability associated with brain cognitive performance in mice


1 Departments of Veterinary Preclinical Sciences, Faculty of Veterinary Medicine, Universiti Putra Malaysia, Serdang, Selangor, Malaysia
2 Department of Nutrition, Metabolic and Cardiovascular Research, Institute for Medical Research, Jalan Pahang, Kuala Lumpur, Malaysia
3 Department of Veterinary Pathology and Microbiology, Faculty of Veterinary Medicine, Universiti Putra Malaysia, Serdang, Selangor, Malaysia

Date of Submission20-Nov-2018
Date of Decision01-Jun-2019
Date of Acceptance08-Jul-2019
Date of Web Publication03-Dec-2019

Correspondence Address:
Dr. Hafandi Ahmad
Department of Veterinary Preclinical Sciences, Faculty of Veterinary Medicine, Universiti Putra Malaysia, 43400 UPM Serdang, Selangor Darul Ehsan
Malaysia
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/wjtcm.wjtcm_22_19

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  Abstract 


Background: Edible bird nest (EBN) is a natural food product rich in glycoprotein such as sialic acid, which has been reported to improve brain functions. The EBN is widely consumed due to its higher nutritional contents and antioxidant status; however, an interaction of EBN on brain cell metabolic activity and viability are still unclear. Objective: The objectives of this study were to identify the effect of sialic acid from EBN on the cell viability and to determine the appropriate concentration of sialic acid on cognitive performance in mice. Materials and Methods: The viability of pheochromocytoma and neuroblastoma cell lines were tested using the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide assay. For in vivo study, 7-week-old female BALB/c mice were randomly assigned into four treatment groups and were treated with sialic acid for 21 days. At day 22, all mice were tested on cognitive performance by Y-maze test. Results: Treatment concentration of sialic acid extract and sialic acid standard at 60 μg/mL (0.6 ppm) increased cell viability and showed no cytotoxicity effects in pheochromocytoma and neuroblastoma cell lines. In addition, an administration of higher dose of sialic acid at 0.6 ppm in animals improved Y-maze test performance, which they showed significantly higher number of entries and time spent in the novel arm. Conclusion: Thus, the current study shows that the sialic acid extract at 0.6 ppm improved brain cognitive performance in mice associated with an increased viability of pheochromocytoma and neuroblastoma cell lines.

Keywords: Cell viability, cognitive function, edible bird nest, mice, sialic acid


How to cite this article:
Abdul Khalid SK, Rashed AA, Aziz SA, Ahmad H. Effects of sialic acid from edible bird nest on cell viability associated with brain cognitive performance in mice. World J Tradit Chin Med 2019;5:214-9

How to cite this URL:
Abdul Khalid SK, Rashed AA, Aziz SA, Ahmad H. Effects of sialic acid from edible bird nest on cell viability associated with brain cognitive performance in mice. World J Tradit Chin Med [serial online] 2019 [cited 2019 Dec 8];5:214-9. Available from: http://www.wjtcm.net/text.asp?2019/5/4/214/271962




  Introduction Top


Edible bird nest (EBN) is a natural product made from saliva produced by the male edible-nest swiftlet (Aerodramus fuciphagus) during the breeding season.[1],[2],[3] Variousin vitro andin vivo findings have shown that dietary supplementation of EBN was able to improve physiological human health.[1],[4] It has been suggested that the EBN contains higher bioactive compounds such as sialic acid,[5] which helps in an increased metabolism and physiological functions such as immune[6] and neurological systems in mammals.[7],[8] In addition, sialic acid is one of the eight glyconutrients that can be found in EBN which increased cell tissues' repair and promoted the cell division and proliferation.[3] However,in vitro study is important to identify any toxicity effects of bioactive compounds from the EBN before it is ready to be consumed.[3] Thus, this could indicate that an identification of amount bioactive compounds from the EBN is very important to avoid any toxicity effects after it undergoes cell absorption.

Evaluation of the cell viability using in vitro study is essential for toxicology studies and assessment of the efficiency of nutrient absorption.[9] The reduction in cell viability or cytotoxicity is important in response to the treatment exposes when the metabolic events lead to apoptosis or necrosis. For instance, treatment of EBN which is claimed to be rich in glyconutrient increased the Caco-2 cell viability and proliferation.[3] This could suggest that the cells with active mitochondria absorb the EBN extraction; it will generate the strong signal and raised the absorbance of the cell viability.[10] In addition, sialic acid is an essential component of brain gangliosides that modified the neural adhesion cells[11] and improved the neurotrophic factor of neuron and brain function development.[12],[13] In fact, sialic acid plays crucial roles in cell-to-cell interactions,[12] neuronal outgrowth, and modifying synaptic connectivity in memory formation.[14] In mammals, diet rich in sialic acid increased the levels of brain cells and helps in expression of genes associated with cognitive function.[15],[16]

As EBN is rich in several compounds,[17] this study was to investigate the effects of sialic acid from EBN on cell viability in rat adrenal pheochromocytoma cell (PC-12) and neuroblastoma cell lines such as human bone marrow cell (SH-SY5Y) and human brain cell (SK-N-MC). The PC-12 cells were isolated in 1976[18],[19] and have been exposed to nerve growth factor and neuronal differentiation.[18] The neuroblastoma cell lines were used to study the neuroprotective effects in relation to cognitive function performance and neuronal degeneration in brain.[20] The viability of neuroblastoma cell lines was tested using the 3- [4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assay kit to determine the concentration of uptake sialic acid and their cytotoxicity as well as cell viability levels.[21],[22] The results from the MTT assay kit were used as a guideline in preparing the concentration dosage for dietary supplementation on animals' cognitive performance in the Y-maze test. Therefore, the aim of this study was to measure the cell viability of sialic acid from EBN and to determine the different concentrations of sialic acid from EBN on cognitive performance in animals.


  Materials and Methods Top


Edible bird nest collection and preparation

A total of 14 raw unprocessed EBN samples from four different states of Peninsular Malaysia (e.g., north, south, east, and west regions) were collected during the breeding season of edible-nest swiftlet from April to August 2016. The samples were manually cleaned from gross dirt, and the visible feathers were removed using forceps. The EBN was finely grounded using a grinder. The sialic acid was extracted from raw EBN samples at SIRIM Berhad, Malaysia using the high-performance liquid chromatography [Figure 1]. Two sialic acid fractionation of EBN sample with same retention time (6.7 min) were received directly from SIRIM Berhad in liquid form with different concentrations (e.g., 50 and 32 mg/kg), and the yield percentages of sialic acid extracted were 0.005% and 0.003%, respectively. However, the sialic acid standard was purchased from Sigma-Aldrich, USA.
Figure 1: The sialic acid fractionation of edible bird nest sample with retention time 6.7 min

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Cell lines' culture and conditions

The PC-12, SH-SY5Y, and SK-N-MC were obtained from the American Type Culture Collection (ATCC) and were seeded and maintained in 25 cm2 culture flasks (Constar, Cambridge, MA). The PC-12 and SH-SY5Y cells were grown in Dulbecco's Modified Eagle's Medium (DMEM; GIBCO New York, USA) supplemented with 20% and 15% of v/v fatal bovine serum, while SK-N-MC cell were grown in Eagle's Minimum Essential Medium (EMEM; GIBCO New York, USA) supplemented with 20% of v/v fatal bovine serum. The entire medium contained 1% of v/v nonessential of amino acid (GIBCO New York, USA), 1% antibiotic (penicillin–streptomycin) (GIBCO New York, USA), and 1% v/v L-glutamine (GIBCO New York, USA). Only PC-12 cell was added with 15% horse serum (GIBCO New York, USA). All the cells were maintained in the same conditions, at 37°C in an incubator with 5% carbon dioxide, 95% humidity, and air atmosphere. The medium was replaced every 2–3 days. The cells were maintained until it reached 80% confluency.

3-[ 4, 5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide cell proliferation and viability assay

The effects of sialic acid standard and extract from EBN samples on cell viability were determined on pheochromocytoma and neuroblastoma cell lines using the MTT assay kit purchased from the ATCC. The treatment concentration for both sialic acid extract and sialic acid standard used in this test was 20, 40, 60, 80, and 100 μg/ml. The adherent cell lines (1 × 106 cells/mL) were seeded onto collagen-treated 96 well plates with complete medium for 24 h. The next day, the complete medium was replaced with the serum-free complete medium. The sialic acid treatment samples were added into each plate using the required concentration included the control and a blank well. Then, the plate was incubated for another 24 hrs. Then, 10 μL of a MTT solution (5 mg/ml) were placed in each well plate including controls and incubated for another 4 h at 37°C in a CO2 incubator until the purple precipitate was visible. When the purple precipitate was clearly visible, the medium was discarded and replaced with 100 μL of detergent reagent (dimethyl sulfoxide) into each well including controls. The wells were swirled gently and left at room temperature in the dark for another 2 h. The cells' absorbance was read at 570 nm using a microplate reader (BioTek, Eon Instruments, USA). The entire test was conducted in triplicate. The numbers of viable cells were directly proportional with the levels of formazan absorption. The MTT technique was used as a guideline to prepare the standard amount of treatment concentration for mice in Y-maze performance.

Animals

Seven-week-old female BALB/c mice (n = 40) were purchased from the Laboratory Animal Resource Unit, Institute for Medical Research, Kuala Lumpur. The animal room was setup following the standard protocol of good laboratory practice, and all animal testing were approved by the Animal Care and Use Committee, Institute of Medical Research, Kuala Lumpur, Malaysia (ACUC/KKM/02-18/2015). Before the experiment day, all animals were acclimatized for 1 week and were fed with standard mice pellet (AIN-93G). During the experimental period, all animals were given different concentrations of sialic acid extract by oral gavage for 21 days as follows: Group LD (n = 10; low dose of 0.2 ppm sialic acid), Group MD (n = 10; medium dose of 0.4 ppm sialic acid), Group HD (n = 10; high dose of 0.6 ppm sialic acid), and Group C (n = 10; control group with no sialic acid). The body weight, food, and water intake of animals were recorded weekly.

Y-maze task performance

The Y-maze test was conducted to assess the cognitive performance and the willingness of the animal to explore the new environments of novel arm of the Y-maze[23] which consisted of three symmetrical arms (start, familiar, and novel) with 35 cm length, 8.5 cm width, and 15 cm height [Figure 2]. The tests consisted of 10 min of first trial (T1) followed by 5 min of second trial (T2). In the T1, the mice were placed in the start arm and were allowed to explore only two arms (start and familiar arms) of the maze, while the third arm (novel arm) was blocked. Different visual cues (round, rectangular, and triangle) were placed on the wall at the end of each arm of the maze. After 60 min, the T2 was conducted and the mice were allowed explore three arms of the maze. The tests were recorded using a ceiling-mounted camera to count the number of entries and the time spent in each arm. The results were acceptable when the numbers of entries and the time spent in novel arm were greater and used as indicator for spatial working memory.[23],[24]
Figure 2: Schematic representation of the Y-maze test with (a) the novel arm blocked with a sliding partition during the first trial, but open during the second trial. (b) Different visual cues (square, circle, and triangle) were placed on the external maze walls

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Statistical analysis

All data were analyzed using the SPSS software version 16.0 (IBM Software, Inc., New York, USA). The viability of the cell lines and the Y-maze performance were analyzed using the one-way analysis of variance (ANOVA) with post hoc Tukey's test to show any significant difference between groups. P < 0.05 was considered as statistically significant.


  Results Top


Effects of sialic acid standard and sialic acid extract on cell viability

The MTT results showed that there were no cytotoxicity effects in pheochromocytoma and neuroblastoma cell lines (e.g., PC- 12, SH-SY5Y, and SK-N-MC) when exposed to sialic acid extract and sialic acid standard below 60 μg/mL (0.6 ppm).

The cell viability in 10 × 106 cell lines in [Table 1] started to increase along with the increase in the treatment until at concentration of 60 μg/ml. However, the cell viability of entire cell lines started to decrease in the treatment (sialic acid extract and sialic acid standard) when the concentration was above 60 g/mL and started to show toxicity towards the neuroblastoma cell lines. The highest percentage of cell viability was observed in sialic acid extract compared to the sialic acid standard. The percentages of cell viability in PC-12, SH-SY5Y, and SK-N-MC cell lines were 74.13% ± 1.86%, 85.57% ± 0.96%, and 82.36% ± 1.17%, respectively.
Table 1: Effects of sialic acid from edible bird nest in neuroblastoma cell lines in 10K cell

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In 15 × 106 cell lines [Table 2], the cell viability also started to increase along with an increase in the treatment concentration until it reached concentration of 60 μg/ml. The percentages of cell viability in PC-12, SH-SY5Y, and SK-N-MC cell lines were 72.53% ± 2.67%, 81.93% ± 8.65%, and 86.57% ± 0.85%, respectively. Similar to 10 × 106 cell lines, the cell viability of entire cell lines started to decrease in both sialic acid extract and sialic acid standard in pheochromocytoma and neuroblastoma cell lines. These findings suggested that both 10 × 106 and 15 × 106 cell lines treated with sialic acid extract and sialic acid standard did not show any cytotoxicity effects to the cell line at low concentration of treatment.
Table 2: Effects of sialic acid from edible bird nest in neuroblastoma cell lines in 15K cell

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Overall, the cell viability of sialic acid extract was slightly higher than the sialic acid standard in all cell lines, especially in 10 × 106 cell lines. Based on these results, the highest or maximum concentration of sialic acid extract treatment that was given to the mice was 60 μg/mL (0.6 ppm). The viability of the cell displayed in both sialic acid extract and sialic acid standard (10 × 106 and 15 × 106 cell lines) showed similar absorbance reading for all neuroblastoma and PC-12 lines. However, higher dose of sialic acid treatment induced the cells viability and proliferation in the cells.

Effects of different doses of sialic acid on the Y-maze performance

One-way ANOVA indicated that there were statistically significant differences among all treatment groups [Table 3] on the numbers of arm entries (F3,36 =13.91, P < 0.05). A Tukey post hoc test revealed that the numbers of novel arms entries by animals were significantly higher in HD (37.90 ± 1.85 entries, P < 0.05) and MD groups (32.4 ± 2.37 entries, P < 0.05) compared to the C group (27.70 ± 5.21 entries). However, there was no significant difference in number of arm entries in the LD (30.10 ± 4.31 entries, P > 0.05) animals compared to C and MD animals.

Furthermore, time spent by animals in novel arms indicated that there were statistically significant differences among all groups of treatment in time spent in novel arms (F3,36 =10.22, P < 0.05). A Tukey post hoc test revealed that the time spent by animals in novel arms were significantly higher in HD (8.79 ± 1.78 min, P < 0.005) and MD (7.98 ± 1.34 min, P < 0.005) groups compared to the C group (5.66 ± 0.81 min). However, there was no significant difference on time spent in novel arm in the LD animals (6.20 ± 1.70 min, P > 0.05) compared to C and MD animals.


  Discussion Top


The cell viability was performed by inducing sialic acid extract from EBN and commercial sialic acid standard on neuroblastoma cell line to determine the concentration to seek effects of sialic acid on neuronal cell culture. To the best of our knowledge, there are limited findings reported on the effects of sialic acid on cell viability in neuroblastoma cell lines. In this study, the percentage of cell viability in sialic acid extract was found higher in viability cell compared to sialic acid standard treatment. However, this percentage depended on the sources of EBN that was used[3] such as the habitat and availability of the food sources of the edible-nest swiftlet.[17] In fact, there were significant differences in nutritional values of EBN components among the regions and nest types.[17],[25]

The current study also showed that the stimulation of the cell lines with different amount of sialic acid concentration on cell viability caused an increase in dose dependence in cell viability and proliferation. In SH-SY5Y, the first effect of sialic acid treatment was noted at concentration of 20 μg/mL which increased significantly until concentration of 60 μg/mL for both sialic acid extract and sialic acid standard treatment. Similar dose dependence was also showed in adrenal gland (PC-12) and SK-N-MC lines. This could indicate that the maximal dose just below the threshold for the cell toxicity revealed no cytotoxicity effects to the entire cell lines. The viability of the entire cell lines increased along with the increasing concentration of the treatment. This result is similar with the previous finding which reported that the EBN increased the viability and proliferation in Caco-2 cell line.[3] In fact, the mitogenic properties in EBN assisted in the promotion of cell division and cell growth in rabbit corneal keratocytes.[26] In addition, the cell viability started to decrease at concentration of 80 μg/mL sialic acid in pheochromocytoma and all neuroblastoma cell lines. This could suggest that at this level of treatment, toxicity started to become evident. Moreover, the opening of mitochondria transition pores on the different stimulation treatments lead to the depolarization of mitochondria membrane potential which leads to the apoptosis of the cells.[20] The current finding also showed that the sialic acid standard and extract increased the cell viability in pheochromocytoma and neuroblastoma cell lines, but the effectiveness of the sialic acid extract in the cell viability was higher compared to the sialic acid standard. This could be due to the sialic acid standard and sialic acid extract from EBN have shown to have epidermal growth factor-like activity which appear to play a role in growth-stimulating normal cellular processes such as proliferation, differentiation, and development.[17],[27]

The present study also showed that the dose at concentration of 60 μg/mL is a suitable dose that can be used as guideline to explore the effects of sialic acid on animals in the Y-maze performance. In this experiment, animals treated with higher concentration of sialic acid extract from EBN showed their ability to remember the pathway and the visual cue of the novel arm. With the high proportion of sialic acid, it provided the optimal condition for cell absorption and utilization of sialic acid to achieve the cognitive developmental requirements. A previous study reported that higher administration of sialic acid was associated with an improvement in brain cognitive functions in mice by increasing the level of sialic acid in the brain[13],[28] and influenced the movement of neurotransmitter to alter the synaptic membrane in the brain.[29] In addition, higher concentration of sialic acid in the brain also helps to establish the structural and functional of the synaptic pathway.[13] The synaptic membrane containing sialic acid contributed to the negative charge to the cells upon binding with ganglioside and glycoprotein.[28],[30] Furthermore, sialic acid helps to regulate the affinity of receptors and involves in transmembrane signaling, growth, and differentiation on early brain development in young mammals when fed with suitable amount presented in human milk.[28] Thus, it is possible that high administration of sialic acid is associated with the brain development and function in mammals.


  Conclusion Top


An administration of high dose of sialic acid concentration is important for better absorption on cell viability. However, an appropriate concentration of sialic acid treatment should be considered to avoid any toxicity effects in the cell lines. The differences in percentage of cell viability and proliferation in the pheochromocytoma and neuroblastoma cell lines when exposed with different concentrations of sialic acid are expected. Thus, the sialic acid extract showed the highest increment in cell viability in pheochromocytoma and neuroblastoma cell lines. Taken together, we confirmed that the higher amount of sialic acid extract increased the neuron function associated with cognitive performance in mice. However, it is important to study the safety and effectiveness of dietary sialic acid supplementation before it can be consumed to avoid being harmful to human health. This finding suggested that amount of sialic acid extract supplementation correlated with the increase in cognitive performance and neuronal tissue development.[31] Thus, further study is needed to study the stimulation and inhibition of the sialic acid in other neuroblastoma cell lines related with the viability and proliferation of cell.

Acknowledgment

The authors would like to thank the staff of the Laboratory of Physiology Faculty of Veterinary Medicine, Nutrition Units of IMR and SIRIM for their assistance during the study. The authors wish to acknowledge the contribution of all Malaysian EBN farmers in the completion of this study.

Financial support and sponsorship

This research is funded by a research grant provided by the Centre of Excellence (CoE) Swiftlets, Faculty of Veterinary Medicine, Universiti Putra Malaysia, Department of Veterinary Services Malaysia, and Ministry of Science and Technology (MOSTI) Malaysia (Project Number: 6371401-10301).

Conflicts of interest

There are no conflicts of interest.



 
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