Swallow Birds Nest mainly
comprises a secretion of the salivary gland of different Swiftlet species has
been used as a symbol of wealth and power as well as an crucial ingredient in
traditional Chinese Medicine. The MTT assay and flow cytometry analysis were
carried out to evaluate the effect of Swallow Birds Nest extract in treated
Influenza A virus infected MDCK cells. MTT assay was performed to determine the
cell viability and Ha assay was used to determine the virus titer after 24 hour
of post treatment with Swallow Birds Nest extract towards MDCK cells challenged
with influenza A virus. The results showed that there was a significant
increase in cell viability (p < 0.05) of Swallow Birds Nest extract
treatment compared to positive infected control. Annexin V-fitc double staining
method was carried out to identify the mode of cell death in MDCK cells by
calculating the means of each apoptosis stage. The results showed that there
was a significant reduced early apoptotic injury in infected cells treated with
Swallow Birds Nest extract. In summary, the current finding suggests that Swallow
Birds Nest may be have the potentiality to be as an apoptotic inhibitor and
ameliorate the infected cells caused by virus.
Introduction
Apoptosis is an important
physiological necessary for development and maintenance of tissues homeostasis
including tissue atrophy, the immune system development and biological tumor
[1-2]. It also plays an important function in the pathogenesis of many
infectious diseases including those caused by viruses [3-5]. Many virus
infections result in apoptosis of host cells, and several viruses have evolved
mechanisms to inhibit apoptosis. Influenza viruses induce apoptosis through
mechanism then both cellular and viral factors depending on the cell type.
However, the precise mechanism still remain unclear.
Edible Swallow Birds Nest (Swallow
Birds Nest) is natural food product made from Aerodramus genus Swiftlet’s
saliva. Studies show its unique glycoprotein properties provide health
benefits, cure many illnesses and rejuvenate cells. The nests contain almost
all of the bessential ingredients for body maintenance. Amino acids, minerals,
and others keep the body healthy and increase immunity against a host of
illness. Another studies have shown that this animal saliva previously found to
contain bioactive compounds that are powered with anti-apoptotic and
antioxidant properties [6-7]. As apoptosis have been suggested as crucial
events in Influenza virus infection, Swallow Birds Nest, the salivary secretion
of Swallow Birds Nest, may have anti-apoptosis relevance in the therapeutic
context of viral infection. Hence, this study aimed to investigate the
anti-apoptosis effect of Swallow Birds Nest.
 |
| The evaluation of Swallow Birds Nest Extract Reduced Influenza A virus Induced Apoptosis on Cultured Cells in vitro study |
Materials
and Methods
Preparation of Swallow Birds Nest
Extracts
Raw Swallow Birds Nest from the swiftlet of Aerodramus genus collected from Swallow Birds Nest house in Teluk Intan, Perak, Malaysia. The cleaning process was carried out by first soaking after the unprocessed Swallow Birds Nest in distilled water until softened. Debris and feathers were removed after which the Swallow Birds Nest was subjected to drying process at 70 oC for 16 hrs and followed by grounding and sifting through a mesh (60μM in pore size). The grounded samples were kept in distilled water at 3oC in about 16 hours and continued heated for 30-60 min at 60oC. The extract was then filtered and freeze-dried after 48 hours freezing at -800C, and stored for the following experiment.
Madin Darby Canine Kidney (Mdck) Cell Culture
MDCK cell line was purchased from the American Type Culture Collection (ATCC,CCL-34TM) and grown in Dulbecco’s Modified Eagle’s Medium (Gibco,UK) supplemented with 10% fetal bovine serum (Gibco, UK), antibiotic-antimycotic (Gibco,UK). The cells then were seeded into a sterile 96-well flat bottom plate (Nunc, USA) and maintained at 37oC humidified incubator with 5% CO2 (Galaxy, UK) for 2-3 days until 70%-80% confluency is achieved.
Virus Propagation
Influenza A virus, strain A/Puerto Rico/1934 (H1N1)was purchased from the American Type Culture Collection (ATCC,® VR-95TM) and propagated in MDCK cells, then the stock virus was titrated by TCID50. In obtained 100 TCID50, the resultant virus titer was further diluted with 100 μl taken from the amount ofTCID50, then it was to be used as a constant positive control in the following procedure
Cell Viability Based On Combination Treatment
This procedure was performed by using Maximal Non- Cytotoxic Concentration of Swallow Birds Nest and constant virus titer (100 TCID50). Briefly,the virus was first inoculated into the MDCK cells and later followed by the Swallow Birds Nest extract replacement for 1 hrs followed by change with media. All the samples were incubated at 37oC in 5% CO2 incubator.
Raw Swallow Birds Nest from the swiftlet of Aerodramus genus collected from Swallow Birds Nest house in Teluk Intan, Perak, Malaysia. The cleaning process was carried out by first soaking after the unprocessed Swallow Birds Nest in distilled water until softened. Debris and feathers were removed after which the Swallow Birds Nest was subjected to drying process at 70 oC for 16 hrs and followed by grounding and sifting through a mesh (60μM in pore size). The grounded samples were kept in distilled water at 3oC in about 16 hours and continued heated for 30-60 min at 60oC. The extract was then filtered and freeze-dried after 48 hours freezing at -800C, and stored for the following experiment.
Madin Darby Canine Kidney (Mdck) Cell Culture
MDCK cell line was purchased from the American Type Culture Collection (ATCC,CCL-34TM) and grown in Dulbecco’s Modified Eagle’s Medium (Gibco,UK) supplemented with 10% fetal bovine serum (Gibco, UK), antibiotic-antimycotic (Gibco,UK). The cells then were seeded into a sterile 96-well flat bottom plate (Nunc, USA) and maintained at 37oC humidified incubator with 5% CO2 (Galaxy, UK) for 2-3 days until 70%-80% confluency is achieved.
Virus Propagation
Influenza A virus, strain A/Puerto Rico/1934 (H1N1)was purchased from the American Type Culture Collection (ATCC,® VR-95TM) and propagated in MDCK cells, then the stock virus was titrated by TCID50. In obtained 100 TCID50, the resultant virus titer was further diluted with 100 μl taken from the amount ofTCID50, then it was to be used as a constant positive control in the following procedure
Cell Viability Based On Combination Treatment
This procedure was performed by using Maximal Non- Cytotoxic Concentration of Swallow Birds Nest and constant virus titer (100 TCID50). Briefly,the virus was first inoculated into the MDCK cells and later followed by the Swallow Birds Nest extract replacement for 1 hrs followed by change with media. All the samples were incubated at 37oC in 5% CO2 incubator.
After 24 hour of post-treatment
incubation, the plates were exposed to the MTT assay. Treated cells were
subjected to MTT reagent (Sigma Aldrich, USA) in which reacts to quantify the
viable cells. Pure DMSO solution (Merck, Germany) was added to the cells after
2-3 hr exposure to the MTT reagent to solubilise the formazon precipitation.
Absorbance of the solution was measured spectrophotomerically by using ELISA
reader at 540nm wavelength (Bio Tek Instruments EL800,USA).
Apoptotic Cells Analysis
The protocol of Annexin V-FITC Apoptosis Detection Kit (BD Pharmingen, USA) by Rieger et al was performed in this procedure. Briefly, upon completion of treatments, cells were harvested and washed with binding buffer. Then, Annexin V and propidium iodide (PI) were added and incubated in dark for 15 min. Subsequently, washing was performed twice with binding buffer which then incubated for 15 min at 37oC. Finally, the samples were reads for analysis with Becton Dickson FACS Calibur Flow Cytometer (BD Biosciences, USA).
Statistical Analysis
Data was collected as triplicate for each experiments. The results were expressed as mean ± standard deviation. Statistical significance was assessed with Anova test and P value < 0.05 was considered significant.
Apoptotic Cells Analysis
The protocol of Annexin V-FITC Apoptosis Detection Kit (BD Pharmingen, USA) by Rieger et al was performed in this procedure. Briefly, upon completion of treatments, cells were harvested and washed with binding buffer. Then, Annexin V and propidium iodide (PI) were added and incubated in dark for 15 min. Subsequently, washing was performed twice with binding buffer which then incubated for 15 min at 37oC. Finally, the samples were reads for analysis with Becton Dickson FACS Calibur Flow Cytometer (BD Biosciences, USA).
Statistical Analysis
Data was collected as triplicate for each experiments. The results were expressed as mean ± standard deviation. Statistical significance was assessed with Anova test and P value < 0.05 was considered significant.
Result
And Discussion
Cytotoxic Profile Of Edible Swallow
Birds Nest (Swallow Birds Nest) Extract
Toxicity study was first performed with addition of Swallow Birds Nest extract to MDCK cells to determine the concentration side effect as well as MNCCs of the extract on cultured cells. MNCC is maximal non-cytotoxic concentration of the Swallow Birds Nest extract.
Toxicity study was first performed with addition of Swallow Birds Nest extract to MDCK cells to determine the concentration side effect as well as MNCCs of the extract on cultured cells. MNCC is maximal non-cytotoxic concentration of the Swallow Birds Nest extract.
Based on the [Figure 1],
cytotoxicity percentage in MDCK cells showed an increasing trend of optimal
density (OD) of cell viabilities along the concentration. As determined from
the bar graph, MNCCs was 12.5 mg/ml.
The Swallow Birds Nest extract in
water soluble form is likely less cytotoxic, thus same as previous report that
pointed out the water soluble subtances possess less cytotoxic effect compared
to the crude Swallow Birds Nest extract. This is due to compound solubility and
stability are major factors that contribute to varied activities in different
extract [8] such as the application of high temperature during the water
extraction process caused affected the potency of proteins within the Swallow
Birds Nest, possibility through denaturation [9].
Swallow Birds Nest extracts improves cell viability in H1N1 viruschalleged MDCK cells
The MTT was performed in order to quantify the cell viability based on the apoptotic reduction activity. Upon challeged with 100TCID50/ul HINI virus for 24 hour post treatment with Swallow Birds Nest extract, cell viability increased to about 20% of that of infected control [Figure 2]. Meawhile, the Swallow Birds Nest alone treatment generally did not affect the infected group. This could be due to the mitogenic property of Swallow Birds Nest that promoted cell growth, as made evident a study by Zainal Abidin et el. Which shows that Swallow Birds Nest promoted cell divison in rabbit corneal keratocytes [10].
Swallow Birds Nest Extracts Reduce Early Apoptotic Event in H1N1 Virus Challeged MDCK Cells
Apoptotic cells resulted due to phosphatidylserine (PS) translocation towards the outer leaflet of the plasma membrane caused the apoptotic or death cells and membrane integrity become loss. Therefore, the annexin V-FITC double stainig was performed in this study to calculate the percentage of cell death in different stages of apoptosis. In this assay, cells residing at different level of apoptosis upon exposure to the virus infection (A/Puerto Rico/8/1934 (H1N1)) by differential staining of DNA and also PS, whereby cells are grouped and revealed in dot plot. Annexin V-FITC-/PI- (lower left quadrant) are classified as healthy cells, the cells that are considered early apoptotic showed Annexin V-FITC+/PI- (lower right quadrant), whereas late
Swallow Birds Nest extracts improves cell viability in H1N1 viruschalleged MDCK cells
The MTT was performed in order to quantify the cell viability based on the apoptotic reduction activity. Upon challeged with 100TCID50/ul HINI virus for 24 hour post treatment with Swallow Birds Nest extract, cell viability increased to about 20% of that of infected control [Figure 2]. Meawhile, the Swallow Birds Nest alone treatment generally did not affect the infected group. This could be due to the mitogenic property of Swallow Birds Nest that promoted cell growth, as made evident a study by Zainal Abidin et el. Which shows that Swallow Birds Nest promoted cell divison in rabbit corneal keratocytes [10].
Swallow Birds Nest Extracts Reduce Early Apoptotic Event in H1N1 Virus Challeged MDCK Cells
Apoptotic cells resulted due to phosphatidylserine (PS) translocation towards the outer leaflet of the plasma membrane caused the apoptotic or death cells and membrane integrity become loss. Therefore, the annexin V-FITC double stainig was performed in this study to calculate the percentage of cell death in different stages of apoptosis. In this assay, cells residing at different level of apoptosis upon exposure to the virus infection (A/Puerto Rico/8/1934 (H1N1)) by differential staining of DNA and also PS, whereby cells are grouped and revealed in dot plot. Annexin V-FITC-/PI- (lower left quadrant) are classified as healthy cells, the cells that are considered early apoptotic showed Annexin V-FITC+/PI- (lower right quadrant), whereas late
Conclusion
It has been successfully demonstrated
that Swallow Birds Nest extracts confer apoptotic protection in H1N1-challenged
MDCK cells. Particularly, Swallow Birds Nest demonstrated apoptotic protection
potential by improving cell viability. Thus, it can be concluded that Swallow
Birds Nest extracts shown to inhibit apoptosis.
Acknowledgement
Our sincere gratitude to Dr
Fadzillah A’ini, from Department of Veterinary Services, Putrajaya, Malaysia,
who kindly provided Swallow Birds Nest sample. This project was supported by
Coe Swiftlet grants no.6371400 from Ministry of Agriculture,Putrajaya,Malaysia.
