The evaluation of Edible Bird Nest Extract Reduced Influenza A virus Induced Apoptosis on Cultured Cells in vitro study

Edible Bird 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 Edible Bird 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 Edible Bird 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 Edible Bird 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 Edible Bird Nest extract. In summary, the current finding suggests that Edible Bird 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 Bird 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, Edible Bird Nest, the salivary secretion of swiftlets, may have anti-apoptosis relevance in the therapeutic context of viral infection. Hence, this study aimed to investigate the anti-apoptosis effect of Edible Bird Nest.

The evaluation of Edible Bird Nest Extract Reduced Influenza A virus Induced Apoptosis on Cultured Cells in vitro study

Materials and Methods

Preparation of Edible Bird Nest Extracts
Raw Edible Bird Nest from the swiftlet of Aerodramus genus collected from Edible Bird Nest house in Teluk Intan, Perak, Malaysia. The cleaning process was carried out by first soaking after the unprocessed Edible Bird Nest in distilled water until softened. Debris and feathers were removed after which the Edible Bird 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 Edible Bird Nest and constant virus titer (100 TCID50). Briefly,the virus was first inoculated into the MDCK cells and later followed by the Edible Bird 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.

Result And Discussion

Cytotoxic Profile Of Edible Bird Nest Extract
Toxicity study was first performed with addition of Edible Bird 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 Edible Bird 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 Edible Bird 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 Edible Bird 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 Edible Bird Nest, possibility through denaturation [9].
Edible Bird 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 Edible Bird Nest extract, cell viability increased to about 20% of that of infected control [Figure 2]. Meawhile, the Edible Bird Nest alone treatment generally did not affect the infected group. This could be due to the mitogenic property of Edible Bird Nest that promoted cell growth, as made evident a study by Zainal Abidin et el. Which shows that Edible Bird Nest promoted cell divison in rabbit corneal keratocytes [10].
Edible Bird 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 Edible Bird Nest extracts confer apoptotic protection in H1N1-challenged MDCK cells. Particularly, Edible Bird Nest demonstrated apoptotic protection potential by improving cell viability. Thus, it can be concluded that Edible Bird 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 Edible Bird Nest sample. This project was supported by Coe Swiftlet grants no.6371400 from Ministry of Agriculture,Putrajaya,Malaysia.

Edible Bird Nest Nutrition Facts: A Variety of Health Benefits

For hundreds of years, Chinese culture has hailed the nutritional benefits of Edible Bird Nest soup.

Virility, better skin, stronger pregnancies and healthier babies – all have been claimed as benefits of consuming soups featuring Edible Bird Nests.

However, as the soup has become a global phenomenon, those old notions of health benefits have come under scrutiny: Is Edible Bird Nest soup as healthy as they say it is?

Turns out the Chinese were right – edible swallow’s nests are not only a delicious delicacy, but they’re also a nutritious meal that can benefit multiple age groups.

Your Immune System Gets Stronger

Whenever cancer patients undergo chemotherapy, both good and bad cells are killed. Some of those good cells are called “B cells”, which are white blood cells that produce antibodies that protect you from sickness.
Because these B cells take a hit during chemo, patients become more susceptible to all kinds of sicknesses.
A group of Chinese researchers was curious as to the ability of Edible Bird Nests to boost the immune system, so they conducted a study in which they subjected mice to radiation and then gave them Edible Bird Nest to recover.
The results confirmed what they thought – Edible Bird Nests have a certain type of protein in them that accelerates the creation of B cells, which boosts your immune system.

Contains Six Important Hormones

The saliva found in Edible Bird Nest soup contains a series of hormones, two of which are very familiar to humans: testosterone and estradiol.

Testosterone

Testosterone is a hormone men produced in considerable quantities. Women also produce it, but at far lower levels.
Research has found that testosterone increases libido, promotes muscle growth, contributes to fat loss and, according to AskMen.com, improves “cognitive function and mood” and fights off depression.

Estradiol

Edible Bird Nests also contain estradiol, which is a type of estrogen.
As a drug on its own, estradiol, according to Drugs.com, is “used to treat symptoms of menopause such as hot flashes … burning and irritation.”
Estradiol is also used to prevent osteoporosis and has been implemented into treatment plans for cancer patients.
The levels of estradiol found in Edible Bird Nests are much lower – certainly not at the same potency or concentration as what you’d find in prescription drugs.
For this reason, Edible Bird Nests are safe for pregnant women and men to consume.

Can Promote Eye Health

Injuries to the cornea, the protective layer on the surface of the eye, can be very painful because the healing process is slow.
Corneal keratocytes are responsible for that healing – they keep the cornea strong and prevent it from getting cloudy via injury or other ailments.
In 2011, researchers from the Department of Anatomy at the National University of Malaysia wanted to know if certain bird hormones in Edible Bird Nests could help the body create more corneal keratocytes.
So, they condensed Edible Bird Nests into serum and injected rabbits with it. The results were positive – doctors noticed that the rabbits who received the concentrated Edible Bird Nests produce more corneal keratocytes than those who didn’t.
While this area needs more research, the preliminary study confirms what centuries of Chinese families have already known – Edible Bird Nests can improve vision.

Only the Best Edible Bird Nests Will Do

While Edible Bird Nests have many health benefits, they can also be harmful if they aren’t processed correctly. A great example of this is blood Edible Bird Nests.
Some of these nests get their brilliant color from chemical reactions that take place between air, moisture and bird waste.
Some Edible Bird Nest companies will purposely leave bird waste in their nests to maintain red coloring, a practice that jeopardizes your health instead of helping it.
At Golden Nest, we condemn these health-hazardous practices and protect the well-being of our customers by ensuring high-quality, health-focused processing steps that emphasize cleanliness and precision.
Our red Edible Bird Nests are sourced in the most responsible way possible, not only to protect your health but to promote safe harvesting practices that protect the well-being of Southeast Asia swallow population.
Our products are known for their excellent taste, beautiful colors and time-honored health benefits. Head to our online store to learn more about our Edible Bird Nests.