Content uploaded by Gallant Kar Lun Chan
Author content
All content in this area was uploaded by Gallant Kar Lun Chan on Feb 24, 2015
Content may be subject to copyright.
Ph ton
620
The Journal of Ethnobiology and Traditional Medicine. Photon 120 (2013) 620-628
https://sites.google.com/site/photonfoundationorganization/home/the-journal-of-ethnobiology-and-traditional-medicine
Original Research Article. ISJN: 6642-3194
The Journal of Ethnobiology and Traditional Medicine
Ph ton
Determination of free N-acetylneuraminic acid in edible bird nest: A
development of chemical marker for quality control
Gallant Kl Chan
a
, Ken Yz Zheng
a,b
, Kevin Y Zhu
a
, Tina Tx Dong
a
, Karl Wk Tsim
a*
a
Division of Life Science and Center for Chinese Medicine, The Hong Kong University of Science and
Technology, Clear Water Bay Road, Hong Kong, China
b
Hanshan Normal University, Chaozhou, Guangdong 521041, China
Article history:
Received: 4 February, 2013
Accepted: 15 February, 2013
Available online: 26 October, 2013
Abbreviations:
NANA: N-acetylneuraminic acid, EBN: Edible bird’s nest,
LC-MS/MS, QQQ: Triplequadrupoles liquid
chromatography tandem mass spectrometry
Keywords:
Aerodramus fuciphagus, Edible Bird Nest, LC-MS/MS
QQQ, Authentication, Quality control, Sialic acid; Anti-
tyrosinase
Corresponding Author:
Tsim K.Wk.*
Email: botsim@ust.hk
Phone: 85223587332
Fax No: 85223581559
Abstract
Edible bird nest (EBN) is a common health food
consumed in Asia. The exaggerated skincare
functions resulted in the abnormally high market
value. Fake EBN started to appear in the market for
the sake of its profits. A reliable authenticate
method is in urgent need. Here, the determination
of a free form of sialic acid, N-acetylneuraminic acid
(NANA), was developed to distinguish EBN
according to their grading. High amount of free
NANA was revealed in red and yellow EBN: both
white and grass EBN contained free NANA at low
level. Unlike total NANA content measurement, fake
EBN did not show detectable amount of free NANA.
Moreover, the water extract of EBN showed anti-
tyrosinase activity, and which was in line to amount
of free NANA. Thus, the amount of free NANA could
differentiate fake EBN from the genuine one, as well
as its grading. In conclusion, the quantitation of free
NANA by triplequadrupoles liquid chromatography
tandem mass spectrometry (LC-MS/MS QQQ) was
shown to be the best method in EBN authentication.
In addition, the amount of free NANA in EBN was in
accord to anti-tyrosinase activity of EBN.
Citation:
Chan G. Kl., Zheng K. Yz., ZhuK.Y., Dong T. Tx., Tsim
K.Wk., 2013. Determination of free N-acetylneuraminic
acid in edible bird nest: A development of chemical marker
for quality control. The Journal of Ethnobiology and
Traditional Medicine. Photon 120, 620-628.
1. Introduction
1.1 Adulteration of edible bird’s nest due to the
lacking of quality control marker
Edible bird nest (EBN) has been served as a
valuable delicacy in Asia for over 1,000 years.
The official record of EBN has only been found
since 16th century from ancient Chinese
literatures. According to the traditional
application, the intake of EBN could repair
lung function, strengthen digestive system,
enhance skin repairing and improve immune
system (Zhao, 1765); however, neither clinical
trial nor basic mechanistic study has proven
those beneficial effects. Although the
functional role of EBN is still largely unknown,
it becomes a very popular health food
supplement for its exaggerated skincare
function promoted by the media. Recently,
fake EBN appeared commonly in the market
(Leung, 2004). The unscrupulous traders may
illegally sell EBN adulterants in the market,
e.g. white fungus and pig skin. On the other
hand, the market prices of EBN varied by
grading of EBN. The grading of EBN mainly
depends on the genuine color, e.g. red, yellow
and white, and the packaging forms, e.g. cup,
stripe and piece. EBN contains impurities such
as seaweeds (grass) and feathers will be
determined as lower grade. Apart from
grading, the scarcity of specific type of EBN
produced from those Southeast Asian
countries such as Vietnam, Malaysia and
Thailand also seriously affects the market
price of EBN in Hong Kong. Refer to the
previous report, the market price of EBN is
ranged from $2,000 to $10,000 USD per
kilogram (Ma & Liu, 2012). The disparity in
market price between different grades of EBN
is even more obvious now. For each kilogram
Ph ton
621
of EBN, the local price of Grass EBN ranged
from $1,000 to $2,000 USD. For Indonesian
EBN, White EBN ranged from $1,000 to
$3,000 USD, Yellow EBN ranged from $1,500
to $3,500 USD and Red EBN ranged from
$2,000 to $4,000 USD. The price for EBN from
Thailand and Malaysia reaches $6,500 and
7,500 respectively. The high quality EBN from
Vietnam even reached $15,000 USD per
kilogram. Some of the traders may modify the
colour appearance of EBN as to achieve a
higher price. Currently, microscopic and
genetic methods were being applied in
authenticating EBN (Lin et al., 2006b; Lin et
al., 2009). However, the ability in
differentiating different grading of EBN was in
doubt. Thus, a fast and reliable method for
EBN authentication and/or classification is
urgently needed.
1.2 N-acetylneuraminic acid as a potential
quality marker for EBN
EBN comes from the nest of swiftlet species,
such as Aerodramus fuciphagus (formerly
named Collocalia fuciphagus), which is made
by their saliva secretion (Green, 1885). EBN
composes of up to 60 % of soluble protein and
about 20 % of water, which also contains fat,
carbohydrate and trace amount of minerals
including sodium, calcium and magnesium
(Norhayati et al., 2010). More importantly,
EBN consists of ~9% of sialic acid (Kathan et
al., 1969). Sialic acid is a family of more than
20 compounds derived from neuraminic acid,
and N-acetylneuraminic acid (NANA) is the
predominant form (Ham et al., 2007). Due to
the highly charged property, NANA is involved
in retaining water on cell surface and
enhances cellular fluid uptakes (Simons &
Fuller, 1985). Moreover, NANA is proposed to
be a major component for anti-influenza
function of EBN (Guo et al., 2006; Yagi et al.,
2008),as well as its effect on the proliferation
of Caco-2 cells (Rashed & Nazaimoon, 2010).
Thus, NANA could serve as a good quality
control marker.
The majority of NANA exists in conjugated
form (i.e. oligosaccharides and glycoproteins).
After the actions of neuraminidase or acid
hydrolysis, a free form of NANA will be
released, and the function of free NANA is
unclear (Sillanaukee et al., 1999).Different
EBN authentication methods have been
developed by quantifying the amount of NANA
(Yu et al., 1998; Huang et al., 2003; Wang et
al., 2006), but all of these methods are using
total amount of NANA (i.e. sum of conjugated
and free NANA) as an authentication marker.
Unfortunately, the conjugated NANA can be
found in many other food sources including
dairy products (Wang et al., 2001). Using total
NANA as a marker for EBN authentication will
be resulted in a highbackground and obtained
larger false positive error. Another drawback of
the current established EBN authentication
methods is that they are incapable of
differentiating different grades of EBN.
2. Objective of Research
Several methods in authenticating EBN such
as electron microscopy (Lin et al., 2006b) and
DNA sequence analysis (Lin et al., 2009) are
proven to be failed in differentiating different
grades of EBN. Thus, total NANA and free
NANA are now under examination as quality
marker of EBN.
2. Materials and Methods
2.1 Chemicals
NANA was purchased from Sigma-Aldrich (St.
Louis, MO), and 10 mM was used as standard
stock solution. The volume was measured
accurately from the stock, and diluted with
fresh Milli-Q water to produce a series of
solution standards (1, 2, 5, 10, 15, 20 µM).
Tyrosinase (EC 1.14.18.1) from mushroom
and L-3, 4-dihydroxyphenyl-alanine (L-DOPA)
was from Sigma-Aldrich. The specific activity
of the enzyme was ≥1,000 U/mg.
2.2 LC-MS/MS QQQ system
The liquid chromatograph is equipped with an
Agilent 6410 Triple Quad MS/MS (Agilent,
Waldbronn, Germany), and an Eclipse XDB-
C18 column (2.1 X 100 mm; 3.5 µm particle
size). The injection volume was 2 µL. A 5 min
linear gradient at flow rates of 0.4 ml/min
between solvent A (Milli-Q water, 0.1% formic
acid) and solvent B (acetonitrile, 0.1% formic
acid) was used. After reaching 80% B, the
system returned to 100% A in 0.5 min. For
column equilibration, a total cycle time of 10
min was needed. Retention time of NANA was
at 0.65 – 0.69 min. The MS was operated in
negative electron spray ionization mode. A
capillary voltage of 3.5 kV and a cone voltage
of 10 V were applied. The source temperature
was 100° C, and the desolvation temperature
was 325° C. Ultra high purity nitrogen was
used for cone gas (3.0 L/min), desolvation gas
(10.0 L/min) and nebulising gas (35 psi). For
collision induced dissociation (CID), collision
energy of 5 eV was used. Negatively single
charged ions [M-H]- of NANA (m/z 307.9) were
selected as precursor ions for CID. The
precursor ion was dissociated into two major
product ions (m/z 87.0 and 170.0), and the
Ph ton
622
product ions m/z 87.0 was the most abundant
from NANA. No internal standard was applied
in this study as the reference for free NANA
was limited, and the relevant chemical could
not be easily accessible. 7 For multiple
reaction monitoring, the transitions m/z 307.9
87.0 and m/z 307.9 170.0 were chosen
as the qualifiers, whilst the transitions m/z
307.9 87.0 was measured for quantification.
For the full scanning of total ion
chromatograms, spectra from m/z 50 to m/z
1000 were recorded.
2.3 Quantification of free NANA
EBN samples were grounded into powder
(approximately 1 – 3 mm) and mixed
thoroughly. 10 mg of each was weighed, and
free sialic acid was extracted by 1 ml of fresh
Milli-Q water under sonication for 10 min.
Followed by centrifugation at full speed for 5
min, the supernatants were filtered, and the
filtrates were collected for LC-MS/MS QQQ
analysis. A 5-point calibration curve with
concentrations of 1, 2, 5, 10 and 20 µM NANA
was made. All calibrators were prepared in
fresh Milli-Q water. Triplicate results were
taken for each sample.Genuine Indonesia
white EBN was used for method validation.
Linearity of free NANA was tested using the
calibration curve of NANA. All calibrators were
prepared in fresh Milli-Q water. The correlation
coefficient, better than 0.9992, was obtained.
2.4 Total NANA assay
The total NANA content was determined by
sialic acid quantitation kit - SIALICQ (Sigma).
Ten mg of powder EBN were weighed
accurately in screw cap microtubes, 0.5 ml of
1 M sulphric acid in methanol was added to
the each tube. The tubes were then heated to
90° C for 90 min in a heating block, and then
which was cool down in room temperature for
10 min. Barium carbonate (0.5 g) was added
to neutralize the solution. The tube was then
sonicated for 5 min and spin down at 1,500 Xg
for 5 min. Supernatants were transferred into
clean tubes and evaporated to dryness at 37
oC. The tubes were then reconstituted by 600
µL of pH 7.5 Tris-HCl, and 1 µL of NANA
aldolase was added. The tubes were
incubated at 37° C for 16 hours. The solution
was diluted 5 times by Tris-HCl buffer at pH
7.5, and a diluted buffer was measured as
blank. Then, 20 β-NADH was added to the
each solution. The solution was mixed well,
and the absorbance reading at 340 nm was
recorded from the initial reading to the final
reading. The NANA content was calculated by
the equation provided in the kit.
2.5 Tyrosinase activity assay
The activity of mushroom tyrosinase was
monitored by dopachrome formation at 492
nm through the oxidation of L-DOPA. The
reaction medium (200 µl) contained 0.5 mM L-
DOPA in 50 mM sodium phosphate buffer (pH
6.8), 5 mM vitamin C was used as a control
inhibitor. The final concentration of mushroom
tyrosinase was 0.2 mg/ml. In this method, 0.1
ml of different concentration of effectors,
including EBN extracts, adulterants of EBN
and NANA, was added to the reaction. The
reaction mixtures were loaded on a 96-well
plate, and the formation of dopachrome was
measured in optical density at 492 nm after 20
min of incubation under dark. The reaction
was carried out under room temperature.
Absorption was recorded using micro-plate
spectrophotometer.
2.6 Statistical tests
Statistical tests were done by using student t
test and one-way ANOVA provided in
GraphPad Prism 5.0. Statistically significant
changes were classed as [*] where p<0.05, [**]
where p<0.01 and [***] where p<0.001.
3. Results
3.1 Authentication methods of EBN
Figure 1: Photos of different grades of EBN
Grass, white, yellow and red EBN are the
major grades of EBN commonly found in the
market (Fig. 1): they are greatly different in
their price, i.e. red > yellow > white > grass.
Ph ton
623
Under normal circumstance, EBN could be
easily differentiated by their physical
appearance. However, different ways of
mimicking red EBN by white EBN had been
developed by illegal traders for better profit.
The amount of total NANA was reported to
serve as a quality control marker of EBN
(Wang et al., 2006). Here, we aimed to
determine the amounts of total NANA in EBN.
Fig. 2 shows the amount of total NANA in
different grades of EBN, and they were in an
order of red > yellow > white > grass EBN.
However, the commonly used dopants, or the
fake EBN, showed significant high amount of
total NANA. Thus, total NANA could not be
served as an authenticating marker here, in
particular to identify genuine EBN.
Figure 2: Amount oftotal NANA in EBN and its
adulterants
The amount of total NANA content was
extracted by water from 27 batches of genuine
EBN (i.e. grass EBN (n=3), white EBN (n=9),
yellow EBN (n=6) and red EBN (n=9) and 21
batches of known fake EBN (n=6) and five
adulterants of EBN (n=3) by SIALIC-Q kit. The
total NANA in mg/kg of dry material was
presented as Mean + SEM (n ± 3).
3.2 Free NANA for EBN authentication
In order to search possible chemical markers
for EBN, the extracts deriving from EBN and
its common dopants were subject to LC-
MS/MS QQQ analysis. The representing total
ion chromatograms of different grading of EBN
and EBN adulterants/dopants (agar, gelatin
and pig skin) were achieved (Fig. 3A). The
retention time of all characteristic peaks from
those different samples were compared. The
mass spectrums from each peak were also
analyzed as a reference for identity. From the
result, all EBN chromatograms showed
distinguishable peak 1 at the retention time of
0.523 – 0.538 min, but peak 1 was also found
in pig skin at the same retention time. From
the mass spectrum information, peak 1 could
be an alanine-rich compound. On the other
hand, a well separated peak 2 at 0.655 min
was noticed in EBN chromatograms. The
mass spectrum suggested a compound having
an ion of m/z 307.9: this was identified as
NANA (Fig. 3A). The NANA peak (peak 2) in
the grass EBN was merged into peak 1 due to
its low level. A characteristic peak 3 was only
observed in red EBN, and the mass
spectrometry result revealed that as an
unknown compound with a m/z 191.0. In
conclusion, an identified free form of NANA
could be considered as a chemical marker of
EBN.
The characteristics of NANA in the MS/MS
were revealed (Table 1). Two product ions
were produced here, 87 and 170 m/z: the ion
at 87 m/z was used for quantitation
(Supplementary Fig. 1). In MS/MS analysis,
each calibration curve was obtained from
different concentrations of marker chemical.
The correlation coefficient (r
2
) of the calibration
curve was higher than 0.999. The LOD and
LOQ were determined at 3.188 µg/kg and
10.626 µg/kg, respectively. The precision,
repeatability and recovery were determined as
described previously (Zhu et al., 2010), and
the results were satisfactory (Table 1). The
results showed that the LC-MS/MS QQQ
method was precise, accurate and sensitive
enough for simultaneous, quantitative
evaluation of NANA from EBN.
Figure 3: The chromatographic fingerprint of EBN
and its adulterants
Ph ton
624
(A): Different water extracts of EBN and its
adulterants, as shown in the figure, were
detected using the MRM scan mode in
MS/MS. The ions with m/z 50 to m/z 1000
were recorded. Ions fractions acquired at
specific time were marked with different
numbers according to their compositions.
Peak 1 is unknown small peptide, peak 2 is
corresponding to NANA and peak 3 is
unknown. Representative chromatograms are
shown, n=3.
(B): The amount of free NANA in different
grades of EBN (i.e. grass EBN (n=3), white
EBN (n=9), yellow EBN (n=6) and red EBN
(n=9)) and its adulterants (n=21) were
determined. Free NANA was extracted from
10 mg of each sample by 1 ml MilliQ water
and measured by LC-MS/MS QQQ system.
The amount of free NANA in µg/kg of dry
material was presented as Mean + SEM (n ≥
3).
After the establishment and validation of our
EBN authentication method, the quality of EBN
available in Hong Kong market was examined.
The extraction of free NANA from EBN was
optimized at sonication for 10 min in water
(Supplementary Fig. 2).
Table 1: Mass spectra properties and results of validation tests on the assay of free NANA in EBN
Mass spectra properties Validation tests results
Formula C
11
H
19
O
9
Calibration curve
h
y=17444.31x+729.72
Calculated mass [M] 309.1 Correlation coefficient (r
2
) 0.9992
Precursor ion [M-H]
a
307.9 Linear range (ng) 0.001 – 0.050
Fragmentor energy
b
135 V
Collison energy
c
5 eV Intra-day precision (n=6)
i
RSD (%) 4.24
Product ion
d
87.0, 170.0 Inter-day precision (n=6)
j
RSD (%) 4.57
Retention time (min)
e
0.672 Repeatability (n=5) RSD (%) 0.66
LOD f (
µ
g/kg) 3.188 Reproducibility (n=5) RSD (%) 3.35
LOQ g (µg/kg) 10.626 Recovery
k
(n=5) Mean (%) 87.33
RSD (%) 7.34
a
The detected chemicals had the greatest responses under the negative mode: the [M-H]- was used as the
precursor ion.
b
The fragmentor energy was optimized to have the greatest ionize efficiency.
c
The collision energy was optimized to have the greatest product ion intensity, which was the key factor in the
MRM mode.
d
Two product ions were used for the MRM analysis. The upper one was used for quantitative analysis and the
lower one was for qualitative analysis, which could guarantee the precision of analytes.
e
The retention time was determined by 5 different individual analyses (n = 5).
f
LOD refers to the limits of detection.
g
LOQ refers to the limits of quantification.
h
These calibration curves were constructed by plotting the peak area versus the concentration of each analyte.
Each calibration curve was derived from eight data points, n = 3.
I
The intra-day analysis refers to the sample examined for six replicates within one day.
j
The inter-day analysis refers to the sample examined in duplicates over three consecutive days.
k
Recovery (%)=100×(amount found−original amount)/amount spiked. The data was presented as average of five
independent determinations, and the SD was < 8% of the Mean, which was not shown for clarity.
The free NANA was determined in EBN and
commonly reported EBN dopants, including
agar, white fungi, pig skin, tora and gelatin.
The free NANA was not detected in EBN
dopants/adulterants (Fig. 3B). In contrast,
NANA was revealed in all EBN having an
order of red > yellow > white > grass EBN. The
median of the free NANA in different EBN
groups were 702.51 µg/kg, 351.44 µg/kg,
170.46 µg/kg and 54.31 µg/kg respectively.
In the market, EBN was clustered according to
their form of packaging (Fig. 4A). EBN sold in
a form of their original “bird’s nest” shaped
were regarded as “cup”; EBN comprised of
long stripes broke down horizontally from the
nest were regarded as “stripe”; and EBN sold
in the form of powder-like or small pieces
within 5-mm diameter were regarded as
“piece”. No significant differences in the
content of free NANA between different forms
of packaging was observed among white EBN
(Fig. 4B). In contrary, the differences in
amount of free NANA between varies
packaging forms of red EBN was obvious. Cup
EBN showed the highest content of free
NANA, followed by the stripe EBN, and the
piece EBN contained the lowest content of
free NANA.
Ph ton
625
Figure 4: Free NANA in different packaging forms
of EBN
(A): Photos of different packaging forms of EBN.
Scale bar, 10 mm
(B): The amount of free NANA in different
packaging forms of EBN was determined. 18 EBN
purchased from Hong Kong market were
categorized into three forms of packaging (i.e. piece
EBN, stripe EBN and cup EBN) from white (n=9)
and red EBN (n=9). Free NANA was extracted from
10 mg of each sample by 1 ml MilliQ water and
measured by LC-MS/MS QQQ system. The amount
of free NANA in µg/kg of dry material was
presented as Mean + SEM (n ≥ 3). Statistical
significant differences were indicated. *** P < 0.001
versus reference group.
3.3 EBN inhibits tyrosinase activity
The inhibitory effect of EBN on tyrosinase
could serve as an indicator for skin whitening
function. Both white and red EBN inhibited the
formation of dopachrome and reduced the
content by at least 50% (Fig. 5). As compared
to the control, a slight reduction of
dopachrome content was found with the
extract of grass EBN; however, the difference
was not statistical significant. None of the
adulterants of EBN could inhibit tyrosinase
activities. Oppositely, the extracts from agar or
pig skin enhanced the oxidation of L-DOPA. In
parallel, free NANA was able to inhibit the
tyrosinase activity, even though the
concentration used was rather high as
compared to that contained within EBN (Fig.
5). The application of vitamin C served as a
control.
Figure 5: Anti-tyrosinase activity of EBN
The dopachrome formed by the oxidation of
0.5 mM of L-DOPA incubated for 20 min with
0.2 mg/ml mushroom tyrosinase was served
as control. Vitamin C (5 mM) was served as an
inhibitor control. The dopachrome
concentration, after treatment of EBN or
others, was determined, all at 10 mg / ml water
extract. Free NANA was at 20 mM. The
contents were presented as Mean + SEM (n =
3). Unpaired one-tailed student t test was
performed on the data set by Graph Pad 5.0.
Statistical significant differences were
indicated. *** P < 0.001 versus control group.
4. Discussions
The authentication methods of EBN can be
divided into four major directions including
physical examination, DNA authentication,
proteomics analysis and glycan and monose
determination. The physical examination on
EBN was mainly based on different ways of
Ph ton
626
microscopy (Lin et al., 2006b). The
advantages of this method are that the nature
of EBN can be maintained or with minimized
interruption, and also no extraction procedure
is required. However, this EBN authentication
is qualitative and not able to distinguish the
grading of EBN.
DNA fragment has been used for EBN
authentication (Lin et al., 2009; Aowphol et al.,
2008). The DNA sequence in EBN is unique,
thus these authenticating properties will not be
cheated easily. However, the DNA
authentication could not reflect the actual
nutrient composition in EBN. Moreover, the
resolution power of using DNA authentication
is not enough for differentiating EBN grading
(Lin et al., 2006b).
Proteomics analysis has been developed to
study EBN including 1-D (Lin et al., 2006a)
and 2-D polyacrylamide gel electrophoresis
(Goh et al., 2001). On the other hand,
epithelial growth factor-like activity (Kong et al.
1987) and mitogenic activities (Ng et al., 1986;
Roh et al., 2011; Hou et al., 2010; Fadhilah et
al., 2011) of EBN were reported. In addition,
the effects of EBN on bone and skin have
been reported (Matsukawa et al., 2011). All of
those reports suggested that a hormone or
growth factor–like substance in protein nature
should be found in EBN. Recently, an antibody
raised against an EBN glycoprotein was
employed for authentication (Zhang et al.,
2012). The database of proteomics of EBN still
under developed, and only a few proteins have
been identified in EBN so far (Goh et al., 2000;
Ou et al., 2001). Moreover, the protein
identification could not classify the grading of
EBN.
Many different types of specific glycans have
been reported to be presented on EBN surface
(Yagi et al., 2008; Oda et al., 1998; Nakagawa
et al., 2007). Glycan was proposed to
responsible for the biological functions of EBN,
e.g. anti-influenza effect (Guo et al., 2006).
Indeed, five sugar markers, including
mannose, galactose, N-acetylgalactosamine,
N-acetylglucosamine and total NANA, were
introduced in EBN. The detection of these
sugar markers however was unable to
differentiate different types of EBN (Yu et al.,
2000).
Here, we introduce a new EBN authentication
method by revealing the level of free NANA in
EBN. Actually, scientists discovered free form
of NANA in EBN since 1960s (Howe et al.,
1961) andthe application of free NANA
quantitation was widely used by diagnostic
study (Ham et al., 2007).However, it is the first
report in combining the two distinct areas of
research and accurately quantifying the free
form of NANA by advance technology for the
authentication of traditional Chinese medicine
or nutraceuticals. The method could be done
within 30 min, and which showed highly
repeatable and reproducible. More important,
this method distinguished genuine EBN as
well as to classify the grading of different EBN.
The form of packaging of EBN is another
parameter that varies the market price
seriously. The intact EBN having “nest
shaped” shows the highest price: this is also
named as cup EBN. According to history in
China, only cup EBN could be used by the
king of Southeast Asian countries as gifts to
Chinese Empires, thus which was named as
officer EBN. As supported by our studies, the
free NANA content in cup EBN is the highest
among different forms of packaging, which
matches with the traditional classification. This
difference could only be revealed here in red
EBN: white EBN, according to packaging
forms, was not significant. The processing of
EBN may be one of the possible reasons in
reducing the amount of free NANA, which
involves the removal of feather and other
foreign matters, air-drying and baking. These
processing steps for strip and pieces of EBN
require a long period of time of water soaking
and molding. In order to preserve white EBN
color after longer time of storage, white EBN
was subjected to additional hot air drying or
chemical spraying (Law et al., 2011). Free
NANA is highly water soluble and degrades
rapidly under acidic environment, and thus
free NANA could be decreased after prolong
processing, as suggested previouslyYu et al.,
1998). In contrast, red cup EBN has lesser
extent of processing.
The skincare functions of EBN have been
widely accepted by the public; however, there
is no scientific supporting evidence until now.
In the past, the unproven skincare functions of
EBN was relied mainly on the high protein
content and the finding of the epidermal
growth factor-like activity (Kong et al., 1987).
Here, we provided a direct report to show the
potential skin whitening function of EBN, and
the adulterants of EBN cannot replace EBN.
Hence, EBN with higher quality (e.g. red EBN)
may have better whitening effect than EBN
with low quality (e.g. grass EBN). Interesting,
the whitening function of EBN was in parallel
to the amount of free NANA. The action
Ph ton
627
mechanism of NANA on the skincare functions
should need further investigation.
Limitations
Honestly speaking, all authentication methods
have advantages but also drawbacks on their
own. Like other drugs with only single quality
assurance marker, free NANA could be
externally added on fake EBN by those
dishonest traders.
Recommendations
Thus, a combination of multi-disciplinal
methods including genomic analysis,
microscopic identification and analytical
analysis should be applied together to
establish a quality control of EBN.
Conclusion
Definitely, due to the correlation of the
bioactivity, this result strengthens the role of
NANA as a quality control marker of EBN.
References
Aowphol A., Voris H.K., Feldheim K.A.,
Harnyuttanakorn P., Thirakhupt K., 2008. Genetic
homogeneity among colonies of white-nest swiftlet
(Aerodramus fuciphagus) in Thailand. Zoological
Science, 25, 372-380.
Fadhilah Z.A., Chua K.H., Ng S.L., Elvy S.M.R., Lee
T.H., Norzana A.G., 2011. Effects of edible bird’s
nest (EBN) on cultured rabbit corneal keratocytes.
BMC Complementary and Alternative Medicine, 11,
1-11.
Goh D.L.M., Chew F.T., Chua K.Y., Chay O.M., Lee
B.W., 2000. Edible bird's nest induced anaphylaxis:
An under-recognized entity? Journal of Pediatrics,
137, 277-279.
Goh D.L.M., Chua K.Y., Chew F.T., Seow T.K., Ou
K.L., Yi F.C., Lee B.W., 2001. Immunochemical
characterization of edible bird’s nest allergens.
Journal of Allergy and Clinical Immunology, 107,
1082-1088.
Green J.R., 1885. The edible bird’s-nest, or nest of
the Java swift (Collocalia nidifica). Journal of
Physiology, 6, 40-45.
Guo C.T., Takahashi T., Bukawa W., Takahashi N.,
Yagi H., Kato K., Hidari KIPJ., Miyamoto D., Suzuki
T., Suzuki Y., 2006. Edible bird's nest extract
inhibits influenza virus infection. Antiviral Research,
70, 140-146.
Ham M., Prinsen B.H.C.M.T., Huijmans J.G.M.,
Abeling N.G.G.M., Dorland B., Berger R., Koning
TJ., Sain-van Der Velden M.G.M., 2007.
Quantification of free and total sialic acid excretion
by LC-MS/MS. Journal of Chromatography B, 848,
251–257.
Hou Y., Xian X., Lin J., Lai X., Chen J., 2010. The
effects of edible birds' nest (Aerodramus) on Con A-
induced rat lymphocytes transformation. Chinese
Modern Medicine, 17, 9-11.
Howe C., Lee L.T., Rose H.M., 1961. Collocalia
mucoid: a substrate for myxovirus neuraminidase.
Archives of Biochemistry and Biophysics, 95, 512-
520.
Huang H., Xi X., Chen W., Chen J., 2003.
Determination of content of bird nest by
spectrophotometer. Guangzhou Food Science and
Technology, 19, 68-69.
Kathan R.H., Weeks D.I., 1969. Structure studies of
Collocalia mucoid. I. Carbohydrate and amino acid
composition. Archives of Biochemistry and
Biophysics, 134, 572-576.
Kong Y.C., Keung W.M., Yip T.T., Ko K.M., Tsao
S.W., Ng M.H., 1987. Evidence that epidermal
growth factor is present in swiftlet's (Collocalia)
nest. Comparative Biochemistry and Physiology B-
Biochemistry & Molecular Biology, 87, 221-226.
Law C.L., Ong S.P., Yong L.C., 2011. Minimising
colour change of cleaned edible birds’s nest by
applying appropriate processing strategy. ICOTOS,
1, 1-3.
Leung C.Y., 2004. Three billions market competition
for edible bird's nest shops. Economic Digest, 1197,
68-69.
Lin J.R., Dong Y., Zhou H., Lai X.P., Wang P.X.,
2006a. Identification of edible bird's nest by
electrophoresis. Modernization of traditional
Chinese Medicine, 8, 30-32.
Lin J.R., Zhou H., Lai X.P., 2006b. Application of
Stereoscopy on edible bird's Nest Identification.
Journal of Chinese Medicine, 29, 219-221.
Lin J.R., Zhou H., Lai X.P., Hou Y., Xian X.M., Chen
J.N., Wang P.X., Zhou L., Dong Y., 2009. Genetic
identification of edible birds’ nest based on
mitochondrial DNA sequences. Food Research
International, 42, 1053-1061.
Ma F., Liu D., 2012. Sketch of the edible bird’s nest
and its important bioactivities. Food Research
International Doi: 10.1016/j.foodres.2012.06.001
Matsukawa N., Matsumoto M., Bukawa W., Chiji H.,
Nakayama K., Hara H., Tsukahara T., 2011.
Improvement of bone strength and dermal thickness
due to dietary edible bird's nest extract in
ovariectomized rats. Bioscience Biotechnology and
Biochemistry, 75, 590-592.
Nakagawa H., Hama Y., Sumi T., Li SC., Maskos
K., Kalayanamitra K., Mizumoto S., Sugahara K., Li
Ph ton
628
YT., 2007. Occurrence of a non-sulfated chondroitin
proteoglycan in the dried saliva of Collocalia
swiftlets (edible bird’s-nest). Glycobiology, 17, 157–
164.
Ng M.H., Chan K.H., Kong Y.C., 1986. Potentiation
of mitogenic response by extracts of the swiftlet's
(Collocalia) nest. Biochemistry International, 13,
521-531.
Norhayati M.K., Azman O., Nazaimoon W.M.W.,
2010. Preliminary study of the nutritional content of
Malaysian edible bird’s nest. Malaysian Journal of
Nutrition, 16, 389-396.
Oda M., Ohta S., Suga T., Aoki T., 1998. Study on
Food Components: The structure of N-linked asialo
carbohydrate from the edible bird's nest built by
Collocalia fuciphaga. Journal of Agricultural And
Food Chemistry, 46, 3047-3053.
Ou K., Seow T.K., Liang R.C.M.
Y., Lee B.W., Goh D.L.M., Chua K.Y., Chung
M.C.M., 2001. Identification of a serine protease
inhibitor homologue in bird's nest by an integrated
proteomics approach. Journal of Food Technology,
22, 3589-3595.
Rashed A.A., Nazaimoon W.M.W., 2010. Effect of
edible bird’s nest on Caco-2 Cell proliferation.
Journal of Food Technology, 8, 126-130.
Roh K.B., Lee J., Kim Y.S., Park J., Kim J.H., Lee
J., Park D., 2011. Mechanisms of edible bird’s nest
extract-induced proliferation of human adipose-
derived stem cells. Evidence-Based
Complementary and Alternative Medicine, 2012, 1-
11.
Sillanaukee P., Pönniö M., Jääskeläinen IP., 1999.
Occurrence of sialic acids in healthy humans and
different disorders. EuropeanJournalof Clinical
Investigation, 29, 413-425.
Simons K., FullerSD., 1985. Cell surface polarity in
epithelia. Annual Review of Cell Biology, 1, 243-
288.
Wang B., Brand-Miller J., McVeagh P., Petocz P.,
2001. Concentration and distribution of sialic acid in
human milk and infant formulae. American Journal
of Clinical Nutrition, 74, 510-515.
Wang H., Ni KY., Wang Y., 2006. Determination of
sialic acid in edible bird’s nest. Chinese Journal of
Pharmacology Analysis, 26, 1251-1253.
Yagi H., Yasukawa N., Yu S.Y., Guo C.T.,
Takahashi N., Takahashi T., Bukawa W., Suzuki T.,
Khoo K-H., Suzuki Y., Kato K., 2008. The
expression of sialylated high-antennary N-glycans
in edible bird’s nest. Carbohydrate Research, 343,
1373-1377.
Yu Y.Q., Xue L., Wang H., Zhou H.X., Zhu X.F., Li
B.S., 2000. Determination of edible bird's nest and
its products by gas chromatography. Journal of
Chromatography Science, 38, 27-32.
Yu Y.Q., Xue L., Zhu X.F., Li B.S., 1998.
Characterization of edible bird's nest by gas
chromatography. Journal of Instrumental Analysis,
17, 33-36.
Zhang S., Lai X., Liu X., Li Y., Li B., Huang X.,
Zhang Q., Chen W., Lin L., Yang, G., 2012.
Competitiveenzyme-linked immunoassay for
sialoglycoprotein of edible bird’s nest in food and
cosmetics. Journal of Agricultural and Food
Chemistry, 60, 3580-3585.
Zhao X., 1765. A supplement to the compendium of
materia medica. People’s medical publishing house,
377-379.
Zhu K.Y., Fu Q., Xie H.Q., Xu S.L., Cheung A.W.H.,
Zheng K.Y.Z., Luk W.K.W., Choi R.C.Y., Lau
D.T.W., Dong T.T.X., Jiang Z.Y., Chen J.J., Tsim
K.W.K., 2010. Quality assessment of a formulated
Chinese herbal decoction, Kaixinsan, by using rapid
resolution liquid chromatography coupled with mass
spectrometry: a chemical evaluation of different
historical formulae. Journal of Separation Science,
33, 3666-3674.
