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ISSN: 0017-3134 (Print) 1651-2049 (Online) Journal homepage: http://www.tandfonline.com/loi/sgra20
Air pollution exacerbates effect of allergenic
pollen proteins of Cassia siamea: a preliminary
report
Varsha Hinge, Jaykiran Tidke, Biswadeep Das, Shrikant Bhute, Pradeep
Parab & Kishori Apte
To cite this article: Varsha Hinge, Jaykiran Tidke, Biswadeep Das, Shrikant Bhute, Pradeep
Parab & Kishori Apte (2016): Air pollution exacerbates effect of allergenic pollen proteins of
Cassia siamea: a preliminary report, Grana, DOI: 10.1080/00173134.2016.1193221
To link to this article: http://dx.doi.org/10.1080/00173134.2016.1193221
Published online: 25 Jul 2016.
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Air pollution exacerbates effect of allergenic pollen proteins of Cassia
siamea: a preliminary report
VARSHA HINGE
1
, JAYKIRAN TIDKE
1
, BISWADEEP DAS
2
, SHRIKANT BHUTE
3
,
PRADEEP PARAB
2
& KISHORI APTE
2
1
Department of Botany, Sant Gadge Baba Amravati University, Amravati, India,
2
APT Research Foundation, Pune, India,
3
Department of Zoology, Savitribai Phule Pune University, Pune, India
Abstract
Pollinosis caused by several allergenic proteins in pollen grains has been a major problem of healthcare in most parts of the
world. Environmental factors such as air pollution are known to alter the release of allergenic pollen proteins from a variety of
the plant species. Cassia siamea is commonly planted along roadsides and in industrialised areas in many parts of India. The
present study reports the findings of animal experiments demonstrating the effect of air pollution on the allergenicity of Cassia
siamea pollen proteins. Total white blood cell (WBC) count and lymphocyte count were significantly higher in animals that
received protein extract of pollen collected from a polluted site compared to those that received protein extract of pollen
collected from a non-polluted site. This was concomitant with increased production of IgE antibodies; followed by marked
degranulation of mast cell leading to heighten type I hypersensitivity in these animals. These results are important for the
development of a consensus linking ever-increasing pollution due to industrialisation and an increase in associated pollinosis.
Keywords: air pollution, Cassia siamea, pollen proteins, allergenicity
The pollen of many trees, weeds and grasses are
major causes of allergies in several parts of the
world (D’Amato et al. 2013) including India (Singh
& Shahi 2008). Although pollen themselves are not
allergenic, they carry several proteins, glycoproteins
and non-protein substances such as pollen associated
lipid mediators (PALMs) within them during their
flight that make them allergenic (Gilles et al. 2009;
Songnuan 2013). Several proteins essential to make
pollen a male gametophyte are strategically located
and released from various sites of pollen like exine,
intine or cytoplasm (Behrendt & Becker 2001).
Some of these proteins belonging to protein families
such as profilin (Tehrani et al. 2011); polcalcin and
expansin (Radauer & Breiteneder 2006) act as
potent aeroallergens. Similarly, by using one-dimen-
sional (1D) and two-dimensional (2D) electrophor-
esis and Western blotting, allergenic pollen
glycoprotein have been characterised (Chow et al.
2005; Manduzio et al. 2012). In addition, it has
been demonstrated that pollen release excessive
amounts of lipids called PALMs upon hydration,
which independent of proteins present in pollen
shows immuno-stimulatory and/or immune-modula-
tory properties (Behrendt et al. 2001; Gilles et al.
2009). Pollen particles thus act as a carrier of com-
plex allergenic substances, which may lead to allergic
rhinitis (Taketomi et al. 2006).
The effect of air pollution on human health has a
long history; there has been a rapid increase in air
pollution during the last century with increased global
population, excessive energy consumption in the form
of petroleum and its derivatives in motor vehicles and
industrial activities (Bartra et al. 2007). The preva-
lence of atopy and other allergenic disorders has been
found rising parallel with increased air pollution,
especially in industrialised countries (D’Amato et al.
2014). Pollen grains of gymnosperms and angios-
perms are major contributors of aeroallergens (Séné-
chal et al. 2015) and some studies have indicated the
Correspondence: Jaykiran Tidke, Department of Botany, Sant Gadge Baba Amravati University, Amravati, Maharashtra 444602, India. E-mail:
varsha11hinge@gmail.com
(Received 4 December 2015; accepted 30 April 2016)
Grana, 2016
http://dx.doi.org/10.1080/00173134.2016.1193221
© 2016 Collegium Palynologicum Scandinavicum
Downloaded by [223.196.18.139] at 20:27 25 July 2016
possible role of air pollution in increased allergenecity
of pollen under polluted conditions over non-polluted
conditions (Armentia et al. 2002; Cortegano et al.
2004). This rise in plant derived respiratory allergies
could be partly explained by the fact that different air
pollutants interact with pollen and alter their antigenic
potency (Arbabian et al. 2011; Rezanejad et al. 2003).
Air pollutants such as carbon dioxide (CO
2
)present
in the air is known to affect fertilisation and pollen
production as well as allergen contents of pollen
grains (Reid & Gamble 2009). Diesel exhaust and
nitrogen oxide have been observed to act as adjuvant
to pollen allergen both in animals (Zijverden et al.
2000) as well as humans (Diaz-Sanchez et al. 2000).
Traffic-related pollutants such as nitrogen dioxide
(NO
2
) and ozone (O
3
) are linked with the release of
pollen cytoplasmic granules (PCGs) which are likely
to contain allergens (Motta et al. 2006). Thus, there is
a direct link between air pollution and altered and/or
enhanced pollen allergies of pollen of many plants.
Cassia siamea L. (Fabeaceae) has attracted ethno-
pharmacology for various medicinally important com-
ponents purified from different parts of the plant
(Kumar et al. 2010;Nsondeetal.2010; Deguchi
et al. 2012). Medium size trees of C. siamea are
often planted along roadsides and in industrialised
areas as afforestation programmes to control pollu-
tion. These plants densely flower during winter, thus
increased pollen loads are observed near plantations
during the flowering period (Singh & Shahi 2008).
Using skin prick tests, Parui et al. (2002)haveshown
the presence of three potent allergenic proteins in the
pollen of C. siamea corresponding to the molecular
weight of 45, 53 and 97 kDa. Hussain et al. (2013)
extended the work of identifying pollen allergens from
three different Cassia species, namely C. fistula L.,
C. occidentalis L. and C. tora L., and showed that the
pollen of these plants are important sources of aero-
allergens near to plantations. However, to date, spe-
cific reports on the effects of air pollution on the
allergenicity of C. siamea pollen are not available.
The present study was therefore carried out to evalu-
ate the effect of air pollution on the allergenicity of C.
siamea pollen proteins.
Material and methods
Collection of Cassia siamea pollen
Two sites, one polluted and one non-polluted, were
selected according to the data on ambient air quality,
monitored by the Pune City Maharashtra Pollution
Control Board. Several hundred anthers of Cassia
siamea were collected from nearby plants for both
sites at the time of anthesis. Anthers were immedi-
ately transported to the laboratory, crushed to obtain
pollen and sieved through different grade of meshes
(100, 200 and 300 µm). Purity of pollen was checked
by microscope observation (shape and size; Parui
et al. 2002) and stored at –80 °C until the protein
extraction.
Pollen protein extraction and quantification
Pollen proteins were extracted as described following
the procedures of Noir et al. (2005). Briefly, 100 mg of
pollen from polluted and non-polluted sites were care-
fully ground in liquid nitrogen and mixed with Tris-
HCl buffer containing 50 mM Tris-HCl (pH 8.8),
EDTA 5 mM, 20 mM DTT, 100 mM KCl and
2mM PMSF. The mixture was further ground for
20 min and centrifuged at 12 000 RPM for 30 min at
4 °C. Total proteins in the supernatant were precipi-
tated with five volumes of acetone and incubated at –
20 °C for 2 h. After the incubation a protein pellet was
obtained by centrifugation at 12 000 RPM for 30 min
at 4 °C. The resulting protein concentration was quan-
tified using the Bradford method (Bradford 1976).
One-dimensional (1D)-gel electrophoresis
Sodium dodecyl sulphate polyacrylamide gel electro-
phoresis (SDS-PAGE) was performed as described
by Laemmli (1970) and Parui et al. (2002). Briefly,
the concentration of protein extracts from polluted
and non-pollutes sites was adjusted to 50 µg in 10 µl
mixed with sample buffer [0.06 M Tris-HCl (pH
6.8), 1% SDS, 10% sucrose, 0.5% β-mercaptoetha-
nol, 0.01% bromophenol blue] and heated at 100 °C
for 3 min. This mixture was loaded onto a 12%
SDS-PAGE gel along with the PageRuler Plus Pre-
stained Protein Ladder (Thermo Scientific, Wal-
tham, MA, USA) and electrophoresed in buffer
containing 0.05 M Tris-HCl, 0.192 M glycine and
0.1% SDS (pH 8.4) at 70 V for 150 min. After
electrophoresis, the gel was stained with 0.1% Coo-
massie Brilliant Blue R250 and destained with
methanol/acetic acid/water (4:1:5).
Animals used
All animal experiments were performed at the
National Toxicology Centre, Pune, India. The experi-
mental protocols (No. 201 on 19 January 2013) were
approved by the Institutional Animal Ethical Com-
mittee (IAEC) following the guideline of the Com-
mittee for the Purpose of Control and Supervision of
Experiments on Animals (CPCSEA), Ministry of
Environment, Forest and Climate Change, Govern-
ment of India, New Dehli, India. Total of 24 Swiss
albino mice (18–20 g) and six Wistar rats (150–170 g)
were obtained from the in-house animal facility of the
2V. Hinge et al.
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National Toxicology Centre, Pune. Throughout the
experiment, animals were fed with standard pellet
food and water ad libitum and maintained at an opti-
mum temperature and relative humidity with 12 h
light/dark cycle. The mice were randomly divided
into four experimental groups having six animals in
each group as follows:
Group 1. –Normal control (NC), received 100 µl/
ml saline.
Group 2. –Positive control (PC), received triple
antigen.
Group 3. –Polluted (P), received 100 µg/ml pro-
tein extract from the polluted site.
Group 4. –Non-polluted (NP), received 100 µg/
ml protein extract from the non-pol-
luted site.
Haematological assays
During the 14-day study, blood samples were with-
drawn at an interval of 0, 7 and 14 days from the
retro orbital plexus of every animal in each group.
Haematological assays were performed to record the
differences in percentage changes of each cell type in
response to allergenic challenge with polluted and
non-polluted pollen extracts. All haematological
assay were performed on Mindray BC-2800 Hema-
tological Analyser.
In vitro mast cell degranulation (MCD)
On day 14, sera from active anaphylaxis induced ani-
mals from all groups was obtained except for the posi-
tive control group. Animals in the positive control
group were injected intraperitoneally with a com-
pound 48/80 at the concentration of 8 mg per kg of
body weight. Mast cells were immediately obtained
from these animals and used as positive control. In
addition, mast cells were obtained from a donor Wis-
tar rat by intraperitoneally injecting 10 ml normal
saline and used for mast cell degranulation (MCD)
assays as described by Das and Chauhan (2013). Fifty
microlitres peritoneal mast cell suspension was mixed
with 100 µl blood sera of each animal from all four
groups and 10 µl antigen from the polluted and the
non-polluted sites, respectively. This mixture was
incubated for 3 min at 37 °C. After incubation, freshly
prepared 2% glutaraldehyde solution in 0.2 M sodium
phosphate buffer was added to the mixture and the cell
pellet was obtained by centrifugation at 300gfor
15 min. The remaining pellet was re-suspended in a
minimum amount of supernatant and smear was made
with 50 µl of the suspension. The smear was air-dried
and stained with 0.1% toluidine blue and mast cells
were counted. Final results were noted as a proportion
of morphologically altered cells to that of the total
number of cells counted using the following formula
%MCD ¼Number of degranulated mast cells
Total number of mast cells 100
Quantification of pollen allergen-specific antibodies
Three Wistar rats in each group (polluted and non-
polluted) were used for antibody production as per the
following dose: On the first day, a subcutaneous injec-
tion of 10 µl of pollen protein extract containing 10 μg
of total proteins + 490 µl saline + 500 µl FCA
(Freund’s Complete Adjuvant) was given to each ani-
mal in the group. On the seventh day, a booster dose
was given containing 5 µl of pollen protein extract
containing 10 μg of total proteins + 495 µl saline +
500 µl FCA. After the similar booster dose on the
tenth day, immune sera were collected and stored at
–20 °C and were used for confirming antibody pro-
duction using ELISA (enzyme-linked immunosorbent
assay). Levels of polyclonal antibody production were
measured by comparing antibody levels on the tenth
day of the immune sera to the pre-immune blood sera
from the first day as described in Sridhara et al.
(2002). Thus, 10 µg/ml pollen protein extract from
polluted and non-polluted samples were applied to a
microtiter plate by overnight incubation at 4 °C and
blocked with 100 µl 1% BSA (bovine serum albumin)
at room temperature for 1 h. The wells were washed in
0.05% Tween in PBS (phosphate-buffered saline)
three times and 1:100 dilution of primary antibodies
(anti-sera obtained from animals) was added to
respective wells and incubated for 3 h at 37 °C for
antibodies to bind to the antigen. After incubation, the
plate was washed with 0.05% Tween in PBS three
times to remove any unbound antibodies and 1:1000
dilution of anti-rat immunoglobulin G (IgG) antibo-
dies as secondary antibodies labelled with horseradish
peroxidase was added to each well. The plates were
incubated for 3 h at 37 °C for secondary antibodies to
bind to primary antibodies and washed with 0.05%
Tween in PBS three times to remove any unbound
antibodies. A substrate 2 mg/ml ABTS was added and
incubated for 10 min to form a colour complex and
optic density (OD) was taken at 410 nm using a
spectrophotometer. Fold increase in antibody produc-
tions was calculated using the following formula:
fold increase ¼OD of polluted sample
OD of non-polluted sample 100
Statistical analysis
Statistical analysis was performed in GraphPad Prism
(version 5). Analysis of variance (one-way ANOVA)
Allergenic Pollen Proteins of Cassia siamea 3
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test was used to compare both the blood cell types
and MCD among the four study groups.
Results
SDS-PAGE
An SDS-PAGE profile revealed a pattern of around
10 bands in both pollen extracts ranging in molecu-
lar weight 25–120 kDa. We observed the qualitative
difference in band intensities of polluted and non-
polluted pollen extracts, polluted pollen extracts
showed intense bands as compared to non-polluted
pollen extracts (Figure 1). Notably, a band at around
50 kDa was found to be highly intense in pollen
extract from the polluted site.
Polluted pollen extract has profound effect on
haematological counts
We applied one-way ANOVA test followed by
Tukey’s HSD (honest significant difference) post
hoc test to check whether pollen extracts from the
polluted site and the non-polluted site have any
effect on haematological cell counts (Figure 2,
Table I). We observed no effect on monocyte and
granulocyte counts throughout the study in any of
the animal groups. However, on day 14, the total
white blood cell (WBC) count (p-value = 0.00074)
and lymphocyte count (p-value = 0.00021) were
found significantly elevated in mice that received
pollen protein extract from the polluted site as com-
pared to the mice that received pollen protein extract
from the non-polluted site as well as with positive
and negative control.
Polluted pollen extract enhanced mast cell degranulation
(MCD)
Mast cells are among the first cells to come in con-
tact with inhaled particulate antigens and play an
important role in immediate hypersensitivity. There-
fore, we hypothesised that pollen extract collected
from the polluted site to have profound effects on
activation of mast cells. We observed that in normal
control group the percentage MCD was
17.1 ± 3.2%, whereas in the positive control group,
it was 83.0 ± 2.7%. Whereas, percentage MCD
observed with non-polluted and polluted pollen
extracts were 69.5 ± 2.9% and 79.0 ± 2.5%, respec-
tively. A t-test revealed that the percentage degranu-
lation of mast cell was significantly higher in the
pollen extract from polluted site than from non-pol-
luted (pvalue < 0.01; Figure 3,Table II). These
results suggest that pollution is responsible for the
release of more allergenic components from pollen
and subsequent rise in mast cell activation/degranu-
lation.
Antibodies against pollen allergen increased in polluted
samples
We next compared the fold increase in antibody
production of the polluted site and the non-polluted
site. We observed 2.98 and 2.25 fold increase in
antibody production for the protein samples col-
lected from the polluted site and the non-polluted
site, respectively. Thus, our results confirm that the
protein extract collected from the polluted site led to
a substantial increase in humoral immune response
compared to protein extract collected from the non-
polluted site.
Discussion
Our results demonstrate that pollen extract of Cassia
siamea collected from both the polluted site and the
non-polluted site elicits type I hypersensitivity. But
the extent of hypersensitivity observed in animals
receiving pollen extract from the polluted site was
higher. We base our conclusion on two facts: first,
pollen extracts from the polluted site evoked produc-
tion of IgE antibodies, and second, the MCD was
higher upon stimulation with pollen extract from this
Figure 1. SDS-PAGE protein profile of Cassia siamea –A. Mole-
cular weight marker. B. Pollen extract collected from polluted
site. C. Pollen extract collected from non-polluted site.
4V. Hinge et al.
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site. Both features are the hallmark of type I hyper-
sensitivity reactions marked by two phases: the sen-
sitisation phase and the effector phase. The
sensitisation phase is marked by the production of
allergen-specific IgE antibodies that eventually bind
to Fc receptors on mast cells. The effector phase is
the pathological phase, which is marked by the
degranulation of mast cells if a Fab region of anti-
bodies cross-linked with the specific allergen during
the subsequent exposure. Our results are in accor-
dance with previous studies reporting similar find-
ings with the pollen extract of other plant species
(Klein et al. 2000; Katayama et al. 2005). We further
speculate that this exacerbation of the effect of air
pollutant on allergenicity of pollen proteins could be
due to altered morphological features of pollen (such
as breaks in exine wall) leading to the excessive
release of pollen proteins as evident from our SDS-
PAGE analysis.
The severity and prevalence of pollen associated
respiratory allergic diseases is more common in
industrialised versus non-industrialised and urban
verses rural areas (Amato 2000). Several studies
conducted on pollen allergies from industrialised
and non-industrialised areas have highlighted the
possible role of air pollution in heightened pollinosis
for several plant species (Arbabian et al. 2011;
Armentia et al. 2002; Ghiani et al. 2012). The pollen
proteins from Cassia siamea, a plant which is abun-
dantly found in most parts of India, have been shown
to possess allergenic properties (Parui et al. 2002).
Out of the 11 bands observed on the 1D-gel, Parui
et al. (2002) found a total of nine protein bands to be
allergenic in susceptible individuals, of which three
bands were major and the rest were minor allergenic
proteins. In contrast to the 11 bands reported in an
earlier study, we were able to observe ten distinct
protein bands on the 1D-gel. This difference could
be attributed to the minor differences during the
protein extraction steps or due the differences in
sensitivity of staining dye used. Other members of
genus Cassia (C. tora, C. occidentalis and C. fistula)
were found to have 10–11 protein bands on the 1D-
gels, some of which found to be allergenic during the
skin prick test in susceptible individuals (Hussain
et al. 2013). In addition, a study based on light and
scanning electron microscopy has revealed morpho-
logical changes in pollen of two Cassia species
Figure 2. Variation in haematological parameters. A. Granulocyte. B. Lymphocytes. C. Monocytes. D. Total white blood cell (WBC) count
in different groups of animals. Abbreviations: NC, negative control; PC, positive control; NP, non-polluted pollen extract; P, polluted
pollen extract; at 0, 7 and 14 days.
Allergenic Pollen Proteins of Cassia siamea 5
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(C. siamea, C. fistula; Kalkar & Jaiswal 2014). The
study reports that the pollen from the polluted site
was shrunken with broken exine and deposition of
particulate matters on their surface as compared to
pollen from the non-polluted site. Hence, the result
presented here, are in accordance with these pre-
vious studies and suggests the allergenic nature of
C. siamea pollen, which further exacerbates due to
pollution.
Strictly controlled laboratory experiments are gen-
erally conducted to assess the effect of air pollutants
on allergenicity of pollen (Motta et al. 2006). How-
ever, extrapolation of the results of such experiments
to atmospheric in situ conditions is often limited due
to the use of a single pollutant at a time and use of
matured dehiscent pollen. In the present study, we
have adopted a more realistic field approach to reveal
the effect of air pollution on allergenicity of Cassia
siamea pollen protein extracts. Such an approach is
now becoming more popular in the relevant studies
(Armentia et al. 2002; Ghiani et al. 2012).
While the use of crude pollen protein extracts can
be viewed as a limitation of our study, the broader
aim of our work was to check the effect of air pollu-
tion on the allergenicity of pollen as a whole and not
of specific pollen protein derived from Cassia siamea.
Furthermore, many components in crude protein
extracts such as carbohydrates, lipids and leptins,
etc. may act as allergens themselves or as an adjuvant
to the allergenic action of the protein fraction (Bashir
et al. 2013). Thus, the heightened allergenic
response that we observed could be a cumulative
one largely due to the allergenic pollen proteins
and partly due to the other components of pollen
such as pollen associated lipid mediators (Gilles
et al. 2009).
Conclusion
Our results suggest an association between traffic
related or industry associated air pollution and the
allergenicity of pollen proteins. Considering the ever
increasing pollution and the extent of Cassia siamea
plantation in India, our result may facilitate policy-
makers to adopt new strategies to control air pollution
and associated allergies.
Figure 3. Percentage mast cell degranulation (MCD) observed per
group. Abbreviations: NC, negative control; PC, positive control;
NP, non-polluted pollen extract; P, polluted pollen extract.
Table I. Summary of a one-way ANOVA of different white blood cell (WBC) types with Tukey’s HSD post hoc test.
Cells and group SS MS F p-Value
Monocyte
NC 0.047778 0.023889 2.905405 0.085806*
PC 0.423333 0.211667 2.744957 0.096413*
NP 0.435039 0.21752 1.099509 0.360143
P 0.04 0.02 1.666667 0.222011
Lymphocyte
NC 0.347778 0.173889 0.289333 0.752849
PC 27.00444 13.50222 3.806183 0.046035**
NP 21.04651 10.52325 2.815067 0.09385*
P 59.61 29.805 15.60199 0.000217**
Granulocyte
NC 0.413333 0.206667 1.660714 0.223095
PC 4.853333 2.426667 1.599883 0.234526
NP 1.231111 0.615556 2.613208 0.106241
P 0.687778 0.343889 1.238991 0.317687
Total WBC
NC 29.03444 14.51722 5.799414 0.013621**
PC 55.76333 27.88167 1.575255 0.239342
NP 10.93525 5.467627 0.579745 0.572934
P 75.00444 37.50222 12.08753 0.000747**
*p-Value below 0.1. **p-Value below 0.05. Values in bold indicate p-values below 0.05.
6V. Hinge et al.
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Acknowledgements
The authors are thankful to Tania Paul (APT Research
Foundation), and the APT Research Foundation,
Pune, India, for helping in the animal experiments.
Disclosure statement
No potential conflict of interest was reported by the
authors.
References
Arbabian S, Doustar Y, Entezarei M, Nazeri M. 2011. Effects
of air pollution on allergic properties of wheat pollens (Tri-
ticum aestivum). Advances in Environmental Biology 5:
1339–1341.
Armentia A, Lombardero M, Callejo A, Barber D, Gil FJM,
Martín-Santos JM, Vega JM, Arranz ML. 2002.IsLolium
pollen from an urban environment more allergenic than rural
pollen?. Allergologia et Immunopathologia 30: 218–224.
doi:10.1016/S0301-0546(02)79124-6.
Bartra J, Mullol J, Del Cuvillo A, Dávila I, Ferrer M, Jáuregui I,
Montoro J, Sastre J, Valero A. 2007. Air pollution and aller-
gens. Journal of Investigational Allergology & Clinical Immu-
nology 17: 3–8.
Bashir MEH, Lui JH, Palnivelu R, Naclerio RM, Preuss D. 2013.
Pollen lipidomics: Lipid profiling exposes a notable diversity in
22 allergenic pollen and potential biomarkers of the allergic
immune response. PloS One 8: e57566. doi:10.1371/journal.
pone.0057566.
Behrendt H, Becker W-M. 2001. Localization, release and bioa-
vailability of pollen allergens: The influence of environmental
factors. Current Opinion in Immunology 13: 709–715.
doi:10.1016/S0952-7915(01)00283-7.
Behrendt H, Kasche A, VonEschenbach CE, Risse U, Huss-Marp J,
Ring J. 2001. Secretion of proinflammatory eicosanoid-like sub-
stances precedes allergen release from pollen grains in the initia-
tion of allergic sensitization. International Archives of Allergy
and Immunology 124: 121–125. doi:10.1159/000053688.
Bradford MM. 1976. A rapid and sensitive method for the quantisa-
tion of microgram quantities of protein utilizing the principle of
protein-dye binding. Analytical Biochemistry 72: 248–254.
Chow L-P, Chiu -L-L, Khoo K-H, Peng H-J, Yang S-Y, Huang
S-W, Su S-N. 2005. Purification and structural analysis of the
novel glycoprotein allergen Cyn d 24, a pathogenesis-related
protein PR-1, from Bermuda grass pollen. The FEBS Journal
272: 6218–6227. doi:10.1111/EJB.2005.272.issue-24.
Cortegano I, Civantos E, Aceituno E, Del Moral A, Lopez E,
Lombardero M, Del Pozo V, Lahoz C. 2004. Cloning and
expression of a major allergen from Cupressus arizonica pollen,
Cup a 3, a PR-5 protein expressed under polluted environment.
Allergy 59: 485–490. doi:10.1046/j.1398-9995.2003.00363.x.
D’Amato G. 2000. Urban air pollution and plant-derived respira-
tory allergy. Clinical and Experimental Allergy 30: 628–636.
doi:10.1046/j.1365-2222.2000.00798.x.
D’Amato G, Baena-cagnani CE, Cecchi L, Annesi-maesano I,
Nunes C, Ansotegui I, D’Amato M, Liccardi G, SofiaM,
Canonica WG. 2013. Climate change, air pollution and
extreme events leading to increasing prevalence of allergic
respiratory diseases. Multidisciplinary Respiratory Medicine
8: 1–9. doi:10.1186/2049-6958-8-12.
D’Amato G, Bergmann KC, Cecchi L, Annesi-Maesano I, San-
duzzi A, Liccardi G, Vitale C, Stanziola A, D’Amato M. 2014.
Climate change and air pollution: Effects on pollen allergy and
other allergic respiratory diseases. Allergo Journal International
23: 17–23. doi:10.1007/s40629-014-0003-7.
Das B, Chauhan R. 2013. Anti-histaminic and mast cell stabilizing
activity of the fern Lygodium flexuosum. International Journal of
Life Sciences Biotechnology and Pharma Research 2: 152–162.
Deguchi J, Hirahara T, Hirasawa Y, Ekasari W, Widyawaruyanti
A, Shirota O, Shiro M, Morita H. 2012. New tricyclic alka-
loids, cassiarins G, H, J, and K from leaves of Cassia siamea.
Chemical & Pharmaceutical Bulletin 60: 219–222.
doi:10.1248/cpb.60.219.
Diaz-Sanchez D, Penichet-Garcia M, Saxon A. 2000. Diesel
exhaust particles directly induce activated mast cells to degra-
nulate and increase histamine levels and symptom severity.
The Journal of Allergy and Clinical Immunology 106: 1140–
1146. doi:10.1067/mai.2000.111144.
Ghiani A, Aina R, Asero R, Bellotto E, Citterio S. 2012. Ragweed
pollen collected along high-traffic roads shows a higher aller-
genicity than pollen sampled in vegetated areas. Allergy 67:
887–894. doi:10.1111/all.2012.67.issue-7.
Gilles S, Mariani V, Bryce M, Mueller MJ, Ring J, Behrendt H,
Jakob T, Traidl-Hoffmann C. 2009. Pollen allergens do not
come alone: Pollen associated lipid mediators (PALMS) shift
the human immune systems towards a T
H
2-dominated
response. Allergy, Asthma, and Clinical Immunology 5: 1–6.
Table II. Summary of t-test between different groups for mast cell degranulation (MCD)
Paired differences
Pair Mean Standard deviation Standard error of the mean tdf p-Value
NC 17.09 3.18 1.31 −38.4347 10 3.39E-12
PC 82.96 2.7 1.11
NC 17.09 3.18 1.30 −35.2094 9 5.94E-11
P 78.94 2.49 1.11
NC 17.09 3.18 1.31 −29.9093 10 4.08E-11
NP 69.45 2.68 1.17
NP 69.45 3.18 1.31 −5.78288 9 0.000265
P 78.94 2.49 1.11
PC 82.96 2.7 1.11 8.355218 10 8.03E-06
NP 69.45 2.68 1.17
PC 82.96 2.7 1.11 2.526686 9 0.032411
P 78.94 2.49 1.11
Allergenic Pollen Proteins of Cassia siamea 7
Downloaded by [223.196.18.139] at 20:27 25 July 2016
Hussain MM, Mandal J, Bhattacharya K. 2013. Airborne load of
Cassia pollen in West Bengal, eastern India: Its atmospheric
variation and health impact. Environmental Monitoring and
Assessment 185: 2735–2744. doi:10.1007/s10661-012-2744-4.
Kalkar S, Jaiswal R. 2014. Effects of industrial pollution on pollen
morphology of Cassia species. International Journal of Life
Sciences 2: 17–22.
Katayama G, Nabe T, Fujii M, Kohno S. 2005. Mast cell hyper-
plasia induced by multiple challenge with cedar pollen in sen-
sitised guinea pigs. Inflammation Research 54: 370–374.
doi:10.1007/s00011-005-1369-2.
Klein JA, McEuen AR, Dijkstra MD, Buckley MG, Walls AF, Fok-
kens WJ. 2000. Basophil and eosinophil accumulation and mast
cell degranulation in the nasal mucosa of patients with hay fever
after local allergen provocation. The Journal of Allergy and Clinical
Immunology 106: 677–686. doi:10.1067/mai.2000.109621.
Kumar S, Kumar V, Prakash O. 2010. Antidiabetic and anti-
lipemic effects of Cassia siamea leaves extract in streptozotocin
induced diabetic rats. Asian Pacific Journal of Tropical Med-
icine 3: 871–873. doi:10.1016/S1995-7645(10)60209-X.
Laemmli UK. 1970. Cleavage of structural proteins during the
assembly of the head of bacteriophage T4. Nature 227: 680–
685. doi:10.1038/227680a0.
Manduzio H, Fitchette A-C, Hrabina M, Chabre H, Batard T,
Nony E, Faye L, Moingeon P, Gomord V. 2012. Glycopro-
teins are species-specific markers and major IgE reactants in
grass pollens. Plant Biotechnology Journal 10: 184–194.
doi:10.1111/pbi.2011.10.issue-2.
Motta AC, Marliere M, Peltre G, Sterenberg PA, Lacroix G.
2006. Traffic-related air pollutants induce the release of aller-
gen-containing cytoplasmic granules from grass pollen. Inter-
national Archives of Allergy and Immunology 139: 294–298.
doi:10.1159/000091600.
Noir S, Bräutigam A, Colby T, Schmidt J, Panstruga R. 2005.
A reference map of the Arabidopsis thaliana mature pollen
proteome. Biochemical and Biophysical Research Communi-
cations 337: 1257–1266.
Nsonde NGF, Banzouzi JT, Mbatchi B, Elion-Itou RDG, Etou-
Ossibi AW, Ramos S, Benoit-Vical F, Abena AA, Ouamba JM.
2010. Analgesic and anti-inflammatory effects of Cassia siamea
Lam. stem bark extracts. Journal of Ethnopharmacology 127:
108–111. doi:10.1016/j.jep.2009.09.040.
Parui S, Mondal AK, Mandal S. 2002. Identification of the aller-
genic proteins of Cassia siamea pollen. Grana 14: 37–41.
Radauer C, Breiteneder H. 2006. Environmental and occupa-
tional respiratory disorders pollen allergens are restricted to
few protein families and show distinct patterns of species dis-
tribution. Journal of Allergy and Clinical Immunology 117:
141–147. doi:10.1016/j.jaci.2005.09.010.
Reid CE, Gamble JL. 2009. Aeroallergens, allergic disease, and
climate change: Impacts and adaptation. EcoHealth 6: 458–
470. doi:10.1007/s10393-009-0261-x.
Rezanejad F, Majd A, Shariatzadeh SMA, Moein M, Aminzadeh
M, Mirzaeian M. 2003. Effect of air pollution on soluble
proteins, structure and cellular material release in pollen of
Lagerstroemia indica L. (Lytraceae). Acta Biologica Cracovien-
sia 45: 129–132.
Sénéchal H, Visez N, Charpin D, Shahali Y, Peltre G, Biolley JP,
Lhuissier F, Couderc R, Yamada O, Malrat-Domenge A,
Nhân PT, Poncet P, Sutra J-P. 2015. A review of the effects
of major atmospheric pollutants on pollen grains, pollen con-
tent, and allergenicity. The Scientific World Journal 2015:
1–29. doi:10.1155/2015/940243.
Singh AB, Shahi S. 2008. Aeroallergens in clinical practice of
allergyinIndia-ARIAAsiapacific work- shop report.
Asian Pacific Jorunal of Allergy and Immunology 26: 245–
256.
Songnuan W. 2013. Wind-pollination and the roles of pollen
allergenic proteins. Asian Pacific Journal of Allergy and Immu-
nology 31: 261–270.
Sridhara S, Kumar L, Verma J, Arora N, Gaur NS, Singh BP.
2002. Immunobiochemical characterization of Sorghum vul-
gare pollen allergens prevalent in Tropical countries. Indian
Journal of Allergy, Asthma and Immunology 16: 33–39.
Taketomi EA, Sopelete MC, Moreira PFS, Vieira FAM. 2006.
Pollen allergic disease: Pollen and its major allergens. Brazilian
Journal of Otorhinolaryngology 72: 562–567. doi:10.1016/
S1808-8694(15)31005-3.
Tehrani M, Sankian M, Assarehzadegan MA, Falak R, Noor-
bakhsh R, Moghadam M, Jabbari F, Varasteh A. 2011. Identi-
fication of a new allergen from Amaranthus retroflexus pollen,
Ama R 2. Allergology International 60: 309–316. doi:10.2332/
allergolint.10-OA-0279.
Zijverden MV, Pijl AVD, Bol M, Pinxteren FAV, Haar CD,
Penninks AH, Loveren HV, Pieters R. 2000. Diesel exhaust,
carbon black, and silica particles display distinct Th1 /Th2
modulating activity. Toxicology and Applied Pharmacology
168: 131–139. doi:10.1006/taap.2000.9013.
8V. Hinge et al.
Downloaded by [223.196.18.139] at 20:27 25 July 2016
