Aerobiological, clinical, and immunobiochemical studies on Alstonia scholaris pollen from eastern India

Aerobiological, clinical, and immunobiochemical studieson Alstonia scholaris pollen from eastern India

Mir Musaraf Hussain & Jyotshna Mandal & Kashinath Bhattacharya

Received: 12 April 2013 /Accepted: 10 August 2013 /Published online: 21 August 2013# Springer Science+Business Media Dordrecht 2013

Abstract Alstonia scholaris or Indian devil tree is acommon, evergreen, tropical tree of the Apocynaceaefamily. The objectives of this study were (a) to observethe seasonal variation of A. scholaris pollen in the atmo-sphere of an industrial and rural area of West Bengal,India by conducting a 2-year aerobiological survey witha Burkard personal volumetric sampler, (b) to study itsallergenicity in the local population by in vivo (skin-prick test) and in vitro tests (enzyme-linked immunosor-bent assay and dot blotting), (c) to identify the immuno-globulin E (IgE)-binding proteins present in the pollenextract (sodium dodecyl sulphate-polyacrylamide gelelectrophoresis and immunoblotting), (d) to study itschemical composition. A. scholaris pollen were presentin the air from September until November. Theycontained 14.3 % carbohydrate, 9.2 % lipid, and 4.3 %protein. Among 140 respiratory allergic local patients,28.57 % showed positive skin reaction to A. scholarispollen extract. Twelve protein bands in the range of

94.4–13.3 kDa were observed in the pollen extract.Seven IgE-binding proteins were found. Among them,one component of 29.9 kDa was the most important in A.scholaris pollen extract. This component could be puri-fied and would be helpful in the diagnosis and therapy ofA. scholaris pollen-susceptible patients.

Keywords Alstonia scholaris pollen . Aerobiology .

IgE-binding proteins . Respiratory allergy . Skin-pricktest

Introduction

Pollen grains as aeroallergens are well established fromacross the world and are reported to be a well-knowncause of pollinosis (Peternel et al. 2003; Singh andMathur 2012). In recent times, the incidence of allergicdiseases is about 10–30 %, out of which 50 % of theallergic patients are sensitized to specific proteins presentin the pollen (D'Amato et al. 1998; Bousquet et al. 2001;Xiao et al. 2013). Hence, regional aeroallergen monitor-ing would be imperative as it would provide usefulinformation to the local respiratory allergic sufferersand the clinicians (Hussain et al. 2013).

West Bengal is an eastern state of India and is thefourth most populous state (e-Census India 2011)(Fig. 1). The state has a rich vegetational diversity;primarily because of its climate with the Gangeticplains in the south and the Himalayas in the north.Aerobiological surveys undertaken in different parts ofWest Bengal have shown several pollen grains causing

Environ Monit Assess (2014) 186:457–467DOI 10.1007/s10661-013-3390-1

M. M. Hussain :K. Bhattacharya (*)Department of Botany, Visva-Bharati University,Santiniketan 731235, West Bengal, Indiae-mail: [email protected]

J. MandalDivision of Plant Biology, Bose Institute, 93/1 AcharyaPrafulla Chandra Road, Calcutta 700 009, India

Present Address:J. MandalInstitute of Evolutionary Biology and EnvironmentalStudies, University of Zurich, Winterthurerstrasse 190,8057 Zurich, Switzerland

pollinosis. They are pollen from the Poaceae family,Azadirachta indica, Areca catechu, Cassia sp., Caricapapaya, Cocos nucifera, Delonix regia, Peltophorumpterocarpum, Phoenix sylvestris, and others (Banikand Chanda 1992; Chakraborty et al. 1998a; Boral andBhattacharya 2000; Boral et al. 2004; Mandal et al.2006; Mandal et al. 2008a, b; Hussain et al. 2013).

Alstonia scholaris, commonly called Indian devil treeor Saptaparni of the family Apocynaceae, is an evergreentropical tree native to the Indian subcontinent, southeastAsia andAustralasia (Anonymous 1960). It is declared asthe state tree of theWest Bengal and is a common avenuetree all over the country. Studies indicate that Alstoniatrees are sensitive to air pollution (Tripathi et al. 2009;Jyothi and Jaya 2010; Nayek et al. 2011) and could act asbiomarkers of cadmium and lead in a polluted environ-ment (Shafiq et al. 2011).

The bark is primarily used in the treatment of malaria(Kumar et al. 2011; Sharma et al. 2012). Its leavescontain some alkaloid fraction which is known to haveanti-inflammatory and analgesic effects (Shang et al.2010) and broncho-vasodilatory activity (Channa et al.2005). According to Baliga (2012), there is a need formore research on the chemical and pharmacologicalproperties of A. scholaris. Flowers of A. scholaris areinsect pollinated and release about 5,000 pollen grainsper flower (Mondal et al. 1998). A. scholaris pollengrains were reported in the atmosphere of the capital cityof West Bengal, Calcutta (Mandal et al. 2006; Mandalet al. 2008a). They were known to initiate about 15–20 % sensitization in respiratory allergic patients using

skin-prick test (Banik and Chanda 1992; Mandal et al.2008a). Even though there are reports regarding theirpresence in the air and sensitization, there are no reportsso far regarding its immunoglobulin E (IgE) reactiveprotein components which may be responsible for im-mediate hypersensitivity reaction in susceptible subjects.

The objective of the present study is (1) to determinethe concentration and seasonal variation of A. scholarispollen in the atmosphere of an industrial (Durgapur) andrural area (Sriniketan) of West Bengal, India for twoconsecutive years (January 2003–December 2004), (2)to know the chemical composition of A. scholaris pol-len, (3) to observe the sensitization toA. scholaris pollenon the respiratory allergic patients by in vitro and in vivoassays, and (4) to identify the IgE reactive componentsof A. scholaris pollen for the first time.

Materials and methods

Aerobiological sampling

An aerobiological study was conducted for 2 years(January2003 toDecember2004) inDurgapur (an indus-trial township) and in Sriniketan (a semi-rural area),towns about 160 km and 213 km northwest ofCalcutta, in the state of West Bengal, in easternIndia, respectively. The sites (Fig. 2) have an averageelevation of 65 m (Durgapur, 23.48 N, 87.32 E) and49 m (Sriniketan, 23.68 N, 87.68 E). The flora aroundthe site is typical of that of the Chota Nagpur Plateau.A Burkard personal 1-day volumetric sampler (BurkardManufacturing Co. Ltd., Rickmansworth, Hertfordshire,UK)was placed at a height of 4m above the ground levelin the sampling area to monitor the air for the entiresampling period. It ran for 10min twice in a day between07:00 and 08:00 hours and 17:00 and 18:00 hours. Theexposed slides were scanned in high-resolution light mi-croscope (Leitz, Diaplan, Germany) at 400× as per theguideline of The British Aerobiology Federation (1995)to study the seasonal variation of relevant pollen grains.The daily pollen concentration was expressed as a num-ber of pollen grains per cubicmeter of air. The identifica-tionof air borne pollengrainswas donebycomparing thepollen types with the reference palynological collection(of the Palynology Section, Department of Botany, BoseInstitute, Calcutta) prepared from the local flora and by

Fig. 1 Flowering twig of A. scholaris

458 Environ Monit Assess (2014) 186:457–467

specific bibliographic consultations (Erdtman 1952;Faegri and Iversen 1964; Erdtman 1969; Huang 1972).

For scanning electron microscopic (SEM) studies,pollen grains suspended in a drop of water weredirectly transferred with a fine pipette to a metallicstub using double-sided cello tape and coated withgold in a sputtering chamber. Coating was restrictedto 150 A. The SEM examination was conducted on aQuanta™ Scanning Electron Microscope (FEI, Quanta

200, The Netherlands). The measurements are based onfive readings.

Preparation of pollen extract

The fresh pollen samples were collected from the ma-ture anthers of the A. scholaris flowers. The batchesused throughout the work contained <1 % non-pollenimpurities. They were defatted with diethyl ether and

Fig. 2 Geographical locations of the sampling sites (Durgapur and Sriniketan) in eastern India

Environ Monit Assess (2014) 186:457–467 459

then 10 g were extracted in phosphate-buffered saline(PBS; 0.1 M Na phosphate, 150 mM, pH 7.2) bycontinuous stirring at 4 °C for 16 h in 1:10 (w/v) ratio.After centrifugation at 14,000×g, the clear supernatantwas dialyzed and passed through a 0.22-μm Milliporefilter (Millipore Corp., USA). The filtrate was thenlyophilized and stored at −70 °C in aliquots.

Biochemical analysis

The total soluble protein content of the whole pollenextract of the A. scholaris pollen was determinedaccording to Lowry et al. (1951). Total lipid was esti-mated using the method of Itoh and Kaneko (1974).Total soluble carbohydrate content was determined bythe method of Duboise et al. (1956).

Skin prick test and sera collection

Skin tests were performed with a set of airborneallergenic pollen grains along with A. scholaris pollengrains on polysensitized patients, attending the AllergyUnit of The Institute of Child Health, Calcutta, selectedbecause they all had demonstrated clinical manifesta-tions of seasonal rhinoconjunctivitis and/or bronchialasthma. All these patients reported having A. scholaristree growing in their locality. The detailed history in-cluding age, sex, family history, onset, and duration ofsymptoms were recorded. The allergen extracts wereprepared by extracting the pollen at 95 % purity with0.1 M PBS (1:50, w/v) (Sheldon et al. 1967). Histaminephosphate (1 mg/mL) and PBS (1 mg/mL) were usedas positive and negative controls, respectively. Testswere performed with 20 μL pollen extract placed onthe ventral side of the forearm with a 26-G disposablehypodermic needle. According to International guide-lines, positivity was defined as mean wheal diameterat ≥3 mm compared with negative control (Dreborgand Frew 1993). The reaction was graded from +1 to+3 level (+1=erythema, 20 mm in diameter; +2=wheal and erythema, >20 mm in diameter; and +3=wheal at >3 mm and erythema) according to Stytiset al. (1982). Sera were collected from the patientswith +2- or +3-level skin reaction to whole pollenextract. None of these patients were receiving immu-notherapy during the time of sera collection. Controlsera were collected from non-atopic healthy volunteers

(confirmed by the negative skin reaction to the panelof allergenic pollen extracts and IgE-enzyme linkedimmunosorbent assay (ELISA), as described later)and having no history of allergic or systemic diseasesin them. The study was approved by the EthicsCommittee of the hospital and written informed con-sents were obtained from the subjects before theirparticipation.

ELISA

ELISA was performed to measure the pollen-specificIgE levels with the crude pollen extract against pooledpatients' sera according to Engvall and Pearlman(1971). Antihuman IgE-alkaline phosphatase conju-gate (Sigma Chemical Co., USA) was used as second-ary antibody and pNPP was used as substrate. Theabsorbance was measured with an ELISA reader at405 nm. The individual patient having P/N (the ratioof OD of patient sera with respect to control) value≥3.5 were selected for further study.

Sodium dodecyl sulphate-polyacrylamide gelelectrophoresis

The sodium dodecyl sulphate-polyacrylamide gel elec-trophoresis (SDS-PAGE) of crude pollen extract wascarried out on 12 % acrylamide gel using discontinu-ous buffer system according to Laemmli (1970). Theextraction of the soluble protein was made in a samplebuffer (0.5 M Tris–HCl, pH 6.8, 2 % SDS, 5 % glyc-erol, 5 % β-mercaptoethanol, and 0.1 % bromophenolblue dye) with heating for 5 min at 100 °C beforeloading. The gel run was made in Tris–glycine buffer(pH 8.3) with 0.1 % SDS. The gel was calibrated withmolecular weight markers.

IgE-specific dot blotting

The dot blotting was performed (Singh and Knox1985) on the pieces (3×1 in.) of PVDF membrane tocompare qualitatively the IgE reactivity of the pollenextract with the positive serum to that of the normalserum. Antihuman IgE-alkaline phosphatase conjugate(Sigma Chemical Co., USA) was used as a secondantibody. The phosphatase-positive spots were detectedusing NBT/BCIP as substrate


IgE-specific immunoblotting

The immunoblotting was performed onto PVDF mem-brane according to Sambrook et al. (1989). After thetransfer, one strip of the membrane was stained withCoomassie Brilliant Blue to ensure the complete trans-fer of the protein bands onto the PVDF membrane.These membranes were incubated with blocking so-lution (0.1 M PBS containing 3 % BSA) for over-night. Then the membrane was incubated withpooled patients' sera diluted (1:2, v/v) in blockingbuffer for 16 h at 4 °C, after which the membranewas washed in PBS containing 0.02 % Tween 20(PBST) for three times 10 min under gentle shaking.Then the membrane was incubated with antihuman

IgE-alkaline phosphatase conjugate in blocking so-lution (1:500) for 4 h at room temperature, and thiswas followed by 3×10 min washing with PBST. TheIgE-reactive bands were detected with NBT/BCIP assubstrate.

Results

Aerobiological survey

The pollen grains of A. scholaris are tricolporate, prolatespheroidal in shape, size±30.0/28.0 μm, Exine±2.5 μmthick, sexine±1.5 μm thick, with granulate surface(Fig. 3). The pollen season started from September and

Fig. 3 a Polar and b equa-torial views of A. scholarispollen grains under light mi-croscope. c Scanning elec-tron micrograph of A.scholaris pollen (×3,000)


extended for a short period up to November (Fig. 4). Atotal of 90 pollen grains per cubic meter were recordedin both the sites. During the peakmonth, the A. scholarispollen contributed 0.28 and 0.3 % of the total aeropollenload in Durgapur and Sriniketan, respectively.

Biochemical analysis

The chemical studies ofA. scholaris pollen type revealedthat the percentage of protein, carbohydrate, and lipidwas 4.31, 14.3, and 9.2 %, respectively.

Fig. 4 Averagemonthly concentrationof A. scholaris pollenin the two study sites(January 2003–December2004)

Table 1 Clinical characteristics of the patients selected for the study

Patient no. Age(years)/sex

Symptoms/clinicalhistory

Reaction inSPT

Specific IgE ELISA(P/N value)

FEV1 %predicted

Aeroallergen sensitization

1 44/M AR +++ 3.79 82.4 Cycas circinalis and Cocos nucifera

2 26/F AR+BA ++ 3.71 91.5 C. circinalis

3 37/M AR+BA +++ 3.01 78.4 C. nucifera, Carica papaya, and RN

4 42/M AR+BA +++ 3.84 87.3 Azadirachta indica and Cassia siamea

5 51/M AR+BA ++ 3.29 83.5 C. nucifera and RN

6 32/M AR ++ 3.54 79.7 A. indica and Saccharum officinarum

7 57/F AR ++ 3.52 90.3 A. indica, RS, and S. officinarum

8 46/F AR ++ 2.88 86.4 A. indica, C. nucifera, and HD

9 39/F AR ++ 3.63 90.8 Peltophorum pterocarpum and HD

10 28/M AR +++ 3.50 77.5 Alternaria alternata and C. nucifera

11 35/F AR+BA ++ 3.67 88.6 C. circinalis and S. officinarum

12 49/F AR+BA ++ 3.87 87.2 Eucalyptus citridora

13 38/F AR ++ 3.48 83.2 HD and P. pterocarpum

14 23/M AR +++ 3.26 88.3 P. pterocarpum, Delonix regia,and S. officinarum

15 48/M AR ++ 3.52 82.1 C. nucifera and C. papaya

16 42/F AR+BA +++ 3.62 87.2 Aspergillus flavus and HD

For immunoblotting, patient no. 1, 2, 4, 6, 7, 9, 10, 11, 12, 15, and 16 were chosen (P/N value>3.5)

AR allergic rhinitis, BA bronchial asthma, “+” no wheal and erythema of 20 mm in diameter, “++” wheal and erythema at >20 mm indiameter, “+++”wheal and erythema at >3 mm, “−” no wheal and no erythema, P/N ratio of optical density of patient serum with respectto control, FEV1 forced expiratory volume in 1 s, HD house dust, RN Rhizopus nigricans


Skin-prick test and ELISA

Skin-prick tests were performed with A. scholaris pollenextracts in a population of 140 local respiratory allergicpatients; 28.57 % (40 individuals) showed positive skinreaction exhibiting +1 or higher level of reaction. Amongthem, based on skin reactivity and high A. scholarispollen-specific IgE level, 16 patients were chosen forfurther sera collection along with control patients(Table 1). The serum of each patient was tested individ-ually against the pollen extracts to detect the presence ofIgE in the serum. The result of ELISAwas expressed interms of P/N value (ratio of OD of patient sera withrespect to control) (Fig. 5). The sera with P/N value morethan 3.5 was selected for further study (IgE-specificimmunoblotting).

IgE-specific dot blotting

IgE-specific dot blotting was performed on selected pa-tients to observe the IgE reactivity of A. scholaris pollenallergen. Here, allergic patient's sera gave dark spot

Fig. 5 Results ofELISAwith patient's seraagainst A. scholaris pollenextracts IgE specific

Fig. 6 Dot immunoblotting with A. scholaris pollen allergenicextract. a Positive and b negative spots

Fig. 7 SDS-PAGE at 12 % of total soluble protein of A.scholaris pollen. A Molecular weight marker. B Crude pollenextract of A. scholaris pollen


(Fig. 6a) with the pollen allergen, whereas the controlsera from the non-atopic patients showed a much lighterspot (Fig. 6b).

SDS-PAGE

SDS-PAGE at 12 % of crude pollen extract of A.scholaris pollen showed 12 major bands. The molecularweight of the 12 protein bands were 94.4, 89.1, 63.1,53.1, 47.3, 42.2, 37.6, 33.5, 29.9, 28.2, 15.8, and13.3 kDa (Fig. 7).

IgE-specific immunoblotting

In ELISA, 11 patients' sera showed P/N ratio greaterthan 3.5. These sera were used for IgE-specific immu-noblotting (Fig. 8; Table 2). With serum 1, four IgEreactive protein bands of A. scholaris having molecularweight of 53.1, 37.9, 29.9, and 15.8 kDa were found.With serum 2, three protein bands with molecularweight of 89.1, 42.2, and 29.9 kDa were found to beIgE reactive. Likewise, with serum 3, one band ofmolecular weight 29.9 showed IgE reactivity. TwoIgE reactive proteins of molecular weight of 29.9 and53.1 kDa were found against serum 4. With serum 5,five IgE reactive protein bands were seen with molec-ular weights of 94.4, 53.1, 42.2, 37.6, and 29.9 kDa.Four IgE reactive protein bands at 94.4, 42.2, 37.6, and29.9 kDa were found when blotted against serum 6.With serum 7, two IgE reactive proteins of molecularweight of 94.4 and 29.9 kDa were found. Three IgEreactive proteins of 94.4, 29.9, and 15.8 kDa werefound when blotted against serum 8. With serum 9,two protein bands of molecular weight of 42.2 and29.9 kDa were found to be IgE reactive. Two IgEreactive protein band of molecular weight of 37.6 and29.9 kDa were also found when immunoblottedagainst serum 10. Three IgE reactive proteins werefound against serum 11, their molecular weights being89.1, 37.6, and 29.9 kDa. Thus, only one band of A.scholaris pollen of molecular weight of 29.9 kDashowed IgE-binding epitope against all the sera of

Fig. 8 Immunoblot analysis of A. scholaris pollen extract withsensitive patient's sera. A Molecular weight marker. B Proteinbands transferred to PVDF membrane. C Control serum. Lanes1–11, sera of sensitive patients

Table 2 Major IgE reactive proteins in Alstonia scholaris pollen extract

Protein bands(kDa)

Serum1

Serum2

Serum3

Serum4

Serum5

Serum6

Serum7

Serum8

Serum9

Serum10

Serum11

Major IgE-binding proteincomponent

94.4 √ √ √ √89.1 √ √63.1

53.1 √ √ √47.3

42.2 √ √ √ √37.6 √ √ √ √ √33.5

29.9 √ √ √ √ √ √ √ √ √ √ √ √28.2

15.8 √ √13.3


patients showing positive reaction to SPT and thisprotein can be identified as major IgE reactive proteinof A. scholaris pollen.

Discussion

A. scholaris is a very common avenue tree in India. Itsentomophilous flowers have a very strong fragrance.Although it is insect pollinated, it releases a considerableamount of pollen grain during its short flowering seasonbetween September and November. This has been re-ported by a recent aerobiological survey in the capitalcity of West Bengal, from Calcutta (Mandal et al. 2006;Mandal et al. 2008a). Another, earlier report from WestBengal did observe the presence of A. scholaris pollengrains in the air (Mondal et al. 1998). This could bepossible as many new taxa are being introduced asavenue trees without considering their allergenic poten-tial (Mandal et al. 2008b; Majd et al. 2013).

In the present study, A. scholaris pollen grains havebeen studied for their seasonal periodicity and allergenic-ity and its IgE reacting protein components have beenidentified for the first time. Although the pollen grainswere seasonal, they caused significant allergenicity. In theclinical survey, A. scholaris pollen showed 28.57 % pos-itive skin reactions in 140 respiratory allergic patients.Previous studies on Cassia sp. (Hussain et al. 2013), D,regia (Mandal et al. 2009), P. pterocarpum (Mandal et al.2009), Lantana camara (Mandal et al. 2012), haveshown similar results as their pollen were seasonal toobut still caused high sensitivity among the local respira-tory allergic subjects. Banik and Chanda (1992) andMandal et al. (2008a, b) have shown 22 and 15.7 %positivity in skin-prick test with A. scholaris pollen aller-gen extract. A. scholaris pollen contained high carbohy-drate (14.3 %). Similar results have been seen in theCassia fistula and Cassia tora pollen grains (Hussainet al. 2013). The results of the 12 % SDS-PAGE analysisof the soluble protein of A. scholaris pollen exhibited 12protein bands in the range of 94.4 to 13.3 kDa. IgE-immunoblotting results with 11 individual patient sera(P/N value>3.5) revealed seven IgE-binding protein com-ponents among which the band of 29.9 kDa was the mostimportant. Allergen characterization has been reportedfrom the pollen of Catharanthus roseus from the samefamily, Apocyanaceae, where three IgE reactive proteinswere found to be important (40–66 kDa)(Ghosh et al.2007). These findings open the scope for further work to

determine cross-reactive protein among the phylogeneti-cally related species. From West Bengal, pollen typesfrom several trees such as Areca (Chakraborty et al.2009), Azadirachta (Karmakar and Chatterjee 1994),Borassus (Chakraborty et al. 1998b), Cassia (Paruiet al. 2002), Cycas circinalis (Hussain et al. 2012),Delonix (Mandal et al. 2009), and Peltophorum(Mandalet al. 2011) were immuniobiochemically studied for al-lergen immunotherapy.

In conclusion, it could be said that A. scholaris pollenact as significant aeroallergens of the study area of WestBengal. Its protein profile and IgE reactive protein com-ponents have been identified and studied for the first time.Detailed studies on the purification of these IgE-bindingcomponents shall be useful for the immunotherapy of thesusceptible patients.

Acknowledgments Thanks are due to the Council of Scientificand Industrial Research, India for providing fellowship to the firstauthor (MMH). Authors are also thankful to Dr. I. Roy, Dr. S.Chatterjee, and the staff members of the Institute of Child Health,Kolkata for their kind help in performing clinical investigation. Mr.Chanchal Chakraborty of the Department of Plant Biology, BoseInstitute, Calcutta, India is also acknowledged for his technicalassistance in SEM.

References

Anonymous (1960). The Wealth of India: raw materials, vol43. New Delhi, India: Council of Scientific and IndustrialResearch

Baliga, M. S. (2012). Review of the phytochemical, pharmaco-logical and toxicological properties of Alstonia scholarisLinn. R. Br (Saptaparna). Chinese journal of integrative med-icine. doi:10.1007/s11655-011-0947-0.

Banik, S., & Chanda, S. (1992). Airborne pollen survey of CentralCalcutta, India, in relation to allergy. Grana, 31, 72–75.

Boral, D., & Bhattacharya, K. (2000). Aerobiology, allergenicityand biochemistry of three pollen types in Berhampore townof West Bengal, India. Aerobiologia, 16, 417–422.

Boral, D., Chatterjee, S., & Bhattacharya, K. (2004). The occurrenceand allergising potential of airborne pollen in West Bengal,India. Annals of Agricultural and Environmental Medicine, 11,45–72.

Bousquet, J., Cauwenberge, P., & Khaltaev, N. (2001). Allergicrhinitis and its impact on asthma. ARIA workshop report.Journal of Allergy and Clinical Immunology, 108, 148–S217.

Chakraborty, P., Gupta Bhattacharya, S., Chakraborty, C., Lacey,J., & Chanda, S. (1998a). Airborne allergenic pollen grainson a farm in West Bengal, India. Grana, 37, 53–57.

Chakraborty, P., Chowdhury, I., Gupta-Bhattacharya, S., Roy, I.,Chatterjee, S., & Chanda, S. (1998b). Aerobiologic, clinicaland immunochemical studies on Borassus flabellifer pollen:


http://dx.doi.org/10.1007/s11655-011-0947-0

an evidence for a 90 kD allergen. Annals of Allergy, Asthmaand Immunology, 80, 311–317.

Chakraborty, P., Mandal, J., Sarkar, E., Chowdhury, I., & Gupta-Bhattacharya, S. (2009). Clinico-immunochemical studies onairborne Areca catechu L. pollen, a probable risk factor inemergency asthma hospitalization from Eastern India.International Archives of Allergy and Immunology, 149,305–314.

Channa, S., Dar, A., Ahmed, S., & Atta-Ur-Rahman. (2005).Evaluation of Alstoniascholaris leaves for broncho-vasodilatoryactivity. Journal of Ethnopharmacology, 97, 469–476.

D'Amato, G., Spieksma, F. T., & Liccardi, G. (1998). Pollen-related allergy in Europe. Allergy, 53, 567–578.

Dreborg, S., & Frew, A. J. (1993). Allergen standardization andskin tests. Allergy, 48(14), 49–82.

Duboise, M., Hamilton, J. K., Rebers, P. A., & Smith, F. (1956).Colorimetric method for sugar and related substances.Annals of Chemistry, 28, 350–356.

e-CENSUS India. (2011). Available from http://www.censusindia.gov.in/.

Engvall, E., & Pearlman, P. (1971). Enzyme linked immunosor-bent assay (ELISA): qualitative assay of immuno-globulinG. Immunochemistry, 8, 871–879.

Erdtman, G. (1952). Pollen morphology and plant taxonomy.Stockholm: Alonquist and Wiksells.

Erdtman, G. (1969). Handbook of palynology. Copenhagen:Munksgaard.

Faegri, K., & Iversen, J. (1964). Textbook of pollen analysis.Copenhagen: Munksgaard.

Ghosh, D., Roy, I., Chanda, S., & Gupta-Bhattacharya, S. (2007).Allergy to periwinkle pollen (Catharanthus roseus G. Don.).Annals of Agricultural and Environmental Medicine, 14, 39–43.

Huang, T. C. (1972). Pollen flora of Taiwan (p. 297). Taipei:Botany Deparment National Taiwan University.

Hussain, M. M., Chakraborty, P., & Bhattacharya, K. (2012).Pollen grains of queen sago (Cycas circinalis L.), a sourceof aeroallergen from West Bengal, India: an immunochem-ical approach. Aerobiologia, 28, 39–47.

Hussain, M.M., Mandal, J., & Bhattacharya, K. (2013). Airborneload of Cassia pollen in West Bengal, eastern India: its atmo-spheric variation and health impact.EnvironmentalMonitoringand Assessment, 185, 2735–2744.

Itoh, T., & Kaneko, H. (1974). Yeast lipids in species variation. I.Simple method for estimating the cellular lipids. Yakugaku,23, 350–354.

Jyothi, S. J., & Jaya, D. S. (2010). Evaluation of air pollutiontolerance index of selected plant species along roadsides inThiruvananthapuram, Kerala. Journal of EnvironmentalBiology, 31(3), 379–386.

Karmakar, P. R., & Chatterjee, B. P. (1994). Isolation and char-acterization of two Ig-E reactive proteins from Azadirachtaindica pollen.Molecular and. Cellular Biochemistry, 131(1),87–96.

Kumar, P., Misra, C. S., James, J., Lipin, D. M. S., Veettil, A. K.T., & Thankamani, V. (2011). Ethnobotanical and pharmaco-logical study of Alstonia (Apocynaceae)—a review. Journalof Pharmaceutical Sciences and Research, 3, 1394–1403.

Laemmli, U. K. (1970). Cleavage of structural protein duringassembly of the head of bacteriophage T4. Nature, 227,680–684.

Lowry, C. H., Rosenbrough, M. J., Farr, A. L., & Randall, R. I.(1951). Protein measurement with Folin phenol reagent.Journal of Biological Chemistry, 193, 256–275.

Majd, A., Rezanejad, F., Irian, S., & Mousavi, F. (2013).Hypersensitivity to Ailanthus altissima (tree of heaven) pollen:identification of a major IgE-binding component. Aerobiologia.doi:10.1007/s10453-013-9290-6.

Mandal, J., Chanda, S., & Gupta-Bhattacharya, S. (2006). Currentstatus of airborne pollen grains in Calcutta with specialreference to their allergenic significance. Indian Journal ofAerobiology, 19, 19–30.

Mandal, J., Chakraborty, P., Roy, I., Chatterjee, S., & Gupta-Bhattacharya, S. (2008a). Prevalence of allergenic pollengrains in the aerosol of the city of Calcutta, India—a two yearperspective study. Aerobiologia, 24, 151–164.

Mandal, J., Roy, I., Chatterjee, S., & Gupta Bhattacharya, S.(2008b). Aerobiological and in vitro studies on pollen grainsof two dominant avenue trees from Kolkata (Calcutta), India.Journal of Investigational Allergology and ClinicalImmunology, 18, 22–30.

Mandal, J., Manna, P., Chakraborty, P., Roy, I., & Gupta-Bhattacharya, S. (2009). Clinical and immunobiochemicalcharacterization of airborneDelonix regia (Gulmohar tree)pollen and cross-reactivity studies with Peltophorumpterocarpum pollen: two dominant avenue trees from easternIndia. Annals of Allergy Asthma and Immunology, 103, 515–524.

Mandal, J., Roy, I., & Gupta-Bhattacharya, S. (2011). Clinicaland immunobiochemical characterization of airbornePeltophorum pterocarpum (yellow Gulmohar tree) pollen:a dominant avenue tree of India. Annals of Allergy Asthmaand Immunology, 106, 412–420.

Mandal, J., Chakraborty, P., Roy, I., & Gupta-Bhattacharya, S.(2012). Aerobiological, clinical and immunobiochemicalstudies on Lantana camara pollen and cross reactivity withotherVerbenaceae pollen species.Aerobiologia, 28, 107–119.

Mondal, A. K., Mondal, S., & Mandal, S. (1998). Pollen pro-duction in some plant taxa with supposed role in allergy ineastern India. Aerobiologia, 14, 397–403.

Nayek, S., Satpati, S., Gupta, S., Saha, R. N., & Datta, J. K.(2011). Assessment of air pollution stress on some com-monly grown tree species in industrial zone of Durgapur,West Bengal, India. Journal of Environmental Science &Engineering, 53, 57–64.

Parui, S., Mondal, A. K., & Mandal, S. (2002). Identification ofthe allergenic proteins of Cassia siamea pollen. Grana, 41,39–43.

Peternel, R., Culig, J., Mitic, B., Vukusic, I., & Sostar, Z. (2003).Analysis of airborne pollen concentrations in Zagreb, Croatia.Annals of Agricultural and Environmental Medicine, 10, 107–112.

Sambrook, J., Fritsh, E. F., & Maniatis, T. (1989). Molecularcloning. A laboratory manual (2nd ed., Vol. 18, p. 75). ColdSpring Harbor: Cold Spring Harbor Laboratory Press.

Shafiq, M., Iqbal, M. Z., Saeed, A. M., & Athar, M. (2011).Alstonia scholaris R. Br. and Cassia siamea Lamk as pos-sible biomonitors of lead and cadmium in the pollutedenvironment of Karachi city, Pakistan. Journal of AppliedBotany and Food Quality, 84, 95–101.

Shang, J. H., Cai, X. H., Feng, T., Zhao, Y. L., Wang, J. K., Zhang,L. Y., Yan, M., & Luo, X. D. (2010). Pharmacological


http://www.censusindia.gov.in/

http://www.censusindia.gov.in/

http://dx.doi.org/10.1007/s10453-013-9290-6

evaluation of Alstonia scholaris: anti-inflammatory and an-algesic effects. Journal of Ethnopharmacology, 27, 174–181.

Sharma, B. K., Deb, S., & Shankar, R. (2012). Antimalarial plantsof northeast India: an overview. Journal of Ayurveda andIntegrative Medicine, 3, 10–16.

Sheldon, J. M., Lovell, R. G., & Mathews, K. D. (Eds.). (1967). Amanual of clinical allergy. Philadelphia: W.B. Saunders Co.

Singh, M. B., & Knox, R. B. (1985). Grass pollen allergens:antigenic relationships detected using monoclonal antibodiesand dot blotting immunoassay. International Archives ofAllergy and Applied Immunology, 78, 300–304.

Singh, A. B., & Mathur, C. (2012). An aerobiological perspec-tive in allergy and asthma. Asia Pacific Allergy, 2, 210–222.

Stytis, D. P., Stobo, J. D., Fudenberg, H., & Wells, J. V. (1982).Basic and clinical immunology (4th ed., p. 409). Singapore:Lange Medical.

The British Aerobiology Federation. (1995). A guide to trappingand counting. West Malling: Kimberly Clarke Ltd.

Tripathi, A., Tiwari, P. B., & Singh, D. M. (2009). Assessment ofair pollution tolerance index of some trees in Moradabadcity, India. Journal of Environmental Biology, 30(4), 545–550.

Xiao, X., Fu, A., Xie, X., Kang, M., Hu, D., Yang, P., & Liu, Z.(2013). An investigation of airborne allergenic pollen at differ-ent heights. International Archives of Allergy and Immunology,160, 143–151.


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Aerobiological, clinical, and immunobiochemical studies on Alstonia scholaris pollen from eastern India