Immunopharmacology and In?ammation

Suppression of ovalbumin-induced Th2-driven airway in?ammation by β-sitosterol

in a guinea pig model of asthma

Shailaja G. Mahajan, Anita A. Mehta ?

Department of Pharmacology, L.M. College of Pharmacy, Ahmedabad, Gujarat, India

abstract article info

Article history:

Received 21 June 2010

Received in revised form 1 September 2010

Accepted 23 September 2010

Available online 12 October 2010

Keywords:

Asthma

β-sitosterol

Cytokines

Moringa oleifera

Ovalbumin

In the present study, the ef?cacy of β-sitosterol isolated from an n-butanol extract of the seeds of the plant

Moringa oleifera （Moringaceae） was examined against ovalbumin-induced airway in?ammation in guinea

pigs. All animals （except group I） were sensitized subcutaneously and challenged with aerosolized 0.5%

ovalbumin. The test drugs, β-sitosterol （2.5 mg/kg） or dexamethasone （2.5 mg/kg）, were administered to

the animals （p.o.） prior to challenge with ovalbumin. During the experimental period （on days 18, 21, 24

and 29）, a bronchoconstriction test （0.25% acetylcholine for 30 s） was performed and lung function

parameters （tidal volume and respiration rate） were measured for each animal. On day 30, blood and

bronchoalveolar lavaged ?uid were collected to assess cellular content, and serum was collected for

cytokine assays. Lung tissue was utilized for a histamine assay and for histopathology. β-sitosterol

signi?cantly increased the tidal volume （Vt） and decreased the respiration rate （f）ofsensitizedand

challenged guinea pigs to the level of non-sensitized control guinea pigs and lowered both the total and

differential cell counts, particularly eosinophils and neutrophils, in blood and bronchoalveolar lavaged

?uid. Furthermore, β-sitosterol treatment suppressed the increase in cytokine levels （TNFα, IL-4 and IL-5）,

with the exception of IL-6, in serum and in bronchoalveolar lavaged ?uid detected in model control

animals. Moreover, treatment with β-sitosterol protected against airway in?ammation in lung tissue

histopathology. β-sitosterol possesses anti-asthmatic actions that might be mediated by inhibiting the

cellular responses and subsequent release/synthesis of Th2 cytokines. This compound may have

therapeutic potential in allergic asthma.

© 2010 Elsevier B.V. All rights reserved.

1. Introduction

Allergic asthma, which affects an estimated 100 million

individuals worldwide （Cohn and Ray, 2000）, is caused by chronic

airway in?ammation associated with IgE- synthesis and subsequent

Th2 （T-helper type-2 cell）-responses （Barnes et al., 1998）. Asthma

is characterized by airway in?ammation and airway hyper-

responsiveness to the spasmogens such as histamine, acetylcholine

and 5-hydroxytryptamine （5-HT） （Saria et al., 1983）. The patho-

physiological hallmark of asthma is the in?ltration of in?ammatory

cells, including eosinophils （Wardlaw et al., 1988）, neutrophils,

lymphocytes and macrophages （Bousquet et al., 2000）. These cells

release various in?ammatory mediators, including histamine （Liu

et al., 1991）andcytokines（Chung and Barnes, 1999）.

Numerous studies have also found elevated levels of histamine in

the plasma of patients with asthma （Ind et al., 1983）; similar effects

have been noted in the lung tissues （Bartosch et al., 1932） of guinea

pigs. Elevated levels of tumor necrosis factor （TNF）-α （Coker and

Laurent, 1998）, interleukin （IL）-4 （Gharaee-Kermani et al., 2001）, IL-5

（Egan et al., 1996） and IL-6 （Elias et al., 1997） have been noted in

bronchoalveolar lavaged ?uid from asthmatic patients after allergen

challenge.

Phytosteroids possesses interesting medicinal and pharmacolog-

ical activities （Dinan et al., 2001）. Chemically, these compounds’

structures are steroid-like, and modern clinical studies have shown

that plants containing such steroids are anti-in?ammatory agents.

Among the phytosteroids, β-sitosterol is found in a variety of plants,

including Moringa oleifera Lam. （Moringaceae）. In our previous pre-

clinical studies, we reported the anti-arthritic （Mahajan et al.,

2007a）, anti-anaphylactic （Mahajan and Mehta, 2007） and immu-

nosuppressive （Mahajan and Mehta, 2010） activity of ethanolic

extract from seeds of the plant. Furthermore, we evaluated the

ef?cacy of ethanolic extract in chemical-induced, immune-mediated

in?ammatory responses in rats （Mahajan et al., 2007b）andin

ovalbumin-induced airway in?ammation in guinea pigs （Mahajan

and Mehta, 2008）.We established that the extract inhibits cytokines

and subsequently prevents eosinophilia and neutrophilia. Further-

more, to obtain a potent extract,we fractionated the ethanolic extract

using n-butanol as a solvent and again con?rmed the extract's

European Journal of Pharmacology 650 （2011） 458–464

? Corresponding author. Department of Pharmacology, L.M. College of Pharmacy,

Ahmedabad 380 009, Gujarat, India. .: +91 79 26302746; +91 79 26304865.

addresses: mahajan.shailaja （S.G. Mahajan）,

dranitalmcp （A.A. Mehta）.

0014-2999/$ – see front matter © 2010 Elsevier B.V. All rights reserved.

doi:10.1016/j.ejphar.2010.09.075

Contents lists available at ScienceDirect

European Journal of Pharmacology

journal homepage: www.elsevier.com/locate/ejpharactivity in the ovalbumin-induced guinea pig model of allergic

asthma, where it signi?cantly lowered cytokine and histamine levels

（Mahajan et al., 2009）. Our preliminary clinical studies also showed a

decrease in the severity of asthma symptoms and improvement in

peak expiratory ?ow rate in patients with asthma （Agrawal and

Mehta, 2008）.

Collectively, results from our preceding studies demonstrated that

the individual extract（s） could signi?cantly downregulate the

synthesis and/or the release of cytokines and histamine but did not

alter the lung function parameters. Furthermore, to determine the

extract components, the quantitative estimation was carried out for

marker compounds present in the plant including β-sitosterol. The

ef?cacy of β-sitosterol was evaluated against histamine- and

acetylcholine-induced bronchospasm in guinea pigs. β-sitosterol

produced a signi?cant increase in pre-convulsion dyspnea time

against both the spasmogens compared to control animals, indicating

the possible bronchodilatory activity of β-sitosterol. Therefore, to

verify our previous results and to determine the constituent of the

extract/fraction responsible for the anti-asthmatic activity, we

conducted the present study using a compound; β-sitosterol.

2. Materials and methods

2.1. Reagents

All solvents used in the study were of analytical grade. Diethyl

ether, ethyl acetate, n-butanol, petroleum ether （60–80 °C）, hexane,

hydrochloric acid, n-heptane,methanol and toluene were purchased

from Rankem （New Delhi, India）. Chloroform and carbón tetra

chloride （CCl4） were purchased from Finar Chemicals Pvt. Ltd.

（Ahmedabad, India）. Silica gel （60–120 mesh）, formaldehyde

solution and aluminium hydroxide gel were obtained from S. D.

Fine Chemicals （Mumbai, India）. β-sitosterol, acetylcholine, hista-

mine and ovalbumin （Grade V） were purchased from Sigma-Aldrich

（St. Louis, MO, USA）. Dexamethasone was obtained as a gift sample

from Zydus Research Pvt. Ltd. （Ahmedabad, India）. Perchloric acid,

NaOH and NaCl were purchased from Ranbaxy Fine Chemicals Ltd.

（New Delhi, India）.Thin layer chromatography （TLC） plates silica gel

（GF254） was purchased from Merck （Darmstadt, Germany）. Keta-

mine was purchased from Themis Medicare Ltd. （Goregaon, India）.

Xylazine was obtained from Five Star Pharmaceuticals （Ahmedabad,

India）. Kits for TNFαand IL-5were purchased fromPro LabMarketing

Pvt. Ltd. （New Delhi, India）, and for IL-4 and IL-6 from Cusabio

Biotech Co., Ltd. （Newark, DE, USA）.

2.2. Plant material

Seeds of M. oleifera were obtained from a commercial supplier in

Ahmedabad and were identi?ed and authenticated by the Depart-

ment of Pharmacognosy, L. M. College of Pharmacy, Ahmedabad,

India. A voucher specimen was deposited in the herbarium of the

same department.

2.3. Extraction and isolation of compound

One kilogram of course powder of dried seeds of M. oleifera was

defatted using petroleum ether （60–80 °C）, and then, it was

exhaustively extracted using 95% （v/v） ethanol （500 ml） in a soxhlet

extractor at 55 °C for 6 h. The resulting extract was further

fractionated using the solvent n-butanol. The n-butanol fraction was

?ltered, and the solvent was removed under vacuum. The remaining

n-butanol fraction was then partitioned with CCl4. The CCl4 fraction

（25 g） was loaded on a preparative TLC plate of silica gel （F254） using

the solvent system methanol–toluene–ethyl acetate （1:8:1）. The

fraction band was scraped, collected from TLC plates and dissolved in

methanol concentrated to dryness （yield 4.21 g）. The powder （1 g）

was chromatographed for puri?cation on a silica gel and eluted with a

hexane–ethyl acetate solvent system. The solvent system was

employed starting with hexane （100%） and then increasing the

polarity of the elution solvent with ethyl acetate by 10% （v/v）

increments until pure isolates were obtained. Fractions of 20 ml were

collected. The progress of separation for β-sitosterol was monitored

by TLC using the solvent system of methanol–toluene–ethyl acetate

（1:8:1）. Fractions of hexane and ethyl acetate elutants containing β-

sitosterol were pooled and concentrated to dryness, and the presence

of β-sitosterolwas con?rmed by co-chromatographywith standard β-

sitosterol. The yield of pure β-sitosterol was 0.82 g; hence, the total

yield from the n-butanol fraction was 0.35% （w/w） of the weight of

starting material （Guevara et al., 1999）.

2.4. Characterization of the isolated compound

The melting point of the isolated compound was measured on

Model II/III （Veego Instruments Corporation, Mumbai, India）. The UV

absorption spectrumof the isolated sample inmethanol was recorded

on a UV/Vis spectrophotometer [UV 1601, Shimadzu （Asia Paci?c） Pvt.

Ltd., Sydney, Australia]. Infrared （IR） （Spectrum GX Perkin-Elmer,

USA） and mass spectra （Shimadzu LCMS model 2010, Columbia, USA）

were recorded. The isolated compound was dissolved in CDCl3, and

1

H-NMR and 13

C-NMR spectra were also obtained for the structure

elucidation of the compound （Brucker Advance II 400 NMR Spec-

trometer, Billerica, MA, USA）.

2.5. Animals

Speci?c pathogen-free male Dunkin–Hartley guinea pigs （300–

500 g） were housed in a climate-controlled room （temperature 22±

1 °C; relative humidity 55±5%） on a 12-h light–dark cycle. Animals

had access to standard pellet diet （certi?ed Amrut brand rodent feed,

Pranav Agro Industries, Pune, India） and ?ltered tap water ad libitum.

All experiments were carried out with strict adherence to ethical

guidelines and were conducted according to the protocol approved by

the Institutional Animal Ethics Committee （IAEC） and according to

Indian norms set by the Committee for the Purpose of Control and

Supervision of Experiments on Animals （CPCSEA）, New Delhi, India.

Throughout the entire study period, the animals were monitored for

growth, health status, and food intake capacity to be certain that they

were healthy.

2.6. Sensitization and treatment of animals

Animals were divided into four groups （n=6/group）. Group I,

non-sensitized controls, received distilled water （2.5 ml/kg）; group II,

the model control group, was ovalbumin sensitized and then received

distilled water （2.5 ml/kg） supplemented with dimethyl sulphoxide

（DMSO; vehicle used for dexamethasone [DXM] and β-sitosterol

treatments）; group III, the reference standard group, was ovalbumin

sensitized and then received DXM （2.5 mg/kg）; group IV, the

experimental group, was ovalbumin sensitized and then received β-

sitosterol （2.5 mg/kg）. All animals （except group I） were sensitized

and challenged as previously described （Duan et al., 2003）. Brie?y,

animalswere injected, s.c., with 100 μg of ovalbumin （which had been

adsorbed onto 100 mg of aluminum hydroxide in saline） on day 0 as

the ?rst sensitization. Boosting was then carried out using the same

dose of antigen two weeks later （i.e., on day 14）. The daily doses of

drug or vehicle were initiated on day 18 and continued until day 29;

they were administered orally.

2.7. Ovalbumin exposure

On days 18–29, 2.5 h after receiving the appropriate drug or vehicle

treatment, the animals were challenged with 0.5% （w/v） of aerosolized

459 S.G. Mahajan, A.A. Mehta / European Journal of Pharmacology 650 （2011） 458–464ovalbumin for 10min. For the challenge, conscious animalswere placed

into a plastic circular chamber （diameter=70 cm, and height=40 cm）

connected to a nebulizer （CX4-Omron Healthcare Company Ltd., Kyoto,

Japan）. Animals in the non-sensitized group （group I） were exposed to

aerosolized saline using the same protocol.

2.8. Lung function and bronchoconstriction test

On days 18, 21, 24, and 29, 2 h after a 10-min ovalbumin exposure,

the tidal volume （ml/s） and respiration rate （breaths/min） of the

animals were measured with a Respiromax （Model no.070613-1,

Columbus Instruments, OH, USA） before and after an acetylcholine-

induced bronchoconstriction test. All ovalbumin-sensitized hosts

were exposed （in a conscious state） to a 0.25% （w/v） acetylcholine

solution for 30 s using a nebulizer connected to the animal holder.

Guinea pigs in the non-sensitized control group were exposed to

normal saline in place of acetylcholine.

2.9. Cellular count and serum preparation

On day 30, blood （3 ml） was collected from each animal under

light ether anesthesia. Each sample was then divided into two

portions. The ?rst aliquot （2.5 ml） was placed in a non-heparinized

tube for serum separation; the isolated serum was stored at −80 °C

until quantitative determination of cytokines. The second portion

（0.5 ml） was placed in a heparinized tube and used for leukocyte

counts. Each sample was centrifuged at 500×g for 10 min at 4 °C; the

cells in the pellet were washed in 0.5-ml saline and total cell counts

were then performed in an automated cell counter （Cell Dyne 3500,

Abbott Laboratories, New York）. In order to perform differential

analyses, aliquots of the cellswere placed onto slides and then stained

with Field's stain. After drying, 300 cells/slide were counted using a

compound microscope （Optima X5Z-H） at X400 magni?cation and

cells were identi?ed as eosinophils, lymphocytes, macrophages, or

neutrophils using standard morphologic determinants.

2.10. Bronchoalveolar lavaged ?uid

At the end of the experiment （i.e., day 30）, bronchoalveolar

lavaged ?uid was collected from each animal. An overdose of

ketamine （30 mg/kg） and xylazine （20 mg/kg） was administered s.c.

A polypropylene cannula （24G） was inserted into the trachea, and

then, 0.9% （w/v） normal saline solution （10 ml） was introduced into

the lungs via a 10-ml syringe at 37 °C and then recovered 5 min later.

The recovered lavaged ?uid （5 ml） was centrifuged at 500×g for

10 min at 4 °C; the resulting supernatant was collected and stored at

−80 °C for cytokine determination. The cells in the pellet were

washed in 0.5-ml saline, and the total and differential cell countswere

performed as described for blood analysis （refer to Section 2.9）.

2.11. Cytokines in serum and bronchoalveolar lavaged ?uid

The levels of TNFα, IL-4, IL-5 and IL-6 in each sample of recovered

serum （400 μl） and bronchoalveolar lavaged ?uid （4.5 ml） were

measured using enzyme-linked immunosorbent assay （ELISA） kits

according to the manufacturer's protocol. All plates were analyzed on

an automated plate reader （Lab System Multiscan Model-51118220,

Thermo Bioanalysis Co., Helsinki, Finland）.

2.12. Histamine assay on lavaged lung tissue

Lung tissue lobes from each animal were separay dissected out

immediay following bronchoalveolar lavaged ?uid collection. One

lobe was used for non-lavagable histamine measurements and the

other for the histology of lavaged tissue. For the former, lung tissue

（200±20 mg） was placed in 2.5-ml normal saline for the prepara-

tion of homogenate, and then 2.5–ml, 0.8-N perchloric acid was

added. After mixing and centrifugation （4000×g,7minat4°C）,

2 ml of the resulting supernatant was transferred to a test tube

containing 0.25–ml, 5-N NaOH, 0.75-g NaCl and 5-ml n-butanol. The

mixture was vortexed for 5 min to partition histamine into the

butanol and then centrifuged. The aqueous phase was discarded by

aspiration, and the organic phase was washed with 2.5-ml salt-

saturated 0.1-N NaOH solution to remove any residual histamine.

The mixture was re-centrifuged and the butanol was transferred to a

test tube containing 2-ml, 0.1-N HCl and 5-ml n-heptane. The

Fig. 1. Effect of treatments on histamine and acetylcholine-induced bronchospasm in

guinea pigs. Group I: control （received distilled water）, group II: treated with ketotifen

fumarate （1 mg/kg） or atropine sulphate （2 mg/kg）, and groups III, IV and V: treated

with β-sitosterol （1.25, 2.5 and 5 mg/kg, respectively）. *Pb0.001 compared to the

control. All bars represent the mean±S.E.M. from n=6 guinea pigs per treatment

group.

Table 1

Effect of treatments on the tidal volume of guinea pigs.

Day Values before and

after acetylcholine

exposure

Tidal volume （Vt） in ml/s

I II III IV

Non-sensitized control

（distilled water）

Model control

（vehicle）

OVA+DXM

（2.5 mg/kg）

OVA+β-sitosterol

（2.5 mg/kg）

18 Before 2.78±0.26 3.11±0.18 3.30±0.25 3.04±0.20

After 2.53±0.21 2.97±0.16 3.16±0.23 2.99±0.17

21 Before 2.99±0.19 2.15±0.14a

2.94±0.23d

2.74±0.20d

After 2.79±0.17 2.00±0.13a

2.81±0.23d

2.59±0.13

24 Before 2.73±0.14 1.90±0.17a

3.01±0.13e

2.68±0.25f

After 2.58±0.18 1.73±0.15b

2.93±0.11f

2.66±0.20e

29 Before 2.96±0.10 1.61±0.10c

2.92±0.20f

2.87±0.13f

After 2.92±0.11 1.46±0.07c

2.89±0.19e

2.77±0.14f

Values shown are the mean±S.E.M. （n=6）.

a

Pb0.05,

b

Pb0.01, and c

Pb0.001 compared to the non-sensitized control.

d

Pb0.05,

e

Pb0.01,

f

Pb0.001 compared to the OVA

（ovalbumin）-sensitized vehicle-treated model control.

460 S.G. Mahajan, A.A. Mehta / European Journal of Pharmacology 650 （2011） 458–464mixture was again centrifuged, and the presence of histamine was

determined ?uorometrically （SL-174, Elico, India） as previously

described （Shore et al., 1959）.

2.13. Histological examination

Dissected lung tissues were washed with normal saline （5 ml） and

then placed in 10% （v/v） formaldehyde solution for 1 week. After

?xation, lung specimens were embedded in paraf?n wax, and 5-μm

sections were cut and stained with hematoxylin and eosin dye for

morphology. Images of selected sections were captured at X10

magni?cation using a zoom digital camera （Model C763, Eastman

Kodak Company, Rochester, NY, USA）.

2.14. Statistical analyses

Results are reported as mean±S.E.M. Statistical analyses were

performed using a one-way analysis of variance （ANOVA） followed by

post hoc Tukey's test; differences were considered statistically

signi?cant at Pb0.05. All statistical analyses were performed using

the Graph Pad software （San Diego, CA, USA）.

3. Results

3.1. Characterization and structure elucidation of the isolated compound

The melting point was obtained at 138–140 °C. The UV absorp-

tion spectrum of the isolated sample in methanol was scanned and

showed maximum absorbance at 292.56 nm. The different peaks of

mass spectra were obtained as M-18 （414.3-18）, 397.3, H2O; M-3

（414.3-3）, 411.3, 3H; M-70 （414.3-70）, 344.4, C24H40O; and M-84

（414.3-84）, 330.6, C23H38O. The isolated compound was identi?ed as

β-sitosterol based on IR,

1

H-NMR and 13

C-NMR spectroscopic data

and comparison with those reported in the literature （data not

shown）.

3.2. Effect of treatments on histamine and acetylcholine-induced

bronchospasm in guinea pigs

A pilot study was conducted with three different doses of β-

sitosterol （1.25, 2.5, or 5 mg/kg） to determine the dose dependent

effect in histamine and acetylcholine-induced bronchospasm. It was

observed that β-sitosterol post-treatment at doses of 2.5 and 5 mg/kg

signi?cantly （Pb0.05） increased pre-convulsion dyspnea time com-

pared to the control animals. Hence, a lower dose was chosen for our

subsequent chronic studies （Fig. 1）.

3.3. Effect of treatments on body weight

All animals present in the model control （group II） and drug

regimen （groups III and IV） groups did not show any signi?cant

difference in body weight during the experimental period compared

to the animals in the non-sensitized control group （group I）.

Furthermore, there were no apparent effects on the appetite/water

consumption or on the outward appearance （i.e., fur coat, and eyes） of

animals in each treatment group （data not shown）.

3.4. Effect of treatments on lung function parameters in the

acetylcholine-induced bronchoconstriction test

Lung function parameters were measured by Respiromax during

the experimental period on days 18, 21, and 24 and on day 29 before

and after exposure to acetylcholine （0.25% for 30 s）. Tidal volume

（Table 1） was decreased and respiration rate （Table 2） was increased

signi?cantly （Pb0.05） before and after exposure to acetylcholine in

model control animals compared to non-sensitized animals fromdays

21 to 29. However, dexamethasone- and β-sitosterol-treated animals

showed signi?cant increase in tidal volume [before （Pb0.001,

Pb0.001） and after （Pb0.01, Pb0.001）, respectively] and decrease in

respiratory rate [before （Pb0.001, Pb0.001） and after （Pb0.001,

Pb0.001）, respectively, of acetylcholine exposure] compared to the

model control animals, suggesting improvement in these parameters

on day 29.

Table 2

Effect of treatments on the respiration rate of guinea pigs.

Day Value before and

after acetylcholine

exposure

Respiration rate （f） in breaths/min

I II III IV

Non-sensitized control

（distilled water）

Model control

（vehicle ）

OVA+DXM

（2.5 mg/kg）

OVA+β-sitosterol

（2.5 mg/kg）

18 Before 103.0±1.5 103.7±4.7 108.4±2.2 110.3±5.7

After 110.7±4.2 111.3±4.1 116.3±3.0 118.9±3.7

21 Before 108.5±1.1 127.5±4.8c

107.7±1.3f

111.1±1.9e

After 113.5±2.8 135.9 ±10.6 112.1±2.4d

116.5±2.5

24 Before 105.9±1.4 129.4±1.0c

110.7±1.0f

105.0±1.2

After 110.6±3.9 148.9±7.2c

112.4±3.0f

108.9±3.6f

29 Before 106.5±1.3 130.2±2.0c

114.9±0.9f

107.3±1.5f

After 115.3±2.1 154.1±6.6c

116.2±2.5f

110.5±3.5f

Values shown are the mean±S.E.M. （n=6）.

c

Pb0.001 compared to the non-sensitized control.

d

Pb0.05,

e

Pb0.01, and f

Pb0.001 compared to the OVA （ovalbumin）-sensitized

vehicle-treated model control.

Table 3

Effect of treatments on total cells and differential leukocyte count in blood （×105

cells/ml）.

Groups Total cells Eosinophils Lymphocytes Monocytes Neutrophils

I Non-sensitized control （distilled water） 8.22±0.50 0.44±0.005 6.20±0.038 0.44±0.005 0.22±0.005

II Model control （vehicle） 20.96±2.55c

0.93±0.01 c

12.56±0.34 c

0.93±0.01 c

0.36±0.005 c

III OVA+DXM （2.5 mg/kg） 12.92±0.41e

0.70±0.03e

8.96±0.24 e

0.70±0.03e

0.25±0.01f

IV OVA+β-sitosterol （2.5 mg/kg） 16.94±0.75d

0.83±0.03d

10.83±0.45e

0.83±0.03 d

0.32±0.006 e

Values shown are the mean±S.E.M. （n=6）.

c

Pb0.001 compared to the non-sensitized control.

d

Pb0.05,

e

Pb0.01, and f

Pb0.001 compared to the OVA （ovalbumin）-sensitized

vehicle-treated model control.

461 S.G. Mahajan, A.A. Mehta / European Journal of Pharmacology 650 （2011） 458–4643.5. Effect of treatments on circulating cellular counts

The total number of leukocytes and each differential count in blood

samples recovered from the model control animals were markedly

increased （Pb0.001） compared to the non-sensitized controls.

However, the numbers of circulating eosinophils （Pb0.01 and

Pb0.05）, lymphocytes （Pb0.01）, monocytes （Pb0.01 and Pb0.05）

and neutrophils （Pb0.001 and Pb0.01） in the blood were signi?cantly

decreased in dexamethasone- and β-sitosterol-treated animals,

respectively, compared to those numbers seen in the model control

guinea pigs （Table 3）.

3.6. Effect of treatments on in?ammatory cellular counts in

bronchoalveolar lavaged ?uid

The model control animals showed a signi?cant increase in the

total cell count and differential cellular count in bronchoalveolar

lavaged ?uid compared to the non-sensitized controls. Dexameth-

asone and β-sitosterol treatment signi?cantly decreased these

counts from the model control levels [total cells （Pb0.01 and

Pb0.05）, eosinophils （Pb0.001 and Pb0.01）, lymphocytes （Pb0.05）

macrophages （Pb0.001 and Pb0.01） and neutrophils （Pb0.001）]

（Table 4）.

3.7. Effect of treatments on cytokine production in serum

Themodel control animals showed signi?cant （Pb0.001） increases

in levels of TNF-α, IL-4, IL-5 and IL-6 compared to the non-sensitized

controls. These elevated levels of TNF-α （Pb0.001）, IL-4 （Pb0.05） and

IL-5 （Pb0.05） were signi?cantly decreased in guinea pigs that

received β-sitosterol treatment compared to those levels seen in the

model controls. However, this treatment did not correlate with any

signi?cant reductions in the level of IL-6 （Fig. 2）.

3.8. Effect of treatments on cytokine levels in bronchoalveolar

lavaged ?uid

The signi?cant （Pb0.001） increase in cytokine levels in bronchoal-

veolar lavaged ?uid fromthe model control animals was not present in

β-sitosterol-treated animals [TNF-α （Pb0.01）, IL-4 （Pb0.05）, and IL-5

（Pb0.05）]. Dexamethasone caused a signi?cant （Pb0.05） reduction in

IL-6 levels compared to the model controls. In contrast, there was no

change in IL-6 levels resulting from β-sitosterol treatment （Fig. 3）.

3.9. Effect of treatments on histamine levels

The level of histamine measured in lung tissues from the model

control animalswas signi?cantly higher （Pb0.01） than the level in the

non-sensitized controls. Compared to the model control group,

treatment group IV showed a signi?cant （Pb0.05） β-sitosterol-

induced normalization of elevated histamine levels; this effect was

approximay equal in magnitude to the normalization-induced by

dexamethasone treatment （Fig. 4）.

3.10. Effect of treatments on histopathology of lung tissue

The histological examination of lung tissue fromthemodel control

guinea pigs showed a massive in?ammatory in?ltration of the

peribronchial tissues, reduced lumen size, epithelial desquamation

and angiogenesis. Treatment with dexamethasone and β-sitosterol

showed a protective effect, as evidenced by the presence of milder or

less pathological features （Fig. 5）.

4. Discussion and conclusion

Herbal medicines have been used to treat asthma for hundreds of

years （Chung and Adcock, 2000）. However, so far, very few compounds

have been isolated from such herbal plants and subjected to clinical

studies based on their anti-asthmatic effects in experimental studies.

Table 4

Effect of treatments on total cells and differential leukocyte counts in bronchoalveolar lavaged ?uid （×105

cells/ml）.

Groups Total cells Eosinophils Lymphocytes Macrophages Neutrophils

I Non-sensitized control （distilled water） 8.51±0.17 0.40±0.012 6.4±0.66 0.40±0.012 0.24±0.017

II OVA-control （vehicle） 17.64±0.93c

0.83±0.037 c

11.68±0.65 b

0.83±0.037c

0.39±0.009 c

III OVA+DXM （2.5 mg/kg） 12.74±0.23e

0.62±0.025f

8.04±0.10d

0.62±0.025f

0.27±0.007f

IV OVA+β-sitosterol （2.5 mg/kg） 13.19±0.30d

0.68±0.017e

8.21±0.14d

0.68±0.017e

0.30±0.008f

Values shown are the mean±S.E.M. （n=6）.

b

Pb0.01 and c

Pb0.001 compared to the non-sensitized control.

d

Pb0.05,

e

Pb0.01, and f

Pb0.001 compared to the OVA （ovalbumin）-

sensitized vehicle-treated model control.

Fig. 2. Effect of treatments on serum cytokine levels of guinea pigs. *Pb0.001 compared

to the non-sensitized controls.

@Pb0.001,

#Pb0.01, and $

Pb0.05 compared to the OVA

（ovalbumin）-sensitized vehicle-treated model controls. All bars represent the mean±

S.E.M. from n=6 guinea pigs per treatment group.

Fig. 3. Effect of treatments on bronchoalveolar lavaged ?uid cytokine levels of guinea

pigs. *Pb0.001 compared to the non-sensitized controls.

#Pb0.01 and $

Pb0.05

compared to the OVA （ovalbumin）-sensitized vehicle-treated model controls. All

bars represent the mean±S.E.M. from n=6 guinea pigs per treatment group.

462 S.G. Mahajan, A.A. Mehta / European Journal of Pharmacology 650 （2011） 458–464The exceptions include ephedrine from the plant Ephedra （Berger and

Dale, 1910）, theophylline from tea （Macht and Ting, 1921） and

cromolyn sodium （sodium cromoglycate） from Khellin （Cox, 1967）;

these drugs have been used for the treatment of asthma for several

years. Furthermore, the scienti?c literature is repletewith reports of the

biological activities of sterols or their glycosides in various animal

models of asthma. The possible ef?cacy of β-sitosterol as a therapeutic

drug for immune-mediated disorders has been reported （Bouic and

Lambrecht, 1999）. β-sitosterol and its glycoside have been shown to

reduce carcinogen-induced colon cancer in rats （Raicht et al., 1980）and

to have anti-in?ammatory activity through cytokine inhibition （Aherne

and O'Brien, 2008）. Moreover, in vitro studies showed that β-sitosterol

increased Th1 while dampening Th2-cell activities （Chen et al., 2009）.

In this study, no animals in the model control or drug-treated

groups showed any signi?cant difference in body weight during the

experimental period compared to the non-sensitized control ani-

mals, suggesting that β-sitosterol treatment did not interfere with

the normal growth of the animals. All animals in the model control

group exhibited irritability, sneezing and hyper-rhinorrhea, indica-

tive of the severity of disease. Furthermore, tidal volume in themodel

control animals was decreased signi?cantly before and after

exposure to acetylcholine from days 21 to 29, demonstrative of

bronchoconstriction due to chronic airway in?ammation, which

resembles an asthmatic condition. Similarly, the signi?cant increase

in respiration rate observed in these animals was indicative of

exertional breathing—a symptom of asthma. Treatment with dexa-

methasone and β-sitosterol had a signi?cant protective effect; both

drugs improved tidal volume and respiratory rate. This defensive

effect might be due to the indirect decrease in resistance resulting

from reduction in airway in?ammation.

Thelate-phaseairwayresponseinasthmaisassociatedwiththe

in?ltration of in?ammatory cells to the site of the response

（Williams, 2004）. In the present study, the model control animals

had increased total and differential cellular counts in blood and in

bronchoalveolar lavaged ?uid; these increases correlated with the

level of cellular in?ltration. Guinea pigs that received dexameth-

asone and β-sitosterol treatment had signi?cantly decreased the

numbers of total cells in both blood and bronchoalveolar lavaged

?uid. However, in the differential cell count, β-sitosterol decreased

each cell count in blood but only the eosinophil and neutrophil

count in bronchoalveolar lavaged ?uid compared to the model

control animals. Furthermore, the amelioration of in?ammatory

cell numbers in bronchoalveolar lavaged ?uid was con?rmed by

lung tissue histology. Therefore, these results suggest that β-

sitosterol treatment could possibly be useful to control the

activation of the in?ammatory processes underlying exacerbation

of allergic asthma.

The initial indication for cytokine involvement in the pathogen-

esis of asthma came from studies performed in the early 1990s,

showing that allergic asthma is associated with Th2 cytokine

expression （Boyton and Altmann, 2004）. Mast cells are most likely

an important source of TNF-α. Furthermore, the localization of

cytokines to mast cell subsets reveals preferential IL-4 with

prominent IL-5 and IL-6 expression （Chung and Barnes, 1999）. In

the present study, we con?rmed the existence of the prominent Th2

type cytokines—TNF-α, IL-4, IL-5 and IL-6—in the model control

Fig. 4. Effect of treatments on lung tissue histamine levels of guinea pigs. *Pb0.001

compared to the non-sensitized controls.

#Pb0.01 and $

Pb0.05 compared to the OVA

（ovalbumin）-sensitized vehicle-treated model controls. All bars represent the mean±

S.E.M. from n=6 guinea pigs per treatment group.

Fig. 5. Effect of treatments on the histopathological changes in lung tissue. Representative hematoxylin- and eosin-stained sections of the lung tissue （X10）. A shows a typical normal

lung histology. B shows a typical damaged lung tissue from a model control group animal with total and differential leukocyte in?ltration, reduced lumen size, endothelial shedding

and angiogenesis. C shows a section from a dexamethasone-treated animal. D shows a section from a β-sitosterol-treated animal.

463 S.G. Mahajan, A.A. Mehta / European Journal of Pharmacology 650 （2011） 458–464animals, suggesting persistent airway in?ammation. β-sitosterol

treatment decreased the level of TNF-α, IL-4, and IL-5 in broncho-

alveolar lavaged ?uid and in serum. This reduction in the level of

cytokines correlates with the inhibition of in?ammation （as

determined by decreased histamine levels） by β-sitosterol.

Furthermore, ongoing chronic in?ammation is associated with

mast cell degranulation as evidenced by the increased levels of mast

cell mediators in lung tissues （Bartosch et al., 1932; Foresi et al.,

1990）. In this study, a signi?cant increase in histamine levels inmodel

control animals was indicative of the in?ammation of lung tissues and

the release of mediators. Treatment with dexamethasone and β-

sitosterol signi?cantly decreased histamine levels compared to the

diseased control animals. These data suggest that β-sitosterol might

inhibit the release of in?ammatory mediators such as histamine. In

addition, atopic asthma has been extensively investigated and

involves structural changes in the airways （Amin et al., 2000）. The

results of histopathology study suggest that β-sitosterol treatment

inhibited angiogenesis, epithelial shedding and leukocyte in?ltration

into the airway after ovalbumin challenge. In spite of the results

presented in this study, we still do not know how β-sitosterol

attenuates the airway in?ammation allied with asthma; hence, we

intend to clarify the precise mechanism underlying the antiasthmatic

function of β-sitosterol in future studies.

In conclusion, β-sitosterol exerted anti-in?ammatory effects in

allergen-induced airway in?ammation. We described the potential

mode of action of β-sitosterol by investigating its ef?cacy against Th2-

cell-derived cytokine production and subsequent cytokine-induced

cellular in?ltration （eosinophils and neutrophils）, its protective

potential （counteraction of acetylcholine-induced bronchoconstric-

tion and improvement in lung functions） and its capacity to block the

release of in?ammatory mediators, such as histamine, into the local

lung tissues. Lastly, the results of our study suggest that β-sitosterol

may be a valuable therapy for asthma; however, a well-designed

clinical trial is warranted,which includes persistent,mild ormoderate

asthmatic patients.

Con?ict of interest statement

The authors state no con?ict of interest.

Acknowledgements

This work was supported by the Department of Science and

Technology, New Delhi, India （Grant Ref. SR/SO/HS-09/2004）.

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