Journal of Ethnopharmacology 78 (2001) 85 87

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Short communication

Immunomodulatory activity of Punica granatum in rabbits a

preliminary study
R. Gracious Ross *, S. Selvasubramanian, S. Jayasundar
Department of Pharmacology and Toxicology, Madras Veterinary College, Chennai 600 -007, India
Received 28 August 2000; received in revised form 6 June 2001; accepted 11 June 2001

Abstract
Punica granatum L. (Punicaceae) fruit rind powder (PGFRP) at the dose of 100 mg/kg orally as aqueous suspension was found
to stimulate the cell-mediated and humoral components of the immune system in rabbits. PGFRP elicited an increase in antibody
titer to typhoid-H antigen. It also enhanced the inhibition of leucocyte migration in Leucocyte Migration Inhibition test and
induration of skin in delayed hypersensitivity test with Purified Protein Derivative (PPD) confirming its stimulatory effect on
cell-mediated immune response. 2001 Elsevier Science Ireland Ltd. All rights reserved.
Keywords: Punica granatum; Punicaceae; Immunostimulatory; Cell-mediated immunity; Humoral immunity; Rabbits

1. Introduction

2. Materials and methods

Punica granatum L. (family Punicaceae), popularly

known as dadima in Sanskrit and pomegranate in
English is described for its medicinal properties in
Ayurveda. The root bark as well as stem barks of the
plant are astringent and antihelmintic. The dried flowers are used in haematuria, hemorrhoids, haemoptysis
and dysentery. The powdered flowerbuds are used in
bronchitis. The seeds are considered to be stomachic
and the pulp as cardiac and stomachic. The fruit rind is
valued as an astringent and green leaves are made into
a paste and applied in conjunctivitis (Chopra et al.,
1960).
In preliminary screening, the extracts of the fruit rind
powder exhibited antibacterial activity against many
bacteria especially Bacillus anthracis, B. subtilis,
Salmonella paratyphi and Vibrio cholerae (Trivedi and
Kazmi, 1979). In the absence of any scientific evidence
for its ability to increase the overall resistance of the
body, an attempt was made to undertake immunologic
investigations with fruit rind powder of P. granatum in
rabbits.

2.1. Experimental animals

* Corresponding author.

Crossbred New Zealand White male rabbits weighing

between 800 and 1200 g kept under uniform husbandry
conditions were used. All the animals were fed with
rabbit pellet feed and water ad libitum.

2.2. P. granatum fruit rind powder (PGFRP)

The commercially available powdered fruit rind of P.
granatum was obtained from Vets Consultancy, Mumbai, India and its aqueous suspension was used at the
dose of 100 mg/kg orally (derived from recommended
human dosage on body weight basis) in this study. The
powder was subjected to phytochemical screening
which revealed positive test for tannins, glycosides and
free sugars.

2.3. Humoral immune response

It was evaluated by demonstrating agglutination antibodies to respective antigens using the Widal tube
agglutination test (Cruikshank et al., 1965). A single
dose of 1 ml of typhoid-H antigen obtained from King

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R. Gracious Ross et al. / Journal of Ethnopharmacology 78 (2001) 8587

86

Institute, Chennai, India, containing 1000 million

bacilli per ml, was administered subcutaneously in all
the rabbits. The experimental animals were divided into
two groups. One group served as control, which received equivalent volume of distilled water orally for 10
days. The other group received PGFRP at the dose of
100 mg/kg as aqueous suspension for 10 days by intragastric intubation, starting from the day of the antigen
challenge.

3. Results
Administration of PGFRP at the dose of 100 mg/kg
caused a statistically significant increase in the antibody
titer in Widal tube agglutination test (Table 1).
On cell-mediated immunity, the PGFRP administered group showed a significant enhancement in leucocyte migration inhibition when compared with the
control group (Table 2). In delayed hypersensitivity
test, the PGFRP group evidenced a significant increase
in the skin thickness compared with the control group
(Table 2).

2.4. Cellular immune response

Two groups of rabbits were administered 0.1 ml of
purified protein derivative (PPD) (Span Diagnostics
Ltd., Surat, India) subcutaneously on day 1 among
which one group was kept as control and the other
treated with PGFRP at the dose of 100 mg/kg orally as
aqueous suspension.

4. Discussion
In the present study, P. granatum fruit rind powder
(PGFRP) has been found to stimulate both the humoral and cell-mediated immune responses in rabbits.
Increase in the delayed type hypersensitivity (DTH)
reaction in response to thymus-dependent antigen revealed the stimulatory effect of PGFRP on T-cells and
accessory cell types required for the expression of the
reaction (Luster et al., 1982). Supportive evidence for
confirmation of the stimulation of cell-mediated immune response was obtained from leucocyte migration
inhibition test in which PGFRP group rabbits showed
an increased inhibition of cellular migration.
The augmentation of the humoral immune response
to typhoid-H antigen by increase in the haemagglutination antibody titers in rabbits also indicated the enhanced responsiveness of macrophages and T and B
lymphocyte subsets, involved in the antibody synthesis
(Benacerraf, 1978). In view of the pivotal role played by
macrophages in co-ordination the processing of antigen
to T and B cells, the augmentation of the humoral
response to typhoid-H antigen reveals that PGFRP
may enhance the effect by facilitating those processes.
In conclusion, the results obtained in the present
study have shown that P. granatum fruit rind powder
has appreciable immunostimulatory activity in rabbits.
Further in-depth studies are required to elucidate the
mechanisms responsible for its immunomodulatory
property.

2.4.1. Leucocyte migration inhibition test (LMIT)

Leucocyte migration inhibition test (LMIT) was performed on days 0 and 14 in both groups (Bendison and
Soberg, 1969). The leucocyte migration inhibition was
arrived at using the formula,

! 

Migration index=
1

n"

Migration in the presence of antigen

Migration in the absence of antigen

100

2.4.2. Delayed hypersensiti6ity test

On day 14, 0.1 ml purified protein derivative was
administered intradermally and the grades of induration of skin were measured using vernier callipers after
48 h (Kenneth, 1982).
2.5. Statistical analysis
The data obtained were analysed by Wilcoxon
Mann Whitney test for antibody titre (Siegal and
Castellan, 1988) and Students t-test (Snedecor and
Cochran, 1967).
Table 1
Effect of Punica granatum on antibody titer (mean, S.D.)
Group

Day
0

Control
Punica granatum (PGFRP)

0.00
0.00

14

21

Mean

S.D.

Mean

S.D.

Mean

S.D.

53.33
133.33**

20.7
41.4

80.00
266.67**

0.0
82.8

106.67
373.33**

41.4
186.39

Significant from control: **P50.01; N, eight per group.

R. Gracious Ross et al. / Journal of Ethnopharmacology 78 (2001) 8587

87

Table 2
Effect of Punica granatum on cellular immune response
Group

Control.
Punica grantum (PGFRP)

LMI (Mean9S.E.M.)

Delayed hypersensitivity (mm)

0 day

14th day

12.62 91.45
12.3591.6

25.75 9 2.14
28.47* 9 1.78

3.48 90.18
4.17** 90.23

Significant from control *, P50.05; **, P50.01; N, eight per group.

Acknowledgements
The authors are grateful to the Dean, Madras Veterinary College, India, for the facilities provided to undergo this study.

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