Journal of Ethnopharmacology 78 (2001) 85–87

Short communication

Immunomodulatory activity of Punica granatum in rabbits—apreliminary study

R. Gracious Ross *, S. Selvasubramanian, S. JayasundarDepartment 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 foundto stimulate the cell-mediated and humoral components of the immune system in rabbits. PGFRP elicited an increase in antibodytiter to typhoid-H antigen. It also enhanced the inhibition of leucocyte migration in Leucocyte Migration Inhibition test andinduration of skin in delayed hypersensitivity test with Purified Protein Derivative (PPD) confirming its stimulatory effect oncell-mediated immune response. © 2001 Elsevier Science Ireland Ltd. All rights reserved.

Keywords: Punica granatum ; Punicaceae; Immunostimulatory; Cell-mediated immunity; Humoral immunity; Rabbits

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1. Introduction

Punica granatum L. (family Punicaceae), popularlyknown as ‘dadima’ in Sanskrit and ‘pomegranate’ inEnglish is described for its medicinal properties inAyurveda. The root bark as well as stem barks of theplant are astringent and antihelmintic. The dried flow-ers are used in haematuria, hemorrhoids, haemoptysisand dysentery. The powdered flowerbuds are used inbronchitis. The seeds are considered to be stomachicand the pulp as cardiac and stomachic. The fruit rind isvalued as an astringent and green leaves are made intoa paste and applied in conjunctivitis (Chopra et al.,1960).

In preliminary screening, the extracts of the fruit rindpowder exhibited antibacterial activity against manybacteria especially Bacillus anthracis, B. subtilis,Salmonella paratyphi and Vibrio cholerae (Trivedi andKazmi, 1979). In the absence of any scientific evidencefor its ability to increase the overall resistance of thebody, an attempt was made to undertake immunologicinvestigations with fruit rind powder of P. granatum inrabbits.

2. Materials and methods

2.1. Experimental animals

Crossbred New Zealand White male rabbits weighingbetween 800 and 1200 g kept under uniform husbandryconditions were used. All the animals were fed withrabbit 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 Vet’s Consultancy, Mum-bai, India and its aqueous suspension was used at thedose of 100 mg/kg orally (derived from recommendedhuman dosage on body weight basis) in this study. Thepowder was subjected to phytochemical screeningwhich revealed positive test for tannins, glycosides andfree sugars.

2.3. Humoral immune response

It was evaluated by demonstrating agglutination anti-bodies to respective antigens using the Widal tubeagglutination test (Cruikshank et al., 1965). A singledose of 1 ml of typhoid-H antigen obtained from King* Corresponding author.

0378-8741/01/$ - see front matter © 2001 Elsevier Science Ireland Ltd. All rights reserved.PII: S0378-8741(01)00287-2

R. Gracious Ross et al. / Journal of Ethnopharmacology 78 (2001) 85–8786

Institute, Chennai, India, containing 1000 millionbacilli per ml, was administered subcutaneously in allthe rabbits. The experimental animals were divided intotwo groups. One group served as control, which re-ceived equivalent volume of distilled water orally for 10days. The other group received PGFRP at the dose of100 mg/kg as aqueous suspension for 10 days by intra-gastric intubation, starting from the day of the antigenchallenge.

2.4. Cellular immune response

Two groups of rabbits were administered 0.1 ml ofpurified protein derivative (PPD) (Span DiagnosticsLtd., Surat, India) subcutaneously on day 1 amongwhich one group was kept as control and the othertreated with PGFRP at the dose of 100 mg/kg orally asaqueous suspension.

2.4.1. Leucocyte migration inhibition test (LMIT)Leucocyte migration inhibition test (LMIT) was per-

formed on days 0 and 14 in both groups (Bendison andSoberg, 1969). The leucocyte migration inhibition wasarrived at using the formula,

Migration index=�1−

�Migration in the presence of antigenMigration in the absence of antigen

n�×100

2.4.2. Delayed hypersensiti�ity testOn day 14, 0.1 ml purified protein derivative was

administered intradermally and the grades of indura-tion of skin were measured using vernier callipers after48 h (Kenneth, 1982).

2.5. Statistical analysis

The data obtained were analysed by Wilcoxon–Mann–Whitney test for antibody titre (Siegal andCastellan, 1988) and Student’s t-test (Snedecor andCochran, 1967).

3. Results

Administration of PGFRP at the dose of 100 mg/kgcaused a statistically significant increase in the antibodytiter in Widal tube agglutination test (Table 1).

On cell-mediated immunity, the PGFRP adminis-tered group showed a significant enhancement in leuco-cyte migration inhibition when compared with thecontrol group (Table 2). In delayed hypersensitivitytest, the PGFRP group evidenced a significant increasein the skin thickness compared with the control group(Table 2).

4. Discussion

In the present study, P. granatum fruit rind powder(PGFRP) has been found to stimulate both the hu-moral and cell-mediated immune responses in rabbits.Increase in the delayed type hypersensitivity (DTH)reaction in response to thymus-dependent antigen re-vealed the stimulatory effect of PGFRP on T-cells andaccessory cell types required for the expression of thereaction (Luster et al., 1982). Supportive evidence forconfirmation of the stimulation of cell-mediated im-mune response was obtained from leucocyte migrationinhibition test in which PGFRP group rabbits showedan increased inhibition of cellular migration.

The augmentation of the humoral immune responseto typhoid-H antigen by increase in the haemagglutina-tion antibody titers in rabbits also indicated the en-hanced responsiveness of macrophages and T and Blymphocyte subsets, involved in the antibody synthesis(Benacerraf, 1978). In view of the pivotal role played bymacrophages in co-ordination the processing of antigento T and B cells, the augmentation of the humoralresponse to typhoid-H antigen reveals that PGFRPmay enhance the effect by facilitating those processes.

In conclusion, the results obtained in the presentstudy have shown that P. granatum fruit rind powderhas appreciable immunostimulatory activity in rabbits.Further in-depth studies are required to elucidate themechanisms responsible for its immunomodulatoryproperty.

Table 1Effect of Punica granatum on antibody titer (mean, S.D.)

Group Day

7 21140

S.D.Mean S.D. S.D.Mean Mean

0.00 41.4Control 106.670.080.0020.753.33186.39373.33**82.8266.67**41.4133.33**0.00Punica granatum (PGFRP)

Significant from control: **P�0.01; N, eight per group.

R. Gracious Ross et al. / Journal of Ethnopharmacology 78 (2001) 85–87 87

Table 2Effect of Punica granatum on cellular immune response

LMI (Mean�S.E.M.)Group Delayed hypersensitivity (mm)

14th day0 day

25.75�2.14 3.48�0.18Control. 12.62�1.4528.47*�1.78 4.17**�0.2312.35�1.6Punica grantum (PGFRP)

Significant from control *, P�0.05; **, P�0.01; N, eight per group.

Acknowledgements

The authors are grateful to the Dean, Madras Veteri-nary College, India, for the facilities provided to un-dergo this study.

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