a b s t r a c t
Herbals such as Ixora coccinea, Daemia extensa and Tridax procumbens were selected to screen in vitro
antibacterial and immunostimulant activity against the freshwater fish pathogen Aeromonas hydrophila
using different organic polar and non-polar solvents. Initial screening results revealed that, ethyl acetate
extracts and its purified fraction of I. coccinea was able to suppress the A. hydrophila strains at more than
15 mm of zone of inhibition and positive immunostimulant activity. The purified active fraction, which
eluted from H40: EA60 mobile phase was structurally characterized by GCeMS analysis. Two compounds
such as Diethyl Phthalate (1,2-Benzene dicarboxylic acid, monobutyl ester) and Dibutyl Phthalate were
characterized using NIST database search. In order to study the in vivo immunostimulant influence of the
compounds, the crude extracts (ICE) and purified fractions (ICF) were incorporated to the artificial diets
at the concentration of 400 mg kg1 and fed to the ornamental gold fish Carassius auratus for 30 days.
After termination of feeding experiment, they were challenged with highly virulent A. hydrophila AHV-1
which was isolated from infected gold fish and studied the survival, specific bacterial load reduction,
serum biochemistry, haematology, immunology and histological parameters. The control diet fed fishes
succumbed to death within five days at 100% mortality whereas ICE and ICF fed groups survived 60 and
80% respectively after 10 days. The diets also helped to decrease the Aeromonas load after challenge and
significantly (P 0.01) improved the serum albumin, globulin and protein. The diets also helped to
increase the RBC and haemoglobin level significantly (P 0.05) from the control group. Surprisingly the
immunological parameters like phagocytic activity, serum bactericidal activity and lysozyme activity
were significantly increased (P 0.001) in the experimental diets. Macrophages and erythrocytes were
abundantly expressed in the treated groups and the present work concluded that, the Phthalate derivatives
from I. coccinea helps to stimulate the immune system against A. hydrophila challenge in
C. auratus.