Immunomodulatory potential of extracts, fractions and pure compounds from Phyllanthus amarus and Psidium guajava on striped catfish (Pangasianodon hypophthalmus) head kidney leukocytes.

This study aimed to identify major phytochemical constituents, as well as compare the immunomodulatory effects of Psidium guajava L. and Phyllanthus amarus Schun and Thonn crude ethanol extracts and their fractions on striped catfish (Pangasianodon hypophthalmus) head kidney leukocytes (HKLs). Moreover, pure constituents were also investigated for their effects on those cells: hypophyllanthin, identified as a major constituent of P. amarus crude extracts and its hexane fraction; corosolic acid, ursolic acid, and oleanolic acid, identified in P. guajava crude extract, ethyl acetate and dichloromethane fractions; with other terpenic derivatives, as well as guajaverin and avicularin, identified with other flavonoids by LC-UV-MS in the crude P. guajava extract and its ethyl acetate fraction. Cell viability, respiratory burst assay (RBA), nitric oxide synthase (NOS) and lysozyme activity in HKLs were analyzed after 24 h stimulation with each extract (10, 20 and 40 µg/mL) or pure compound (7.5, 15 and 30 µM). Our results show that the hexane fraction of both plant extracts inhibited the viability of HKLs, while several other fractions enhanced the cell viability. All P. guajava fractions at all or some concentration considerably enhanced the RBA production in HKLs. Similarly, NOS production was also significantly increased by some or all concentrations of P. guajava dichloromethane and ethyl acetate fractions. However, the NOS production was dose-dependently inhibited in HKLs treated with Pa ethyl acetate and both plants aqueous fractions at 10 or 10 and 40 µg/mL respectively. The lysozyme activity in cells treated with P. guajava crude extracts and all its organic solvent fractions were stronger than those in P. amarus treatments. Pure compounds including corosolic acid, guajaverin, ursolic acid, hypophyllanthin inhibited the HKLs viability according to concentration and type of compound. All pure compounds except avicularin significantly stimulated, at certain or all concentrations, the RBA production and/or the lysozyme activity in HKLs. The NOS production was significantly reduced in HKLs treated with oleanolic acid (30 µM) and hypophyllanthin (7.5 µM) while its level was increased by hypophyllanthin at 30 µM. These results highlighted that the crude ethanol extracts of P. guajava and P. amarus, their fractions and some of their pure components at certain concentrations can potentially act as immunomodulators, and could be considered as valuable candidates in fishery sciences.

Screening of immuno-modulatory potential of different herbal plant extracts using striped catfish (Pangasianodon hypophthalmus) leukocyte-based in vitro tests.

Many medicinal plants have been shown to possess biological effects, including immuno-modulatory activities on human and other mammals. However, studies about the potential mechanisms of plant extracts on the humoral and tissular immunities in fish have received less attention. This study aimed to screen the immunestimulating properties of 20 ethanol plant extracts on striped catfish Pangasianodon hypophthalmus leukocytes. The peripheral blood mononuclear cells (PBMCs) and head kidney leukocytes (HKLs) of striped catfish (50 ±â€¯5 g per fish) were stimulated at 10 and 100 µg of each plant extract per mL of cell culture medium. Several humoral immune parameters (lysozyme, complement and total immunoglobulin) were examined at 24-h post stimulation (hps). Furthermore, the responses of four cytokine genes, namely il1ß, ifrγ 2a and b, and mhc class II were assessed by quantitative real-time PCR at 6, 12, 24, and 48 hps. The results showed that lysozyme, complement as well as total immunoglobulin levels in both PBMCs and HKLs were regulated by some of the plant extracts tested in a concentration-dependent manner; some plant extracts induced the highest immune responses at the low dose (10 µg mL-1) while others were more efficient at high dose (100 µg mL-1). Among the extracts, five extracts including garlic Allium sativum L. (As), neem Azadirachta indica A. Juss (Ai), asthma-plant Euphorbia hirta L. (Eh), bhumi amla Phyllanthus amarus Schum. et Thonn (Pa), and ginger Zingiber officinale Rosc (Zo) induced significant changes in the expression of pro-inflammatory cytokine (il1ß), antiviral cytokines (ifrγ 2a and b) and adaptive immune cytokine (mhc class II) in striped catfish cells. Pa always modulated the strongest expression of the four cytokines in PBMCs and HKLs over the whole experimental period (p < 0.05), whereas Zo did not stimulate the mhc class II expression in striped catfish leukocytes throughout experimental periods. These in vitro results demonstrated that some plant extracts could differently modulate great potential immune response in fish, supporting their applications in further in vivo experiments.

Plant extract-based diets differently modulate immune responses and resistance to bacterial infection in striped catfish (Pangasianodon hypophthalmus).

A feeding trial was performed to compare the effects of five ethanol herbal extracts (bhumi amla, Phyllanthus amarus Schum and Thonn [Pa]; guava, Psidium guajava L. [Pg]; sensitive plant, Mimosa pudica L. [Mp]; neem, Azadirachta indica A. Juss [Ai] and asthma plant, Euphorbia hirta L. [Eh]) on the immune response and disease resistance against Edwardsiella ictaluri infection of striped catfish (Pangasianodon hypophthalmus). Fish were fed diets supplemented with two doses of each plant extract (0% [basal diet], 0.4% Eh [Eh0.4], 2.0% Eh [Eh2.0], 0.2% Pa [Pa0.2], 1.0% Pa [Pa1.0], 0.2% Pg [Pg0.2], 1.0% Pg [Pg1.0], 0.4% Mp [Mp0.4], 2.0% Mp [Mp2.0], 0.4% Ai [Ai0.4], 2.0% Ai [Ai2.0]) for 8 weeks. Results showed that hematological parameters (total red blood cells, white blood cells, lymphocytes, monocytes, and neutrophils) of fish fed extract-based diets were significantly higher than in those fed the control diet (p < 0.05) after 4 and 8 weeks. Plasma lysozyme activity increased in fish whose diets contained both doses of Eh (p < 0.05) in week 4 (W4), whereas lysozyme activity increased in fish fed 0.2% Pa and Pg, and 2.0% Ai and Eh (p < 0.05) in week 8 (W8). The lysozyme levels in skin mucus did not significantly differ between treatments (p > 0.05) in W4 and after the bacterial challenge test. At the end of the feeding trial, levels of ACH50 significantly increased in most of extract groups compared to the control group (p < 0.05). Total immunoglobulin increased considerably in both the plasma and skin mucus of fish fed extract-supplemented diets after 8 weeks. In addition, dietary supplementation with Pg, Mp, Pa0.2, Eh2.0, and Ai0.4 for 8 weeks considerably reduced the cumulative mortality against E. ictaluri infection in striped catfish. The results suggest that plant extracts possibly modulate the striped catfish immune response in a time and dose dependent manner. Specifically, diets enriched with extracts of P. guajava at 0.2 and 1.0%, or M. pudica at 2.0% for 8 weeks, have great potential for improving striped catfish health by enhancing the immune system and reducing mortality against bacterial challenges.