Immunosuppressive effects of the standardized extract of Phyllanthus amarus on cellular immune responses in Wistar-Kyoto rats

Abstract

Phyllanthus amarus (family: Euphorbiaceae) is of immense interest due to its wide spectrum of biological activities. In the present study, the standardized 80% ethanol extract of P. amarus was investigated for its modulatory activity on various cellular immune parameters, including chemotaxis of neutrophils, engulfment of Escherichia coli by neutrophils, and Mac-1 expression, in leukocytes isolated from treated/nontreated Wistar-Kyoto rats. The detailed cell-mediated activity of P. amarus was also investigated, including analysis of the effects on T- and B-cell proliferation and CD4(+) and CD8(+) T-cell subsets in splenic mononuclear cells, and estimation of serum cytokine production by activated T-cells. The main components of the extract, phyllanthin, hypophyllanthin, corilagin, geraniin, ellagic acid, and gallic acid were identified and quantitatively analyzed in the extracts, using validated reversed-phase high-performance liquid chromatography (HPLC) methods. N-formyl-methionyl-leucyl-phenylalanine (fMLP)-induced neutrophils isolated from rats administered with the extract of P. amarus, at doses ranging from 100 to 400 mg/kg for 14 days, revealed a significant dose-dependent reduction in neutrophil migration (P<0.05). Similar patterns of inhibition were also observed in phagocytic activity and in fMLP-induced changes in expression of β2 integrin polymorphonuclear neutrophils. The results in P. amarus-treated rats also demonstrated a dose-dependent inhibition of both lipopolysaccharide-stimulated B-cell proliferation and concanavalin A-stimulated T-cell proliferation as compared with sensitized control. At a dose of 400 mg/kg (P<0.01), there was a significant decrease in the (%) expression of CD4(+) and CD8(+) in splenocytes and in serum cytokines of T helper (Th1) (IL-2 and IFN-γ) and Th2 (IL-4). In conclusion, P. amarus showed effective immunosuppressive activities in cellular immune response, by various immune regulatory mechanisms, and may be useful for improvement of immune-related disorders.

Keywords: T- and B-cell proliferation; immunosuppression; leukocytes migration; phagocytic activity; splenic mononuclear cells.

Figure 1

Representative HPLC chromatograms of (A) Phyllanthus amarus for identification of phyllanthin and hypophyllanthin at RT and 27.279 and 28.051, respectively; and of (B) P. amarus for identification and quantification of gallic acid at RT and 7.891 minutes, geraniin at RT and 23.764 minutes, corilagin at RT and 26.416 minutes, ellagic acid at RT and 33.421 minutes, with an unidentified peak at RT (23.860 minutes). Abbreviations: HPLC, high-performance liquid chromatography; RT, room temperature.

Figure 2

Graph of the effect of Phyllanthus amarus (100, 200, and 400 mg/kg) on the chemotaxis of neutrophils isolated from experimental animals. Notes: Results are represented as mean ± SEM, with n=6 in each group. **P<0.01; *P<0.05. The statistical tests employed were ANOVA, followed by post-Dunnett’s test. Abbreviations: ANOVA, analysis of variance; Cyclo, cyclophosphamide; NSC, nonsensitized control; PA, Phyllanthus amarus; SEM, standard error of the mean; VH, vehicle-treated group.

Figure 3

Flow cytometric evaluation of the effect of Phyllanthus amarus (100, 200, and 400 mg/kg) on CD18 and CD11b in neutrophils isolated from experimental animals. Abbreviations: Cyclo, cyclophosphamide; FITC, fluorescein isothiocyanate; NSC, nonsensitized control; PA, Phyllanthus amarus; VH, vehicle-treated group.

Figure 4

Flow cytometric evaluation of the effect of Phyllanthus amarus (100, 200, and 400 mg/kg) on phagocytosis of FITC-conjugated E. coli by neutrophils isolated from treated rats. Abbreviations: Cyclo, cyclophosphamide; FITC, fluorescein isothiocyanate; NSC, nonsensitized control; PA, Phyllanthus amarus; VH, vehicle-treated group; E. coli, Escherichia coli.

Figure 5

Flow cytometric evaluation of the effect of Phyllanthus amarus (100, 200, and 400 mg/kg) on CD4 and CD8 in splenocytes of experimental animals. Abbreviations: Cyclo, cyclophosphamide; FITC, fluorescein isothiocyanate; PA, Phyllanthus amarus; SC, sensitized control; VH, vehicle-treated group.

Figure 6

Evaluation of the effect of Phyllanthus amarus (100, 200, 400 mg/kg) on IFN-γ (A), IL-2 (B), and IL-4 (C) in splenocytes of experimental rats. Notes: Results are represented as mean ± SEM, with n=6 in each group. ***P<0.001; **P<0.01; *P<0.05. The statistical tests employed were ANOVA, followed by post-Dunnett’s test. Abbreviations: ANOVA, analysis of variance; Cyclo, cyclophosphamide; PA, Phyllanthus amarus; SC, sensitized control; SEM, standard error of the mean; VH, vehicle-treated group; IL-4, interleukin 4; IFN-γ, interferon γ.