Immuno-enhancement, Antioxidant and Anti-mutagenic Effects of Hypericum triquitrifolium in vitro and in vivo

The study aimed to investigate anti-oxidant, cytogenetic, hepatoprotective and immunological effects of Hypericum triquetrifolium methanolic extract in vitro and in vivo (albino male mice). Interactions between the extract and
cyclophosphoamide (CYP) or carbon tetrachloride (CCl4) were also made to evaluate in vivo the extract modulating effects. Furthermore, the impact of extract on in vitro cytokine release from cultured mononuclear cells obtained
from acute lymphoblastic (ALL) and acute myelogenous leukemic (AML) Iraqi patients was also evaluated. In addition, total flavonoids content was spectrophotochemically determined in the extract, and it was 115.73 μg/ml. Anti-oxidant activity of methanolic extract the Hypericum triquetrifolium was evaluated in vitro via assessment of reductive ability and DPPH [1, 1-
diphenyl-2-picrylhydrazyl (α,α-diphenyl-β-picrylhydrazyl] radical scavenging activity. At all concentrations tested (0.02, 0.04, 0.08, 0.16, 0.32 and 0.64 mg/ml), the absorbance was significantly increased in a concentration-dependent manner. Moreover, the extract was significantly more effective in DPPH radical scavenging activity than vitamin C at the four concentrations tested (0.0625, 0.125, 0.250 and 0.500 mg/ml).. Cytogenetic analyses included assessment of metaphase index (MI) in bone
marrow and spleen cells, as well as micronucleus (MN) formation in bone marrow cells of mice treated with cyclophosphoamide and H. triquetrifolium methanolic extract. The extract was able to modulate the reducing effects of CYP and restored both MIs almost to their normal percentage at the first dose (50 mg/kg) of plant extract , but at higher doses(100 and 200 mg/kg) , the MIs exceeded blank control values. In addition, treating mice with the extract reduced MN frequency significantly in a dose-dependent manner in compared to blank controls. Immunological evaluations included total leukocyte counts (TLC) and absolute counts of leukocytes, as well as assessment of anti-inflammatory activity in mice treated with CYP and H. triquetrifolium methanolic extract. Treating mice with vitamin C or the first dose of extract (50 mg/kg) was associated with a significant recovery of TLC, and a better recovery was observed at the doses 100 and 200 mg/kg of the methanolic extract. Absolute counts of lymphocytes, neutrophils and monocytes almost behaved in a similar manner. With respect to anti-inflammatory activity, the best percentage of edema inhibition was observed four hour post-induction at the dose 200 mg/kg, followed by the same dose but six hours post-induction. The hepatoprotective evaluations included assessment of malondialdehyde (MDA) and catalase (CAT) activities in liver tissue homogenate and liver function enzymes (aspartate aminotransferase; AST and alanine aminotransferase; ALT and alkaline phosphatase; ALP) in serum, as well as histopathological evaluation of liver tissue, in mice after the administration of CCl4. The extract doses 100 and 200 mg/kg were the best in reducing CCl4- increased MDA activity, and the same doses contributed significantly to an increased activity of catalase in comparison to control activity. The three doses of H. triquetrifolium methanolic extract (50, 100 and 200 mg/kg) were also able to counteract CCl4-effects on AST and ALT in a dose-dependent manner, and similarly, For ALP, the third dose of plant extract (200 mg/kg) was the best in normalizing ALP activity, and at such dose, the histological profile of liver tissue was look-like with normal hepatocytes. Cytokine production (IL-2, IL-4, IL-10, IL-17A and IFN-γ) from acute lymphoblastic (ALL) and acute myelogenous leukemic (AML) cells was also modulated by H. triquetrifolium methanolic extract, and combining phytoheamagglutinin (PHA) and the extract contributed to a significant increase in IL-2 level, especially at the second concentration of extract (1.791 μg/ml) in ALL patients compared to controls or AML patients, while no such effects were observed on IL-4 level. For IL-10, PHA and plant extract were also ffective, and the highest level was observed in supernatants of controls and ALL cells at the concentration 1.791 μg/ml, while the supernatant of AML cells showed a less level and the difference was significant. Treating cultured cells with the plant extract alone induced a similar increased production of IL-10, but it did not reach the synergistic effects of PHA and plant extract. In the case of IL-17A, the cultured cells of AML and controls were better than ALL cells in their production of IL-17A after the different treatments (PHA only, PHA+plant extract or plant extract only). The highest production was observed in AML and control cultures, which were significantly higher than the corresponding cultures of ALL patients. For IFN-γ, the best response of cultured cells was observed in supernatants of cultures treated with PHA+plant extract at the concentration 1.791 μg/ml in ALL and AML patients, and the difference was significant as compared to the corresponding supernatant of controls.