Investigation of the cytotoxicity, anti-oxidant, and anti-inflammatory effects of Ligusticum porteri (Osha) on human peripheral lymphocytes and promyelocytic leukemia cells




Nguyen, Khanh T.P.


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Ligusticum porteri is a traditional Native American herb. The roots of L. porteri have been used in treatments for many kinds of diseases as well as for boosting the immune system. Even though L. porteri has been widely used in traditional remedies, its acclaimed medicinal effects have been barely validated. This study is the first investigation into the medicinal effects of L. porteri on the cytotoxicity, anti-oxidative, and anti-inflammatory activity in human peripheral blood lymphocytes (PBLs) and promyelocytic leukemia (HL-60). This study also investigated the attenuating effects of L. porteri on the cytotoxicity of hydrogen peroxide induced oxidative damage in these cell cultures. Methods: Vacuum-dried ethanolic root extract of L. porteri was dissolved in dimethyl sulphoxide (DMSO) to prepare a stock solution. Appropriate volumes of stock solution of the root extract were added to cultured PBLs and HL-60 cells (1:10 v/v) so that the final concentrations of L. porteri root extract in each batch of cell culture were respectively 0 μg/ml (control), 50 μg/ml, 100 μg/ml, 200 μg/ml, and 400μg/ml. Additionally, to investigate the attenuating effects of L. porteri in oxidative-damaged cell cultures, PBLs and HL-60 cells were challenged with 50 μM of hydrogen peroxide. The cell suspensions were incubated at 37°C humidified with 5% C02. After each day during the incubation period, cell pellets and supernatants were harvested for the investigation of the cytotoxicity, anti-oxidation, and antiinflammation induced by the cell cultures treated with L. porteri root extract. Results: Treatments with L. porteri at concentrations as high as 400 μg/ml could enhance the viability and proliferation of human PBLs and HL-60 cells. After 2 days incubation with 200 μg/ml and 400 μg/ml of root extract, the viability of PBLs was 2 and 2.5 fold higher than the control. The PBLs treated with L. porteri at 200 μg/ml and 400 μg/ml proliferated until day 3 while the untreated and those treated with lower concentrations {50 μg/ml and 100 μg/ml) of the root extract did not survive. After 7 days of incubation with 200 μg/ml and 400 μg/ml of L. porteri root extract, the proliferation of HL-60 cells was two-fold higher than the control {P < 0.05). This study also found that the oxidative stress induced by hydrogen peroxide {H202) reduced the viability of PBLs and HL-60 cells. Data showed that the percentage viability of stress-induced PBLs and HL-60 cells was reduced by 54% and 42%, respectively {P < 0.05). The anti-proliferative effect of H202 was ameliorated by 400 μg/ml L. porteri treatment. This effective dose helped maintain the PBLs' viability at 1.5 times higher than the control after 2 days of incubation while this dose increased the proliferation of stressed HL-60 cells by 42% {P < 0.05). Treatments at lower concentrations did not have a significant proliferative effect which ultimately resulted in growth decline due to H202-exposure. Lipid peroxidation was measured in terms of malondialdehyde {MDA) formed during the stress. Data showed that the root extracts reduced the MDA accumulation in stressed PBLs and HL-60 cells {P < 0.05). The addition of 400 μg/ml L. porteri significantly decreased the lipid peroxidation in stressed PBLs by 94% {P < 0.05). Treatment of stressed HL-60 cells with the root extract concentration equal or higher than 100 μg/ml reduced the lipid peroxidation by 12-13% {P < 0.05). Treatment with 400 μg/ml of the root extract resulted in 26.4% and 29.4% increase of glutathione levels {GSH) in stressed PBLs and HL-60 cells respectively as compared to stressed cell cultures without the root extract {P < 0.05). Studies on the superoxide dismutase {SOD) and catalase {CAT) activities in H202-challenged PBLs and HL-60 cells showed increased activities in response to oxidative stress. Positive modulatory effects of L. porteri to the activities of these enzymes in stressed cells were noted at concentrations as low as 100 μg/ml (P < 0.05). The activities of SOD and CAT increased significantly, by 17.5% and 55.2% respectively, when stressed PBLs were treated with 400 μg/ml L. porteri for 2 days. Treatment with root extract at 100 μg/ml significantly {P < 0.05) increased the activity of SOD in H202-challenged HL-60 cells. This study found that CAT activity in stressed HL-60 cells showed a 2- to 2.5-fold increase after treatment with more than 50 μg/ml L. porteri. Treatment with 400 μg/ml L. porteri significantly {P < 0.05) increased IFN-y and IL-2 in H202- challenged cells. Addition of the root extract did not cause a significant difference in IL-10 levels between stressed PBLs with and without 400 μg/ml L. porteri (P > 0.05). However, treatment with ·400 μg/ml L. porteri diminished the effect of H202-induced decrease in IL-10 in stressed HL-60 cell cultures {P < 0.05). Conclusion: The use of ethanolic root extract of L. porteri at high concentration {400 μg/ml) enhanced the viability of human PBLs and HL-60 cells. Treatment with L. porteri may protect the cells against H202-induced oxidative stress by reducing lipid peroxidation and oxidation of GSH, as well as by elevating the activities of SOD and CAT. Treatment with the L. porteri root extract further enhanced the production of IFN-y and IL-2. Along with the mild enhancement of secretion of IL-10, cytokine stimulation by the addition of L. porteri suggested that the root extract may be a potential immune-modulating agent involving protective effects against oxidative damage.


A Thesis Submitted In Partial Fulfillment of the Requirements for the Degree of MASTER OF SCIENCE IN BIOLOGY from Texas A&M University – Corpus Christi in Corpus Christi, Texas.


Ligusticum porteri, herbal remedy, cytotoxicity, anti-oxidant, anti-inflammatory response, human peripheral blood lymphocytes, human promyelocytic leukemia cells.



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