Dry leaf extracts of Tinospora cordifolia (Willd.) Miers attenuate oxidative stress and inflammatory condition in human monocytic (THP-1) cells

Abstract

Background: Tinospora cordifolia (Willd.) Miers is known for its therapeutic value in Indian traditional medicine for treating diabetes, rheumatoid arthritis, jaundice and cardiac diseases. However, information regarding its protective role against inflammatory diseases at the molecular level is limited.

Purpose: The objective of the present work is to study the antioxidant and anti-inflammatory effect of alcoholic and water extracts of T. cordifolia (Willd.) Miers leaves in activated human monocytic THP-1 cells.

Study design/methods: Phytochemical analyses of the dry leaf extracts of T. cordifolia (Willd.) Miers prepared using the solvents alcohol (TCAE) or water (TCWE) are performed employing spectrophotometric methods for estimating total phenolic and flavonoid content, and the plant material was authenticated by detecting T. cordifolia (Willd.) Miers metabolite biomarkers using LC-MS/MS. Arachidonic acid (AA)- and lipopolysaccharide (LPS)-activated human monocytic (THP-1) cells were used as experimental models to investigate the antioxidant and anti-inflammatory activities of the plant extracts. Arachidonic acid (AA)-induced reactive oxygen species (ROS) in THP-1 cells were monitored by confocal microscopy/spectrofluorimetry and transcript of antioxidant enzyme catalase (CAT), by quantitative real time PCR. Lipopolysaccharide (LPS)-induced proinflammatory marker like TNF-α at transcription and protein levels in THP-1 cells were measured by quantitative real-time PCR or ELISA respectively. Further, the effect of T. cordifolia (Willd.) Miers extracts on LPS-induced NF-κB translocation, and IκB and P-IκB protein levels, were studied by immunoblotting and confocal microscopy.

Results: T. cordifolia (Willd.) Miers extracts exhibited significant amounts of total phenolic and flavonoid content, and LC-MS/MS analyses detected tinosponone, a TC-specific clerodane-derived diterpene. Both types of extracts attenuated AA-induced ROS generation via enhancing catalase enzyme activity in THP-1 cells. Real time PCR and ELISA experiments revealed that the elevated levels of LPS-induced TNF-α was remarkably attenuated in THP-1 cells pretreated with T. cordifolia (Willd.) Miers extracts. Western blot and confocal microscopy showed that the alcoholic extract's anti-inflammatory activity by attenuating NF-κB translocation into the nucleus in LPS-activated THP-1 cells via the inhibition of IκB degradation in the cytosol.

Conclusion: Our findings suggest that T. cordifolia (Willd.) Miers dry leaf extracts possess antioxidant and anti-inflammatory properties via upregulation of antioxidant enzymes and attenuation of NF- κB nuclear translocation in activated human monocytic (THP-1) cells, therefore the present study supports our proposed molecular basis for the traditional use of T. cordifolia (Willd.) Miers for treating various inflammatory diseases.

Keywords: Anti-inflammation; Antioxidant; Arachidonic acid; Lipopolysaccharide; Nuclear factor kappa-B; Tinospora cordifolia (Willd.) Miers; Tumor necrosis factor alpha.