Chemical characterization and cerebroprotective effect of methanolic root extract of Colebrookea oppositifolia in rats.

ETHNOPHARMACOLOGICAL RELEVANCE

Colebrookea oppositifolia Smith is one of the extensively used plants to treat neurological conditions such as epilepsy by the various ethnic communities in sub-Himalayan regions of India such as Bhoxa, Tharu and nomadic Gujjars.

AIM OF THE STUDY

This study was conducted to evaluate the cerebroprotective effect of C. oppositifolia methanolic root (MeCO) extract in Wistar rats.

MATERIALS AND METHODS

The MeCO was characterized for total phenolic content and later subjected for detailed liquid chromatography-mass spectrometry analysis. Further, it was evaluated for in vitro antioxidant activity using 2, 2-diphenyl-1-picrylhydrazyl, ferric reducing antioxidant power and oxygen radical absorbance capacity assays. In addition, the MeCO was investigated on generation of ROS, nitrite, and TNF-α in LPS-stimulated RAW 264.7 cell lines. Finally, the cerebroprotective effect of MeCO was examined against global ischemia and reperfusion (I/R)-induced brain injury in Wistar rats. Male Wistar rats were allocated in to five groups (G-I to G-V, n = 10). G-I and G-II served as sham control and I/R control, respectively, and received only vehicle (0.5% w/v carboxy methyl cellulose, 10 ml/kg, p.o.). G-III served as reference standard and received quercetin (20 mg/kg, p.o.). G-IV and G-V animals received 200 and 400 mg/kg oral doses of MeCO, respectively. All the treatments were given for a period of seven days and the parameters such as neurobehavioral (neurological, and cognitive), and motor functions, biochemical (enzymatic and non-enzymatic antioxidants, TNF-α, IL-6, IL-10, ICAM-I), morphological (cerebral edema and infarct area) and histopathological evaluations were performed.

RESULTS

The MeCO showed a total phenolic content of 137.28 mg gallic acid equivalents/g, and LC-MS/MS analysis of MeCO showed presence of acteoside, gossypin, quercetin and ferulic acid as major ingredients (6680.3, 1.55, 3.52 and 431.1 ng/mg). In in vitro antioxidant assays, the MeCO exhibited potent activity with IC of 49.10 µg/ml in DPPH assay; FRAP and ORAC values of 1180.5 and 2983.5 respectively. Furthermore, the MeCO significantly inhibited generation of ROS, nitrite and TNF-α in LPS-stimulated RAW 264.7 cell lines. Sixty min of global ischemia with 24 h reperfusion produced substantial alterations in neurobehavioral functions in the I/R control group compared to sham control. In addition, a significant reduction in catalase and superoxide dismutase activities was observed. Moreover, lipid peroxidation increased and reduced glutathione levels decreased significantly. Furthermore, the levels of pro-inflammatory cytokines (TNF-α, IL-6, and ICAM-I) increased significantly and those of anti-inflammatory (IL-10) decreased. I/R insult increased the brain volume and aggravated cerebral infarct formation. Histopathological examination of the rat brain revealed vascular congestion, cerebral edema, leukocyte infiltration, and brain tissue necrosis. Interestingly, seven days pretreatment with MeCO (200 and 400 mg/kg) alleviated all the I/R-induced perturbances (neurobehavioral, and motor functions, biochemical, morphological and histopathological) compared with the I/R control.

CONCLUSIONS

The MeCO exhibit potent cerebroprotective activity through its potent antioxidant and anti-inflammatory mechanisms, and hence may be useful in the management of ischemic stroke and associated complications.