Antioxidant potential, total phenolic and total flavonoid contents of Rhododendron anthopogonoides and its protective effect on hypoxia-induced injury in PC12 cells

Linlin Jing, Huiping Ma, Pengcheng Fan, Rongmin Gao, Zhengping Jia

Rhododendron anthopogonoides Maxim, a kind of traditional Tibetan medicine, has been used to remove body heat, body detoxification, cough, asthma, stomachic and swelling, eliminate abundant phlegm and inflammatory for a long time. In the present study, the total phenols and total flavonoid contents as well as antioxidative properties of the crude extract and solvent fractions of R. anthopogonoides were determined using seven antioxidant assays. Additionally, the protective effect of the extracts on hypoxia-induced injury in PC12 cells was also investigated. The content of total flavonoid and total phenolic was determined by the aluminum colorimetric method and Folin-Ciocalteu assay, respectively. In vitro antioxidant study, the effect of the crude extract and solvent fractions on total antioxidant activity, reducing power, DPPH radical scavenging, ABTS radical scavenging, superoxide radical scavenging, hydroxyl radical scavenging and nitric oxide radical scavenging were examined. The correlation between the phenolic and flavonoid content of the extracts and their antioxidant properties also analyzed. Furthermore, the protective effect of extracts on hypoxia-induced damage on PC12 cells was investigated by cell viability, lactate dehydrogenase (LDH) release, malondialdehyde (MDA) production and the activities of antioxidant enzymes. Our results showed that ethyl acetate and n-butanol fractions had higher content of phenolics and flavonoid compounds than other fractions. Except ABTS radical assay, n-butanol fraction exhibited the strongest antioxidant activity. While the hexane fraction showed the lowest antioxidant activity. Ethyl acetate also presented excellent antioxidant activity, which was just lower than n-butanol fraction. Significant correlation between the phenolic, flavonoid content of the extract and fractions with antioxidant assay excluding ABTS, OH scavenging assay was observed. Moreover, ethyl acetate and n-butanol fractions showed protective effect in PC12 cell under hypoxia condition, while crude extract and water fraction had no protective effect. In contrast, hexane fraction exhibited strong cytoprotective effect. Further study indicated that pretreatment of PC12 cells with ethyl acetate and n-butanol fractions, prior to hypoxia exposure, significantly increased the survival of cells and the activities of SOD, CAT, GSH-Px and T-AOC, as well as reduced the level of LDH and MDA. The gathered data demonstrated that ethyl acetate and n-butanol fractions were able to protect PC12 cells against hypoxia induced injury through direct free radical scavenging and modulation of endogenous antioxidant enzymes. These findings suggested that ethyl acetate and n-butanol fractions of R. anthopogonoides had significant antioxidant activity and could prevent PC12 cells against hypoxia-induced injury. So it might be regarded as an excellent source of antioxidants and had great potential to explore as therapeutic agent for preventing hypoxia related sickness in future.