Anti-inflammatory activity of total flavonoids from seeds of Camellia oleifera Abel.

Inflammation is the primary response to infection or injury that functions to clear the injurious material or agent and promote tissue repair. However, when inflammation persists, such as chronic inflammation, it can cause tissue damage and loss of function. Persistent inflammation is closely associated with many chronic diseases, such as cancer, arthritis, osteoporosis, asthma, Alzheimer’s disease, obesity, diabetes, and cardiovascular disease [1]. Numerous molecules such as cytokines, prostaglandins, and nitric oxide (NO) are involved in the induction and maintenance of the inflammatory response. Inhibition and/or down-regulation of these pro-inflammatory molecules may exert anti-inflammatory effects. In conventional therapy, steroidal anti-inflammatory drugs and non-steroidal anti-inflammatory drugs are used to treat acute inflammation. However, they fail to cure chronic inflammatory diseases, such as rheumatoid arthritis and osteoarthritis. Furthermore, these compounds have several undesired side effects. Recently, anti-inflammatory activity of natural bioactive compounds is attracting growing interest because these compounds may offer a safer and effective treatment for inflammation, especially for long-term use [2]. Camellia oleifera Abel. belongs to the Camellia genus in the Theaceae family, which is widely distributed in the central and southern China. Its seeds have been used as oil material in China for more than 1000 years. Camellia seed oil is not only used as cooking oil, but also traditionally applied as a medicine for stomach ache and burning injury in China. Pharmacological studies indicated that seeds of C. oleifera contain various bioactive substances including unsaturated fatty acids, flavonoids, saponins, polysaccharides, and proteins, and possess many bioactivities such as antioxidation, antibacterial, anticancer, hepatoprotection, and anti-inflammation [3]. Flavonoids are members of a class of natural compounds widely distributed in the plant kingdom, and possess many bioactivities including antioxidation, antibacterial, antiviral, and protective effects from many diseases such as cancer, cardiovascular, and inflammation [4]. Kaempferol and several kaempferol glycosides have been isolated from the seeds of C. oleifera, and kaemferol-3-O-[2-O-b-D-glucopyranosyl-6O-L-rhamnopyranosyl]-b-D-glucopyranoside and kaemferol3-O-[2-O-b-D-xylopyranosyl-6-O-a-L-rhamnopyranosyl]-b-Dglucopyranoside were identified as the two main flavonoids [5–10]. Various flavonoids such as genistein, quercetin, daidzein, flavone, isorhamnetin, naringenin, and pelargonidin from fruits, herbs, and spices have been found to possess important activity on the inflammatory process in vitro and in vivo [11]. However, little is known about the antiinflammatory effects of flavonoids from the C. oleifera seeds. In this study, total flavonoids were prepared from the 80% ethanol extract of C. oleifera seeds by semi-preparative HPLC (Waters, Milford, USA), and the composition was characterized by UPLC–UV–MS (Waters) analysis. The antiinflammatory activity of total flavonoids was evaluated by NO inhibitory assay in RAW 264.7 cells. Moreover, the effects of total flavonoids on the mRNA and protein expression levels of pro-inflammatory enzymes and cytokines including inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), tumor necrosis factor-a (TNF-a), and macrophage inflammatory protein-1a (MIP-1a) were determined by quantitative real-time-polymerase chain reaction (qRT-PCR) and western blot analysis, respectively. Four compounds (1–4) were detected in total flavonoid fraction of C. oleifera seeds by UPLC–UV–MS, and numbered corresponding to their order of elution (Supplementary Fig. S1). Quasi-molecular ions [M2H] for compounds 1–4 were observed at 755.19, 739.12, 725.19, and 593.17 in negative ion mode, respectively (Supplementary Fig. S2). The fragment ions at m/z 285 suggested that the aglycone was kaempferol. These results were consistent with the previous reports [5–8,10], and the four compounds were identified as kaemferol-3-O-[2-O-b-D-glucopyranosyl-6-O-L-rhamnopyranosyl]-b-D-glucopyranoside (compound 1), kaempferol 3-Ob-D-glucopyranosyl-(1!4)a-L-rhamnopyranosyl-7-O-a-Lrhamnopyranoside (compound 2), kaemferol-3-O-[2-O-bActa Biochim Biophys Sin 2014, xx: 1–3 |a The Author 2014. Published by ABBS Editorial Office in association with Oxford University Press on behalf of the Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences. DOI: 10.1093/abbs/gmu071.