Anti-inflammatory effects of ethyl acetate fractions from adlay bran extracts

Adlay (Coix lachryma-jobi L.var. ma-yuen Stapf.) bran has been reported that possesses many physiological activities such as immune modulation, anti-inflammation, and antitumor. Our laboratory desires to develope a product that possesses the effects of anti-inflammation, antitumor, and attenuating the side effects of radiotherapy. The aim of this study is to compare the adlay bran ethanolic extract (ABE) and adlay bran ethyl acetate extract (ABEa) that were respectively partitioned into n-hexane, ethyl acetate, n-butanol, and water fractions for the anti-inflammtory effects screening in lipopolysaccharide (LPS)-stimulated RAW 264.7 murine macrophage cell model and the differences of the yields.
The results showed that ethyl acetate fraction (ABE-Ea/ABEa-Ea) has the strongest inhibition effect on nitrite production in both extracts, and the yields of them were 0.91% and 1.16% of wet weight of fresh adlay bran, respectively. The yield of ABEa-Ea was 27.5% higher than that of ABE-Ea. ABE-Ea and ABEa-Ea were further subfractionated into 30% ethyl acetate/n-hexane (Ea/Hex), 80% Ea/Hex, 100% Ea and 95% ethanol (EtOH) by silica gel chromatography. The results showed that ABE-Ea-80% Ea/Hex, ABE-Ea-100% Ea, ABE-Ea-95% EtOH, ABEa-Ea-80% Ea/Hex, and ABEa-Ea-100% Ea could inhibit the nitrite production in LPS-stimulated RAW 264.7 murine macrophage cells. In addition, the above five subfractions could inhibit the inducible nitric oxide synthase (iNOS) and cyclooxygenase (COX)-2 protein expressions at 50 μg/mL. Among of these five subfractions, the ABE-Ea-95% EtOH could completely inhibit iNOS and COX-2 protein expressions. Except for the ABEa-Ea-100% Ea, other four subfracions significantly inhibited the secretion of interleukin (IL)-6. However, only the ABE-Ea-95% EtOH and ABEa-Ea-80% Ea/Hex could significantly inhibit the secretion of tumor necrosis factor (TNF)-α. Based on the HPLC-MS analysis, the more abundant phenolic compounds in ABE-Ea-95% EtOH in orders are p-hydroxybenzoic acid (2770 μg/g sample), p-coumaric acid (750 μg/g sample), and quercetin (433 μg/g sample); those in ABEa-Ea-80% Ea/Hex are tangeretin (1579 μg/g sample), nobiletin (1423 μg/g sample), and p-hydroxybenzoic acid (991 μg/g sample). In conclusion, ABE-Ea-95% EtOH and ABEa-Ea-80% Ea/Hex had the most potential for anti-inflammation, and the different phenolic compounds might attribute to the anti-inflammatory activities.