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In this study, we evaluated anti-hepatitis B virus (HBV) activity of the extracts (dichloromethane, ethyl acetate, n-butanol, acetone, and methanol respectively) of fruit (peels and pulp) of Citrus madurensis Lour. (calamondin) using HBV transected cell line MS-G2. The ethyl acetate extracts from the peels strongly reduced the HBsAg expression of HBV in dose 50 μg/mL. According to the previous result, we chose this extract to elute by column chromatography and then separately eluted into 7 fractions. In dose in 50 μg/mL, fractions 2, 3, and 4 had lower HBsAg expression (compare with control). Furthermore, fraction 3 had a strongest inhibitory ability. The flavonoids (hesperidin, diosmin, neohesperidin, nobiletin, tangeretin, and 5-hydroxy-3’,4’,6,7,8-pentamethoxyflavone) were analysis in each fractions by high-performance liquid chromatography (HPLC). The results shown fraction 2, 3, 4 were detected containing nobiletin, tangeretin, and 5-hydroxy-3’,4’,6,7,8-pentamethoxyflavone. The anti-HBV activity of the flavonoids show that nobiletin, tangeretin, and also had a lower HBsAg expression. The IC50 value were nobiletin (33.9 μM), tangeretin (20.7 μM), and 5F (5.12 μM), respectively. Taken together, we suggest that 5F could be used as a standard marker for the anti-HBV effect of calamondin.

The Flavonoids in Citrus madurensis Lour and their Anti-Hepatitis B Virus Activity