| Hepatitis B virus (HBV) infection causes major public health problems worldwide. Nucleoside analog are the major drugs for HBV infections. However, significant side effects of these drugs and inevitable drug resistance have been noted. Therefore, it is important for our country to research and develop new anti-HBV drugs from the Traditional Chinese Medicine.Anti-HBV activity of the 70% ethanol extract from Euphorbia humifusa Willd. (EHE) has been found for the first time and then two active fractions including EHE-2 and EHE-3 were enriched from EHE under the guidance of the anti-HBV assay in vitro. In this dissertation, the phytochemistry of the active fractions from Euphorbia humifusa was studied deeply and systematically, anti-HBV activities of the flavones from the active fraction were evaluated, structure-activity relationships of these flavones were explained and further antiviral mechanism of the active compounds were discussed.Euphorbia humifusa Willd. (Euphorbiacea) is used for the treatment of bacillary dysentery, enteritis, cough up blood, blood-spitting, bloody stool, uterine bleeding, bleeding wound, jaundice with damp-heat pathogen and so on.The chemical investigation of the active fractions of this plant resulted in the isolation of 40 compounds by a variety of chromatography methods, and 38 compounds were identified on the basis of spectral data including MS, 1D-NMR, 2D-NMR, NOESY, CD and UV, as follows: phyllanthusiin E methyl ester (EHE-2-3-1), methyl brevifolincarboxylate (EHE-2-3-2), brevifolin carboxylic acid (EHE-2-3-3), 1-O-ethyl-3,6-O-(R)-hexahydroxydiphenoyl-(1C4)-β-D-glucopyranoside (EHE-2-3-4), humifusaone (EHE-2-3-5), furosin (EHE-2-3-6), valoneaic acid dilactone (EHE-2-3-7), 1-(2',3',4',5'-tetrahydroxypentyl)-6,7-dimethyl-quinoxaline- 2,3(1H,4H)-dione (EHE-2-3-8), gallic acid (EHE-2-3-10), methyl gallate (EHE-2-3-11), 6,8-di-C-β-D-glucopyranosylapigenin (EHE-2-3-12), 7''-ethyl- sanguisorbic acid dilactone (EHE-2-3-13), (1R,9S,11R)-5(Z),7(Z)-diene-6-carbonyl- 12-hydroxyl-caryophyllane (EHE-2-3-14), 1-O-methyl-6-O-p-digalloyl-α-D- glucopyranoside (EHE-2-3-15), 1-O-ethyl-6-O-p-digalloyl-α-D-glucopyranoside (EHE-2-3-16), 1-O-methyl-6-O-p-digalloyl-β-D-glucopyranoside (EHE-2-3-17), (1R,8S,9S,11R)-5(Z)-ene-6-carbonyl-8,12-dihydroxyl-caryophyllane (EHE-2-3-18), sanguisorbic acid dilactone (EHE-2-4-1), brevifolin (EHE-3-4-1), luteolin-7-O-β-D- glucopyranoside (EHE-3-4-2), luteolin-7-O-(6''-O-trans-feruloyl)-β-D- glucopyranoside (EHE-3-4-3), apigenin-7-O-β-D-glucopyranoside (EHE-3-4-4), apigenin-7-O-(6''-O-galloyl)-β-D-glucopyranoside (EHE-3-4-5), luteolin-7-O-(6''-O -coumaroyl)-β-D-glucopyranoside (EHE-3-4-6), apigenin-7-O-β-D-lutinoside (EHE-3-4-7), apigenin-7-O-β-D-apiofuranosyl(1→2)-β-D-glucopyranoside (EHE-3-4-8), apigenin (EHE-3-4-9), quercetin-3-O-α-L-rhamnosyl(1→6)-β-D- galactoside (EHE-3-4-10), quercetin-3-O-β-D-glucopyranoside and quercetin-3-O-β-D-galactoside (EHE-3-4-11), ellagic acid (EHE-3-4-12), hesperidin (EHE-3-4-13), ethyl brevifolincarboxylate (EHE-3-4-15), 3,3'-di-O-methyl ellagic acid-4-O-β-D- glucopyranoside (EHE-3-4-16), ethyl gallate (EHE-3-4-17), 3,3'-di-O-methyl ellagic acid (EHE-3-5-1), luteolin (EHE-3-5-2) and quercetin (EHE-3-5-3). Among these compounds, EHE-2-3-4, EHE-2-3-5, EHE-2-3-13, EHE-2-3-14, EHE-2-3-16, EHE-2-3-18 and EHE-3-4-5 are new compounds. EHE-2-3-1, EHE-2-3-2, EHE-2-3-3, EHE-2-3-6, EHE-2-3-7, EHE-2-3-8, EHE-2-3-11, EHE-2-3-12, EHE-2-3-15, EHE-2-3-17, EHE-2-4-1, EHE-3-4-3, EHE-3-4-6, EHE-3-4-7, EHE-3-4-8, EHE-3-4-9, EHE-3-4-10, EHE-3-4-11, EHE-3-4-13, EHE-3-4-15, EHE-3-4-16, EHE-3-4-17 and EHE-3-5-1 were isolated from the plant for the first time.Through the anti-HBV assay of flavones from the active fraction, 4 active compounds (EHE-3-4-2, EHE-3-4-3, EHE-3-4-4 and EHE-3-4-5) were reported for the first time. EHE-3-4-2 blocked effectively the secretion of HBsAg with IC50 value of 20μg·mL-1 (44.64μΜ). EHE-3-4-3 appeared to significantly downregulate the secretion of HBeAg with IC50 value of 75.81μg·mL-1 (121.49μΜ) and the inhibition ratio of the secretion of HBsAg by this compound was 18.6% (at 80μg·mL-1). EHE-3-4-4 and EHE-3-4-5 blocked the production of HBeAg and HBsAg from HepG2.2.15 in a dose-dependent manner with IC50 values of 34.71μg·mL-1 (80.34μΜ) and 16.27μg·mL-1 (37.66μΜ), 75.75μg·mL-1 (129.71μΜ) and 36.90μg·mL-1 (63.18μΜ), respectively.The structure-activity relationship of these flavones showed that parent structure was closely relevant to their activities (agigenin > luteolin > quercetin). It was found that the number of glucoside determined their activities (flavone monoglucosides > flavone diglucosides) and cytotoxicities (flavones > flavone monoglucosides > flavone diglucosides). In addition, the substitution of acyl group on glucoside may be important to keep their anti-HBV activities (galloyl > feruloyl > coumaroyl).According to the study on the anti-HBV compounds of Euphorbia humifusa, 38 compounds were isolated and elucidated including 15 flavones, 20 phenols, 2 sesquiterpenoids and 1 alkaloid. Among these compounds, 7 compounds are new compounds and 24 compounds were isolated from this plant for the first time. Anti-HBV activities of 4 flavones were reported for the first time and further the structure-activity relationships were revealed.In the future, further active evaluation in vivo of the active compounds would help to determine anti-HBV candidates. And further officinal drug research on the active fractions would develop the fifth new drugs of the Traditional Chinese Medicine.
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