Anti-hepatitis B virus activities of friedelolactones from Viola diffusa Ging.
Background: Hepatitis B virus (HBV) infection is the major factor of causing hepatitis B, cirrhosis and liver cancer. Interferon and nucleoside drugs, the main drugs to treat HBV infection, have disadvantages of scavenge difficulty and drug resistance respectively. Viola diffusa Ging is used as a traditional Chinese herbal medicine for the treatment of hepatitis.
Purpose: The aim of the study is to investigate the chemical constituents of Viola diffusa Ging and their anti-HBV activity.
Methods: Chemical constituents were extracted and purified by ethanol reflux extraction and chromatographic separation technology including D-101 Macroporous resin, silica gel, Sephadex LH-20 and preparative thin-layer chromatography. Their structures were elucidated on the basis of extensive NMR and MS data. Cytotoxicity and inhibiting effects on HBsAg and HBeAg secretion of HepG2.2.15 of all compounds except 10 were studied by MTT method and ELISA method.
Results: Three friedelolactones with naturally occurring seco-ring-A friedelane triterpenoids, 2/l-hydroxy-3, 4-seco-friedelolactone-27-oic acid (1), 2[beta], 2[beta]-dihydroxy-3,4-seco-friedelolactone-27-oic acid (2) and 2[beta], 30[beta]-dihydroxy-3,4-seco-friedelolactone-27-lactone (3), and a stigmastane, stigmast-25-ene- 3[beta],5[alpha],6[beta]-triol (11) together with nine known compounds were isolated from the whole plant of Viola diffusa G. (Violaceae). Compounds 1-3, 9, 11,12 exhibited significant activities of blocking both HBsAg and HBeAg secretion, and compound 4, 6, 7, 8 selectively inhibited HBeAg secretion while compound 13 selectively inhibited HBsAg secretion. [IC.sub.50] values of compounds 1 and 2, 26.2 [micro]M and 33.7 [micro]M for HBsAg, 8.0 [micro]M and 15.2 [micro]M for HBeAg, was significantly lower than that of positive control lamivudine.
Conclusion: Compounds 1-3, 11 are new compounds never reported before and the promising results demonstrate the potential of compound 1-3, 9, 11, 12 for the treatment of HBV infection.
Viola diffusa G.
HepG 2.2.15 cells
Highly oxidized metabolites are often found particularly among the tetracyclic triterpenes and steroids. In the oxidation process, ring-fission and subsequent modification of the skeleton is frequently accompanied. However, ring-fission is not restricted to these compounds; seco-derivatives have also been found among the pentacyclic triterpenes. However, naturally occurring seco-ring-A friedelane triterpenoids with the A ring expanded to seven-membered ring containing compounds, are relatively rare among secondary metabolites. Apetalactone was the first 3,4-seco-ring-A friedelane lactone, found in the leaf of two Calophyllum species (Govindachari et al. 1968). Two closely related compounds friedelolactone (Katai et al. 2006) and itoaic acid (Chai et al. 2009) were isolated respectively from Euonymus japonicus and ltoa orientalis. Beside 3,4-seco compounds, a natural 2,3-secotriterpenelithocarpic lactone (Hui et al. 1975) was described in the literature. Recently a new type of triterpene anhydride lobatanhydride (Rodriguez et al. 2009) was found from Crossopetalum lobatum. Although the known seco-ring-A friedelane lactones from nature are rare, they have been reported to have significant biological activity that includes, promotion rat mesenchymal stem cells (rMSCs) proliferation (Wang et al. 2010), cytotoxicty (Reyes et al. 2010), anti-inflammatory (Chai et al. 2009) and anti-dermatophytic activities (Kuiate et al. 2007).
Viola of Violaceae, containing about more than 500 species, is widely distributed in temperate zone, tropic and subtropics, and mainly in temperate zone of northern hemisphere. There are about 111 species of Viola in China. They distribute in both southern and northern area and grow in the mountain forest, hillside meadow, roadside shrubs (Delectis Florae Republicae PoPularis Sinicae Agendae Academiae Sinicae Edita 1991). Since Peter et al. (Peter et al. 1980) identified two isomerides of violaxanthin from Viola tricolor by MS, 1R and UV, the researches on the chemical constituents of Viola plants began to rise. At present, flavonoids (Lee et al. 1993), coumarins (Qin et al. 1994), terpenoids (Hu and Ding 1987), sterols (Asilbekova et al. 1999), polypeptides (Ulf et al. 2004), organic acids (Liu et al. 2011) and other components (Peter et al. 1980) have been isolated from Viola genus. Furthermore, the antibacterial (Zhu et al. 1986), anti-inflammatory (Li et al. 2010), anti-viral (Ngan et al. 1988), anti-oxidation (Shen and Xie 2009) and inhibiting K562 leukemia cell (Li et al. 2009) activities of these constituents have also been reported.
Viola diffusa G. is an annual or perennial herb of Viola genus mainly distributed in the southern part of P.R. China, and has been used as a traditional Chinese herbal medicine for the treatment of hepatitis, pleuritis, conjunctivitis, venomous snake bite and ulcerative carbuncle (Fujian Science and Technology Committee 1990). So far, its chemical constituents have not been systematically investigated. Here, we isolated of three new friedelolactones, violaic A (1), violaic B (2) and violalide (3), a new stigmastane, stigmast-25-ene-3[beta],5[alpha], 6[beta]-triol (11), and nine known compounds, epifriedelanol (4), friedelin (5), derosterol (6), derosterol galactoside (7), decorinone (8), decortinol (9), isodecortinol (10), cerevisterol (12) and palmitic acid (13) (Fig. 1), and evaluated their HBV surface antigen (HBsAg) and e antigen (HBeAg) secretion blocking effects on HBV-infected HepG2 2.2.15 cells of compounds 1-9,11,12,13. This is the first report of friedelolactones from the Violaceae.
Material and methods
The whole plants of Viola diffusa G. were collected in Yongchun, Fujian, China, in August 2009 and authenticated by Professor Ji Ma, School of Chinese Medicine, Southern Medical University. A voucher specimen (No. 200908101) was deposited in the Chemistry Department of Traditional Chinese Medicine, Southern Medical University.
Extraction and isolation
The dried materials (10 kg) were successively extracted with 95% EtOH three times under reflux, 1.5 h each time. The extract was concentrated under vacuum to give a residue (0.9 kg), which was suspended in [H.sub.2]O (5.5 L) and sequentially partitioned with petroleum ether (3x4 L), EtOAc (3x4 L), and n-BuOH (3x4 L). The n-BuOH portion was separated by D101 macroporous resin, repeated silica gel and Sephadex LH-20 column chromatography (CC) and preparative TLC to afford violaic A (1), violaic B (2), violalide (3) and palmitic acid (13). Epifriedelanol (4), friedelin (5) and clerosterol (6) was isolated from petroleum ether portion by repeated CC and preparative TLC on silica gel. Clerosterolgalactoside (7), decorinone (8), decortinol (9), isodecortinol (10), stigmast-25-ene-3[beta],5[alpha],6[beta]-triol (11) and cerevisterol (12) was isolated from ethyl acetate portion by repeated silica gel, Sephadex LH-20 CC and preparative TLC on silica gel (Fig. 1).
Compound 1 was obtained as colorless needle crystals (in MeOH). The molecular formula, [C.sub.30][H.sub.48][O.sub.5], was deduced by the positive HRES-IMS (m/z 511.3393 [[M + Na].sup.+]), indicating 7 degrees of unsaturation. The [sup.1]H NMR data (Table 1) exhibited signals of four angle methyls [[[delta].sub.H] 0.87 (3H, s, Me-24), 0.87 (3H, s, Me-25), 1.12 (3H, s, Me-26) and 1.21 (3H, s, Me-28)], two gem-dimethyl [[[delta].sub.H] 0.99 (3H, s, Me-29), 0.93 (3H, s, Me-30)], a doublet methyl [[[delta].sub.H] 1.23 (3H, d, J = 6.5 Hz, Me-23)], and two hydroxymethines [[[delta].sub.H] 4.23 (q, J = 6.5 Hz, H-4), 4.34 (dd, J = 12.0, 2.0 Hz, H-2)]. By the analysis of [sup.13]C NMR (Table 2) and DEPT spectra, compound 1 exhibited 30 carbon atoms, including seven methyls, ten methylenes, five methines and eight quaternary carbons. Two methines ([[delta].sub.C] 84.9,69.3) were attributed to those attached to an oxygen atom, and two of quaternary carbons ([[delta].sub.C] 181.6,177.0) were attributed to carbonyls.
In the HMBC and HSQC spectra, correlations from H-4 to C-23, from H-l, H-2, and H-4 to C-3, from H-10 to C-l, C-2, from H-4 to C-10, and from H-24 to C-4, C-5, and C-10 were observed and suggested a seven-membered lactone ring in compound 1 (Fig. 2). Comparison of the spectroscopic data with those of 2[alpha]-hydroxyfriedelolactone (Patra 1990) demonstrated that compound 1 possessed a skeleton of seco-friedelolactone similar to that of 2[alpha]-hydroxyfriedelolactone except for C-13 substituent and 2-OH configuration. The Me-13 in 2a-hydroxyfriedelolactone was replaced by a COOH group in compound 1. The assignment was supported by the [sup.13]C NMR changes at C-13, C-12 and 30 mass units increase in HRESIMS. The configuration of 2-OH was probably opposite to 2[alpha]-hydroxyfriedelolactone deduced from the chemical shift difference at C-l and C-2 between them. The NOESY (Fig. 2) correlation of H-2 with H-4 and H-10 further supported that the 2-hydroxyl group was [beta]-oriented. Thus the structure of compound 1 was deduced to be 2[beta]-hydroxy-3, 4-secofriedelolactone-27-oic acid, and it was named violaic A.
Compound 2 was obtained as white granular crystals (in MeOH). The molecular formula, [C.sub.30][H.sub.48][O.sub.6], was deduced by the negative HRESIMS (m/z 503.3393 [[M].sup.+], indicating 7 degrees of unsaturation. The [sup.1]H NMR data (Table 1) exhibited signals for five angle methyls [[[delta].sub.H] 0.72 (3H, s, Me-24), 0.79 (3H, s, Me-25), 0.96 (3H, s, Me-26), 0.92 (3H, s, Me-29) and 0.91 (3H, s, Me-30) ], a doublet methyl [[[delta].sub.H] 1-05 (3H, d, J = 6.5 Hz, Me-23)], two hydroxymethines [[[delta].sub.H] 4.38 (q, J = 6.5 Hz, H-4), 4.41 (dd, J = 12.0,2.0 Hz, H-2)], and one hydroxylmethylene [[[delta].sub.H] 3.47 (dd, J = 10.0,4.0 Hz, H-28a), 3.27 (dd, J = 10.0,4.0 Hz, H-28b)]. By the analysis of [sup.13]C NMR (Table 2) and DEPT spectra, compound 2 exhibited 30 carbon atoms, including six methyls, eleven methylenes, five methines and eight quaternary carbons. One of methylenes ([[delta].sub.C] 65.5) and two of methines ([[delta].sub.C] 82.3,68.0) were attributed to those attached to an oxygen atom, two of quaternary carbons ([[delta].sub.C] 175.4,177.5) were attributed to carbonyls.
In the HMBC and HMQC spectra, correlations from H-4 to C-23, from H-l, H-2, and H-4 to C-3 were observed (Fig. 3). The [sup.13]C NMR of compound 2 was similar to that of compound 1, except the disappearance of a methyl (C-28) in compound 1 and the presence of an additional hydroxymethylene (C-28) in compound 2. The assignment was supported by the downfield shift of C-17 and upheld shift of C16, C-18 and C-22 in [sup.13]C NMR and 16 mass units increase in HRESIMS. The [beta]-oriented configuration of 2-OH was the same as the compound 1 and opposite to 2[alpha]-hydroxyfriedelolactone deduced by the chemical shift change at C-1 and C-2 between compound 2 with compound 1 and 2[alpha]-hydroxyfriedelolactone. Thus the structure of compound 2 was deduced to be 2[beta], 28[beta]-dihydroxy-3,4-seco-friedelolactone-27-oic acid, and it was named violaic B.
Compound 3 was obtained as white cluster crystals (in MeOH). The molecular formula, [C.sub.30][H.sub.46][O.sub.6], was deduced by the positive HRESIMS (m/z 525.3189 [[M + Na].sup.+], indicating 8 degrees of unsaturation. The [sup.1]H NMR data (Table 1) exhibited signals for five angle methyls [[[delta].sub.H] 0.88 (3H, s, Me-24), 0.83 (3H, s, Me-25), 0.99 (3H, s, Me-26), 1.13 (3H, s, Me-28) and 0.98 (3H, s, Me-29)], a doublet methyl [[[delta].sub.H] 1.22 (3H, d, J = 6.5 Hz, Me-23), three hydroxymethines [SH 4.34 (br s, H-15), 4.17 (q, J = 6.5 Hz, H-4), 4.29 (dd, J = 12.0, 2.0 Hz, H-2)], and one hydroxylmethylene [[[delta].sub.H] 3.24 (br s, H-30)]. By the analysis of [sup.13]C NMR (Table 2) and DEPT spectra, compound 3 exhibited 30 carbon atoms, including six methyls, ten methylenes, six methines and eight quaternary carbons. One of methylenes ([[delta].sub.C] 74.3) and three of methines ([[delta].sub.C] 84.5, 80.5, 69.1) were attributed to those attached to an oxygen atom, two of quaternary carbons ([[delta].sub.C] 179.9,176.9) were attributed to carbonyls.
[sup.13]C NMR spectrum data of compound 3 was similar to that of both compound 1 and compound 2 (Table 2). The observed difference between compound 3 and compound 1 was the disappearance of a methyl (C-30) in compound 1 and the presence of additional hydroxymethylene (C-30) and hydroxymethine (C-15) in compound 3. The assignment was supported by the downfield shift of C-4, C-15, C-16, C-20, C-30 and upheld shift of C-8, C-13, C-19, C-21, C-29, and HSQC spectra correlations (Fig. 4). The one more degree of unsaturation of compound 3 was attributed to the new lactone ring between C-27 and C-15, it also indicated by the HSQC correlations from H-15 to C27. The [beta]-oriented configurations of 2-OH and 30-C[H.sub.2]OH were also deduced by the NOESY correction of H-2 with H-4 and H-10, H-28 with H-26, H-25 and H-30 (Fig. 5). Thus the structure of compound 3 was deduced to be 2[beta], 30/1-dihydroxy-3,4-seco-friedelolactone-27-lactone, and it was named violalide.
Compound 11 was obtained as white crystals. The molecular formula, [C.sub.29][H.sub.50][O.sub.3], was deduced by EIMS (m/z 446), the positive HREIMS (446.3749, calcd. 446.3754, indicating 7 degrees of unsaturation) and NMR. The [sup.1]H NMR data (Table 1) exhibited signals of three methyls [[[delta].sub.H] 0.60 (3H, s, Me-18), 1.00 (3H, s, Me-19) and 1.52 (3H, s, Me-26)], a doublet methyl [Sh 0.86 (3H, d J = 6.5 Hz, Me-21)], a triplet methyl [[[delta].sub.H] 0.75 (3H, t,/= 7.5 Hz, Me-29), two olehnic protons [[[delta].sub.H] 4.72 (1H, br s, H-27a), 4.63 (1H, br s, H-27b)], and two protons geminal to an oxygen atom [[[delta].sub.H] 3.78 (1H, m, H-3), 3.29 (1H, br s, H-6)]. By the analysis of [sup.13]C NMR (Table 2) and DEPT spectra, compound 11 exhibited 29 carbon atoms, including five methyls, twelve methylenes, eight methines and four quaternary carbons. Two of methines ([[delta].sub.C] 65.8, 74.1) and one of quaternary carbons ([[delta].sub.C] 74.3) were attributed to those attached to an oxygen atom, one of methylenes ([[delta].sub.C] 111.7) and one of quaternary carbons ([[delta].sub.C] 146.9) were attributed to C=C double bond.
[sup.13]C NMR spectrum data of compound 11 was similar to that of compound 6 of steroids with chemical shift of ring B, C and carbon atoms of branch ([C.sub.11]-[C.sub.29]). The main difference between compound 11 and compound 6 was the disappearance of two carbon atoms linked by C=C double bond ([[delta].sub.C]: 140.8, 121.7) in compound 6 and the presence of two additional carbon atoms linked with oxygen atom ([[delta].sub.C]: 74.3, 74.1) in compound 11, inferring that compound 11 was trihydroxyl sterol compound whose hydroxyls were from oxidation of the C=C double bond of compound 6. By further comparison with the [sup.13]C NMR spectrum data of Cholestan-3[beta],5[alpha],6[alpha]-triol (Konno and Hikino 1976), Cholestan-3[beta],5[alpha],6[alpha]-triol (Konno and Hikino 1976) and Cholebrin B (Yang et al. 2000), the similarity of [sup.13]C NMR spectrum data of mother nucleus of steride between compound 11 and Cholestan-3[beta],5[alpha],6[beta]-triol demonstrated that compound 11 possessed mother nucleus structure of 3[beta],5[alpha],6[beta]-trihydroxycholestane (Fig. 6). Thus the structure of compound 11 was deduced to be stigmast-25-ene-3[beta],5[alpha],6[beta]-triol.
Nine known compounds were identified as epifriedelanol (4) (Xie 2006), friedelin (5) (Xie 2006), clerosterol (6) (Guevara et al. 1989), derosterol galactoside (7) (Ahmad et al. 1993), decorinone (8) (Khoo 2005), decortinol (9) (Ahmad et al. 1993), isodecortinol (10) (Ahmad et al. 1993), cerevisterol (12) (Ahmad et al. 1993) and palmitic acid (13) (Yu and Yang 1999) by comparison of their [sup.1]H NMR, [sup.13]C NMR and HRESIMS spectroscopic data with those reported in the literature.
HepG2.2.15 is a human hepatoblastoma cell line stably transfected with cloned HBV DNA to produce viral particles, which was used as the model system. The HepG 2.2.15 cells were routinely cultured in Dulbecco's modified Eagle's medium (DMEM, Gibco, USA) supplemented with 10% (v/v) fetal calf serum (Gibco, USA), 100 [micro]g/ml penicillin G, 100 [micro]g/ml streptomycin, 0.03% (v/v) L-glutamine, and 150 [micro]g [ml.sup.-1] G418 (Sigma) under 5% C[O.sub.2] atmosphere at 37[degrees]C.
The HepG2.2.15 cell suspensions with 3 x [l0.sup.4] cells per well were seeded in 96-well plates and cultured for 24 h. Then sequential dilutions of the purified compounds in DMSO (ranging from 3.125 to 100 [micro]mol/1) were added to the medium and cultured for 3 days. Briefly, 20 [micro]l of MTT (5 g/1) was added to each well and further incubated for 4 h at 37[degrees]C. Then, the culture medium was removed from each well and 150 [micro]l of DMSO was added to dissolve the purple formazan of MTT. The absorbance was measured at 570 nm. The viability of the cultured cells with tested compounds was measured by the MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) method. The percent of cell death (%) was calculated by comparing the treatment group with the tested compounds and the solvent control group with DMSO.
HBsAg, HBeAg assay
After 2.2.15 cells of each group cultured for 3 days as above, the culture supernatants were collected to determine the HBsAg and HBeAg secretions using the enzyme-linked immunosorbent assay (ELISA) kits according to the manufacturer's protocol (Shanghai Kehua Biotech Co., Ltd.). The inhibiting rates (%) were calculated by comparing the treatment group with the tested compounds and the solvent control group with DMSO. The percent of cell death (%) = [1 - OD value of sample well/OD value of DMSO well] x 100%. The percent of inhibition (%) = [1 - OD value of sample well/OD value of DMSO well] x 100%. The [TC.sub.50] (50% toxicity concentration) and [IC.sub.50] (50% inhibition concentration) of the samples was estimated from the Reed-Muench (Reed and Muenc 1938).
All data were expressed as means [+ or -] standard error (SEM). The significance of differences was evaluated by means of LSD-test or Dunnett's T3-test. P values lower than 0.05 were considered to indicate significance.
Results and discussion
Anti-HBV activity and cytotoxicity of compounds
Hepatitis B virus is a partly double-stranded DNA virus with several serological markers: HBsAg and anti-HBs, HBeAg and anti-HBe, and anti-HBcIgM and IgG (Trepo et al. 2014). It has been suggested that HBsAg quantification reflects the concentration of the covalently closed circular DNA (cccDNA), the template of hepatitis B virus transcription, which plays a key role in the life cycle of the virus and permits the persistence of infection (Martinot-Peignoux et al. 2014), and that HBsAg is useful as an on-treatment response marker to predict the natural course of HBV-infection, HBV-coinfection and for treatment guidance (Honer Zu Siederdissen and Cornberg 2014). Furthermore, the high positive rate and contents of HBV-DNA in HBeAg positive patients farther confirmed that HBeAg was a reliable indicator for active HBV replication (Pan 2009). HepG2 2.2.15 is a human hepatoblastoma cell line transfected with cloned HBV DNA, which can secret HBsAg, HBeAg and Dane particles stably in the supernatant of culture, so it was commonly used as the model system for screening anti-HBV activities in vitro. When the secretion of HBsAg and HBeAg in the supernatant of HepG2.2.15 cell lines was inhibited by exogenous substances, it means the substances can reduce the infection and replication of hepatitis B virus, and showed anti-HBV activities. In other words, the inhibitors of HBeAg and HBsAg secretion may be potential candidates of anti-HBV agents. So far, common inhibitors of HBV are divided into interferons such as IFN-[alpha], PEG-IFN-[[alpha].sub.2a], and nucleotide analogs like adefovir dipivoxil, lamivudine and entecavir (Brent et al. 2006). The former are proteins made and released by host cells in response to the presence of viruses which interfere on the viral replication by protecting cells from virus infection, and the latter inhibit the reverse transcriptase of hepatitis B competitively and act as a chain terminator of DNA synthesis of virus. Among the inhibitors of hepatitis B, the nucleotide analogs are used commonly as positive control for the anti-HBV activity evaluation of potential inhibitors (Cao et al. 2013; Guo et al. 2013; Huang et al. 2013).
Compounds 1-9,11,12,13 were evaluated for their anti-HBV activities in the HBV-transfected HepG 2.2.15 cell line. Results including their activities and cytotoxicities were shown in Table 3. Compounds 1-3,9,11,12 inhibited the secretion of both HBsAg and HBeAg, with [IC.sub.50] values of 26.2, 8.0 [micro]M to compound 1, 33.7, 15.2 [micro]M to compound 2, 104.0, 21.6 to compound 3, 62.0, 92.5 [micro]M to compound 9, 32.7, 21.8 [micro]M to compound 11 and 112.8, 35.0 [micro]M to compound 12 respectively. The activities of compound 1, 2 and 11 were stronger than that of positive control lamivudine, a reverse transcriptase inhibitor of hepatitis B, with an [IC.sub.50] value against HBsAg of 41.3 [micro]M and no activity against HBeAg. However, compound 11 showed the highest toxicity. Compound 4, 6,7,8 showed selective inhibition on HBeAg and no activity on HBsAg, while compound 13 selectively inhibited HBsAg secretion. Compound 5 did not exhibit any activity in these experiments. On the basis of the structures and activities of compounds 1-3, it was concluded that the seven-member lactone ring A in friedelanes may be an important part to their anti-HBV activities.
Conflict of interest
The authors declare that there are no conflicts of interest.
Abbreviations: [CC.sub.50], the concentration to inhibit Hep G2.2.15 cell proliferation by 50%; DEPT, distortionless enhancement by polarization transfer; DMEM, Dulbecco's modified Eagle's medium; DMSO, dimethyl sulfoxide; ELISA, enzyme-linked immunosorbent assay; HBeAg, hepatitis B e antigen; HBsAg, hepatitis B surface antigen; HBV, hepatitis B virus; HMBC, [sup.1]H detected heternuclear multiple bond correlation; HMQC, [sup.1]H detected heternuclear multiple quantum coherence; [IC.sub.50], 50% inhibition concentration against HBV synthesis; MTT, methyl thiazolyl tetrazolium; NOESY, nuclear Overhauser enhancement spectroscopy; SI, selectivity index; TLC, thin layer chromatography.
Received 6 May 2014
Revised 30 April 2015
Accepted 4 May 2015
The PubChem CID of chemical compounds:
Cerevisterol, PubChem CID: 10181133
Clerosterol, PubChem CID: 5283638
Epifriedelanol, PubChem CID: 101341
Friedelin, PubChem CID: 91472
Palmitic acid, PubChem CID: 985
The authors are grateful to Xiuhong Zou, Yongchun Forestry Administration, Fujian, China, for collecting the plants. The authors also thank the staff of the analytical group of the Guangdong College of Pharmacy, for measurements of all spectra. This work was financially supported by the Scientific & Technical Program of Guangdong Province (2007B031406004).
Supplementary material associated with this article can be found, in the online version, at doi:10.1016/j.phymed.2015.05.001.
Ahmad, V.U., Aliya, R., Perveen, S., Shameel, M., 1993. Sterols from a marine green alga Codium decorticatum. Phytochemistry 33, 1189-1192.
Asilbekova, D.T., Gusakova, S.D., Glushenkova, A.L., 1999. Composition of the floral waxes of rosegallica, jasminum grandiflorumn and viola odorata. Chem. Nat. Compd. 35, 295-300.
Brent, E.K., Phillip, A.F., Michael, J.O., 2006. Clevudine: a potent inhibitor of hepatitis B Virus in vitro and in vivo. Expert Rev. Anti Infect. Ther. 4, 549-561.
Cao, T.W., Geng, CA, Jiang, F.Q., Ma, Y.B., He, K., Zhou, N.J., Zhang, X.M., Zhou, J., Chen, J.J., 2013. Chemical constituents of Swertia yunnanensis and their anti-hepatitis B virus activity. Fitoterapia 89, 175-182.
Chai, X.Y., Xu, Z.R., Bai, C.C., Zhou, F.R., Tu, P.F., 2009. A new seco-friedelolactone acid from the bark and twigs of Itoa orientalis. Fitoterapia 80, 408-410.
Delectis Florae Republicae PoPularis Sinicae Agendae Academiae Sinicae Edita, 1991. Flora Repubulicae Popularis Sinicae, vol. 51. Science Press, Peking.
Fujian Science and Technology Committee, 1990. Flora Fujianica. Fujian Science and Technology Press, Fuzhou.
Govindachari, T.R., Prakash, D., Viswanathan, N., 1968. Apetalactone, a new triterpene lactone from Catophyllum species. J. Chem. Soc. C 1323-1324.
Guevara, A.P., Lim-sylianco, C.Y., Dayrit, F.M., Finch, P., 1989. Acylglucosyl sterols from Momordica charantia. Phytochemistry 28, 1721.
Guo, R.H., Geng. C.A., Huang, X.Y., Ma, Y.B., Zhang, Q., Wang, L.J., Zhang, X.M.. Zhang, R.P., Chen, J.J., 2013. Synthesis of hemslecin A derivatives: a new class of hepatitis B virus inhibitors. Bioorg. Med. Chem. Lett. 23, 1201-1205.
Honer Zu Siederdissen, C., Cornberg, M., 2014. The role of HBsAg levels in the current management of chronic HBV infection. Ann. Gastroenterol. 27, 105-112.
Hu, D., Ding, X.L., 1987. Isolation and identification of 3-friedelin from violet. Chin. J. Chin. Mater. Med. 12, 47.
Huang, Q,, Huang, R., Wei, L, Chen, Y., Lv, S., Liang, C., Zhang, X., Yin, F., Li, H., Zhuo, L., Lin, X., 2013. Antiviral activity of methyl helicterate isolated from Helicteres angustifolia (Sterculiaceae) against hepatitis B virus. Antivir. Res. 100,373-381.
Hui, W.H., Li, M.M., Lee, Y.C., 1975. Structure of lithocarpic lactone, a new triterpenoid from two Lithocarpus species of Hong Kong. J. Chem. Soc., Perkin Trans. 1 7, 617-619.
Katai, M., Nakamura, Y., Terai, T., 2006. The constituents of the leaves of Euortymus Japonicas Thunb. A new flavonoid glycoside. Osaka Kogyo Daigaku Kiyo, Rikohen. 51, 1-7.
Khoo, G.M., 2005. The Polysaccharide and Chemical Constituents in Green Alga Codium Species and Their Preliminary Biological Activities Research (Msc. D. Thesis). Jinan University. Guangzhou, pp. 35-60.
Konno, C., Hikino, H., 1976. [sup.13]C nuclear magnetic resonance spectra of ethers and glycols. Tetrahedron 32, 325-331.
Kuiate, J.R., Mouokeu, S., Wabo. H.K., Tane, P., 2007. Antidermatophytic triterpenoids from Syzygium jambos (L.) Alston (Myrtaceae). Phytother. Res. 21, 149-152.
Lee, S.W., Chen, Z.T., Chen, C.M., 1993.4[alpha]-methylsterol violasterol A from viola formsana hayata. J. Chin. Chem. Soc. 40, 305-307.
Li, C.Y., Zhong, F., Li, X.H., Lv, J.M., Jia, W., 2010. Anti-inflammatory effects of Viola diffusa Ging in animal models of acute inflammation. Lishizhen Med. Mater. Med. Res. 21, 844-845.
Li, X.H., Hu, X.Q., Long, Y., Cao, P., Zheng, Y.F., Zhang, J., 2009. Effect of the herb serum containing VDG on proliferation and apoptosis of K562 leukemia cell. Chin. J. Ethnomed. Ethnopharm. 15, 27-29.
Liu, J., Luo, J.H., Liu, D.J., Gao, Y., Ma, K.J., 2011. Purification of chlorogenic acids from Viola by macroporous resin. Heilongjiang Med. Pharm. 34, 26-27.
Martinot-Peignoux, M., Lapalus, M., Asselah, T., Marcellin, P., 2014. HBsAg quantification: useful for monitoring natural history and treatment outcome. Liver Int. 34, 97-109.
Ngan, F., Chang, R.S., Tabba, H.D., 1988. Isolation, purification and partial characterization of an active anti-HIV compound from the Chinese medicinal herb viola yedoensis. Antivir. Res. 10, 107-115.
Pan, X.W., 2009. Correlation between serum HBV DNA and HBeAg levels in patients with chronic hepatitis B. J. Radioimmunol. 22, 68-71.
Patra, A., 1990. Applications of two-dimensional NMR inspectral assignments of some friedelanes and secofriedelanes. Mag. Reson. Chem. 28, 85-92.
Pdter, M., J6zsef, S., Lajos, R., 1980. Occurring of 15-cis-violaxanthins in viola tricolor. Phytochemistry (Amsterdam) 19, 623-627.
Qin. B., Chen, Q.P., Lou, Z.C., 1994. Active constituents of Viola prionanthaBge. J. Chin. Pharm. Sci. 3, 91-96.
Reed, L.J., Muench, H., 1938. A simple method of estimating fifty per cent endpoints. Am. J. Hyg. 27, 493-497.
Reyes, B.M., Ramirez-Apan, M.T., Toscano, R.A., Delgado, G, 2010. Triterpenes from Garcia parviflora. Cytotoxic evaluation of natural and semisynthetic friedelanes. J. Nat. Prod. 73, 1839-1845.
Rodriguez, F.M., Perestelo, N.R., Jimenez, LA., Bazzocchi, l.L, 2009. Friedelanes from Crossopetalum lobatum. A new example of a triterpene anhydride. Helv. Chim. Acta 92, 188-194.
Shen, X.Y., Xie, C.X., 2009. Study on antioxidantive activity of extracts from Viola tianshanica Maxim. Food Sci. 30, 139-141.
Trepo, C., Chan, H.L., Lok, A., 2014. Hepatitis B virus infection. Lancet 18.
Ulf, G., Martin, S., Erika, S., Per, C., Lars, B., 2004. Reversible antifouling effect of the cyclotide cycloviolacin [O.sub.2] against barnacles. J. Nat. Prod. 67, 1287-1290.
Wang. H., Xiong, Z.Y., Chen, Z.Y., Zeng, H.P., 2010. Structural characterization and inducing proliferation activity of friedelin-3,4-lactone from Catophyllum polyanthum. Yunnan Daxue Xuebao, Ziran Kexueban 32, 690-694.
Xie, H.G., 2006. Studies on the Chemical Constituents of Inula cappa DC (Msc. D. Thesis). Peking Union Medical College, Peking, pp. 30-50.
Yang, H., Wang, J., Hou, A.J., Guo, Y.P., Lin, Z.W., Song, H.D., 2000. New steroids from Clerodendrum colebrookianum. Fitoterapia 71, 641-648.
Yu, D.Q., Yang, J.S., 1999. Analytical Chemistry Handbook, Nuclear Magnetic Resonance Spectroscopy Analysis. Chemical Industry Press, Beijing.
Zhu, T.Z., Sheng, Z.M., Ru, Q,T., Wu, H.L., Shi, M.Q,, Zhong, A.Q., Ni, S.C., Mo, R.D., Sun, Q.H., 1986. Antibacterial test of Viola diffusa Ging in vitro. Chin. J. Vet. Med. 10, 39-41.
Jiao-Jiao Dai (1), Hua-Ming Tao (1), Qing-Xi Min, Quan-Hong Zhu *
School of Traditional Chinese Medicines, Southern Medical University, Guangzhou 510515, PR China
* Corresponding author. Tel.: +86 20 61648770; fax: +86 20 61648770. E-mail address: email@example.com (Q..-H. Zhu).
(1) These authors contributed equally to this work.
Table 1 [sup.1]H NMR spectroscopic data for 1,2,3 and 11 (500 MHz). (a) Position 1 (b) 2 (c) 1 2.02, 1.62 m 1.72, 1.50 m 2 4.34 dd (12.0, 2.0) 4.41 dd (12.0, 2.0) 1 -- -- 4 4.23 q (6.5) 4.38 q (6.5) 5 -- -- 6 1.52, 1.14 m 1.16, 0.98 m 7 1.64, 1.46 m 8 1.79 m 1.53 m 9 -- -- 10 1.24 m 1.17 m 11 1.70, 1.41 m - 12 2.10, 1.49 m 2.02, 1.99 m 13 -- -- 14 -- -- 15 1.95, 1.52 m 2.01, 1.30 m 16 1.73, 1.26 m 1.38, 1.08 m 17 -- -- 18 1.82 m 1.44 m 19 1.73, 1.22 m 1.74, 1.24 m 20 -- -- 21 1.43, 1.25 m 1.31, 1.21 m 22 1.42, 0.87 m 1.32, 1.08 m 23 1.23 d (6.5) 1.05 d (6.5) 24 0.87 s 0.72 s 25 0.87 s 0.79 s 26 1.12 s 0.96 s 27 -- -- -- -- 28 1.21 s 3.47 dd (10.0, 4.0) -- 3.27 dd (10.0, 4.0) 29 0.99 s 0.92 s 30 0.93 s 0.91 s Position 3 (b) 11 (c) 1 2.07, 1.58 m 1.46, 1.12 m 2 4.29 dd (12.0, 2.0) 1.59, 1.23 m 1 -- 3.78 m 4 4.17 q (6.5) 1.85, 1.37 m 5 -- -- 6 1.56, 1.11 m 3.29 br s 7 1.55, 1.47 m 1.56, 1.29 m 8 1.24 m 1.30 m 9 -- 1.34 m 10 1.25 m -- 11 1.76, 1.19 m 1.25, 1.12 m 12 2.10, 1.24 m 1.91.1.06 m 13 -- -- 14 -- 0.90 m 15 4.34 br s 1.50, 0.97 m 16 1.82, 1.77 m 1.73, 1.28 m 17 -- 1.11 m 18 1.97 m 0.60 s 19 1.47, 1.06 m 1.00 s 20 -- 1.34 m 21 1.73, 1.52 m 0.86 d (6.5) 22 1.93 1.06, m 123 0.9 23 1.22 d (6.5) 1.31, 1.22 24 0.88 s 1.82 m 25 0.83 s -- 26 0.99 s 1.52 s 27 -- 4.72 br s -- 4.63 br s 28 1.13 s 1.28 m -- -- 29 0.98 s 0.75 t (7.5) 30 3.24 br s Table 2 [sup.13]C NMR spectroscopic data for 1,2,3 and 11 (125 MHz). (a) Position 1 (b) 2 (c) 1 29.5, C[H.sub.2] 29.3, C[H.sub.2] 2 69.3, CH 68.0, CH 3 177.0, qC 175.4, qC 4 84.9, CH 82.3, CH 5 40.2, qC 39.9, qC 6 37.8, C[H.sub.2] 37.4. C[H.sub.2] 7 18.3, C[H.sub.2] 17.7, C[H.sub.2] 8 52.6, CH 52.3, CH 9 38.2, qC 37.5, qC 10 60.4, CH 59.7. CH 11 37.9, C[H.sub.2] 37.4, C[H.sub.2] 12 27.7, C[H.sub.2] 27.2, C[H.sub.2] 13 54.5, qC 52.8, qC 14 39.1, qC 38.5, qC 15 33.0, C[H.sub.2] 31.8, C[H.sub.2] 16 35.6, C[H.sub.2] 28.1, C[H.sub.2] 17 30.7, qC 35.3, qC 18 43.2, CH 38.7, CH 19 36.1, C[H.sub.2] 34.5, C[H.sub.2] 20 28.4, qC 28.4. qC 21 32.3, C[H.sub.2] 30.2, C[H.sub.2] 22 38.5, C[H.sub.2] 32.8, C[H.sub.2] 23 16.2, C[H.sub.3] 16.0, C[H.sub.3] 24 13.5, C[H.sub.3] 13.1, C[H.sub.3] 25 18.4, C[H.sub.3] 18.5, C[H.sub.3] 26 22.7, C[H.sub.3] 21.2, C[H.sub.3] 27 181.6, COOH 177.5, COOH 28 30.9, C[H.sub.3] 65.5, C[H.sub.2] 29 30.3, C[H.sub.3] 31.6, C[H.sub.3] 30 35.5, C[H.sub.3] 34.6, C[H.sub.3] Position 3 (b) 11 (c) 1 29.4, C[H.sub.2] 32.0, C[H.sub.2] 2 69.1, CH 31.1, C[H.sub.2] 3 176.9, qC 65.8, CH 4 84.5, CH 40.9, C[H.sub.2] 5 40.0, qC 74.3, qC 6 36.9, C[H.sub.2] 74.1, CH 7 19.7, C[H.sub.2] 34.5, C[H.sub.2] 8 47.7, CH 30.0, CH 9 37.3, qC 44.6, CH 10 59.3, CH 37.8, qC 11 35.2, C[H.sub.2] 20.8, C[H.sub.2] 12 22.5, C[H.sub.2] 39.8, C[H.sub.2] 13 50.5, qC 42.3, qC 14 48.3, qC 55.7, CH 15 80.5, CH 23.9, C[H.sub.2] 16 40.0, C[H.sub.2] 27.8, C[H.sub.2] 17 30.8, qC 55.8, CH 18 43.9, CH 11.9, C[H.sub.3] 19 28.4, C[H.sub.2] 16.3, C[H.sub.3] 20 33.2. qC 34.9, CH 21 28.0, C[H.sub.2] 18.5, C[H.sub.3] 22 34.4, C[H.sub.2] 33.2, C[H.sub.2] 23 16.2, C[H.sub.3] 28.7, C[H.sub.2] 24 13.2, C[H.sub.3] 48.8, CH 25 16.3, C[H.sub.3] 146.9, qC 26 13.6, C[H.sub.3] 17.5, C[H.sub.3] 27 179.9, qC 111.7, C[H.sub.2] 28 29.1, C[H.sub.2] 26.0, C[H.sub.2] 29 25.1, C[H.sub.3] 12.0, C[H.sub.3] 30 74.3, C[H.sub.2] (a) Chemical shifts are expressed in [delta] (ppm), and coupling constants (J) are expressed in Hz. (b) NMR data are measured in CD[Cl.sub.3]. (c) NMR data are measured in DMSO-d6. Table 3 Anti-HBV activities of compounds 1 -9, 11, 12, 13. (a) Compound [CC.sub.50] [IC.sub.50] SI (d) ([micro]M) (b) ([micro]M) (b) HBsAg HBeAg HBsAg HBeAg 1 30.4 26.2 8.0 1.2 3.8 2 84.6 33.7 15.2 2.5 5.6 3 392.5 104.0 21.6 3.8 18.2 4 329.2 NA (e) 88.5 NA 3.7 5 274.2 NA NA NA NA 6 113.8 NA 47.7 NA 2.4 7 77.9 NA 51.0 NA 1.5 8 483.4 NA 49.6 NA 9.7 9 1.1E+09 62.0 92.5 17.7E+06 17.7E+06 11 17.1 32.7 21.8 0.5 0.8 12 89.6 112.8 35.0 0.8 2.6 13 1561.8 90.8 NA 17.2 NA Lamivudine (f) 2460.0 41.3 NA 59.6 NA (a) All values are the mean of three independent experiments. (b) [CC.sub.50]: the concentration to inhibit Hep G2.2.15 cell proliferation by 50%. (c) [IC.sub.50]: 50% inhibition concentration against HBV synthesis. (d) SI: selectivity index, [CC.sub.50]/[IC.sub.50]. (e) Not active. (f) An antiviral agent used as a positive control.