Antiviral activity of a nanoemulsion of polyprenols from ginkgo leaves against influenza A H3N2 and hepatitis B virus in vitro.

UNLABELLED: In order to improve the bioavailability levels of polyprenols (derived from ginkgo leaves (GBP)) in the human body, a GBP nanoemulsion was prepared, and its antiviral activity was evaluated against influenza A H3N2 and hepatitis B virus in vitro. METHODS: A GBP nanoemulsion was prepared by inversed-phase emulsification (IPE). Next, we investigated the antiviral activity of the GBP nanoemulsion on influenza A H3N2 and hepatitis B virus in vitro by the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenlytetrezolium bromide) method. ELISA and the fluorescent quantitative PCR method were used to measure the content of HBsAg, HBeAg and DNA virus in human samples. RESULTS: The GBP nanoemulsion exhibited uniformity at an average particle size 97 nm with a hydrophilic-lipophilic balance (HLB) of 9.5. GBP is non-toxic to normal cells, hepatitis B virus DNA, hepatitis B virus antigen and HepG2215. Furthermore, GBP could reach a 70% virucidal activity and a 74.9% protection rate (*** p < 0.001) on MDCK cells infected with H3N2 virus at a high concentration of 100 µg/mL. GBP had a good inhibition rate on HBsAg (52.11%, ** p < 0.01) at 50 µg/mL and Day 9 of incubation, and a 67.32% inhibition effect on HBeAg at a high concentration of 100 µg/mL and Day 9. GBP had good inhibition on HBV DNA with CT 18.6 and lower copies (** p < 0.01) at a middle concentration of 12.5 to 25 µg/mL. CONCLUSIONS: The GBP nanoemulsion was very stable and non-toxic and had very strong antiviral activity against influenza A H3N2 and hepatitis B virus in vitro. The inhibitory effects and reactive mechanisms were similar to the drug, 3TC; by lengthening the incubation time and increasing the drug concentration, GBP has promising potential as an antiviral drug.

Isolation, synthesis and anti-hepatitis B virus evaluation of p-hydroxyacetophenone derivatives from Artemisia capillaris.

p-Hydroxyacetophenone (p-HAP), as a main hepatoprotective and choleretic constituent of Artemisia capillaris, was revealed with anti-hepatitis B virus (HBV) effects in recent investigation. In addition to p-HAP, four derivatives of p-HAP were also isolated from A. capillaris by various chromatographic methods. Subsequent structural modification on p-HAP and its glycoside led to the synthesis of 28 additional derivatives, of which 13 compounds showed activity inhibiting hepatitis B surface antigen (HBsAg) secretion; and 18 compounds possessed inhibition on HBV DNA replication. The primary structure-activity relationships (SARs) suggested that the conjugated derivatives of p-HAP glycoside and substituted cinnamic acids (2a-2i) obviously enhanced the activity against HBV DNA replication with IC50 values ranged from 5.8 to 74.4 µM.

UFLC/MS-IT-TOF guided isolation of anti-HBV active chlorogenic acid analogues from Artemisia capillaris as a traditional Chinese herb for the treatment of hepatitis.

ETHNOPHARMACOLOGICAL RELEVANCE: Hepatitis B induced by HBV is a serious health problem. Artemisia capillaris (Yin-Chen) has long been used to treat hepatitis in traditional Chinese medicine. Coumarins, flavonoids and organic acids were revealed as its hepatoprotective and choleretic components, but its anti-HBV active components remain unknown. This current study focused on its anti-HBV active constituents by various chromatographic methods. MATERIAL AND METHODS: LC/MS and bioassay-guided fractionation on the active extract of Artemisia capillaris led to the isolation of nine chlorogenic acid analogues. Structures of the isolates were elucidated by MS/MS and NMR techniques. Anti-HBV assay was performed on HepG 2.2.15 cell line in vitro: reduction of HBsAg and HBeAg secretions was measured by an ELISA method; inhibition of HBV DNA replication was monitored by real-time quantitative PCR and cellular toxicity was assessed by a MTT method. RESULTS: The 90% ethanol extract of Artemisia capillaris (Fr. AC) showed significantly inhibitory activity on HBV DNA replication with an IC50 value of 76.1 ± 3.9 µg/mL and low cytotoxic effects (SI>20.1). To clarify its active constituents, the extract was further separated into 3 sub-fractions (AC-1, AC-2 and AC-3), of which Fr. AC-2 was the most active fraction against HBeAg secretion and HBV DNA replication with IC50 values of 44.2 ± 2.8 and 23.2 ± 1.9 µg/mL. Nine chlorogenic acid analogues were detected from the active part (Fr. AC-2) by a LC/MS technique and further separated by a HPLC method. The isolates were determined as chlorogenic acid (1), cryptochlorogenic acid (2), neochlorogenic acid (3), 3,5-dicaffeoylquinic acid (4), 4,5-dicaffeoylquinic acid (5), 3,4-dicaffeoylquinic acid (6), chlorogenic acid methyl ester (7), cryptochlorogenic acid methyl ester (8), neochlorogenic acid methyl ester (9). Compounds 1-6 possessed potent activity against HBV DNA replication with IC50 values in the range of 5.5 ± 0.9-13.7 ± 1.3 µM. Di-caffeoyl analogues (4-6) also exhibited activity against the secretions of HBsAg and HBeAg. Esterified analogues (7-9) showed dramatically decreased anti-HBV activity, indicating that carboxyl group is closely associated to the anti-HBV activity. CONCLUSIONS: This investigation was focused on the active fractions of Artemisia capillaris and their active compositions, which showed that Fr. AC-2 was the main active section of Artemisia capillaris and chlorogenic acid analogues were the main constituents contributing to its anti-HBV activity. These results support the ethnopharmacological use of Artemisia capillaris as anti-HBV agents.

Polyacetylenes and anti-hepatitis B virus active constituents from Artemisia capillaris.

Three new polyacetylenes, 8-(Z)-decene-4, 6-diyne-1, 3, 10-triol (1), 1, 3S, 8S-trihydroxydec-9-en-4, 6-yne (2), 3S, 8S-dihydroxydec-9-en-4, 6-yne 1-O-ß-D-glucopyranoside (3), and one new glucosyl caffeoate, 1-O-ethyl-6-O-caffeoyl-ß-D-glucopyranose (4), together with 34 known compounds were isolated from Artemisia capillaris. The structures of the new compounds were determined by extensive spectroscopic analyses including 1D and 2D NMR, HRESIMS, [α]D and CD experiments. Among them, 19 compounds showed activity inhibiting HBsAg secretion; 20 compounds showed activity inhibiting HBeAg secretion; and 25 compounds possessed inhibitory activity against HBV DNA replication according to our anti-HBV assay on HepG 2.2.15 cell line in vitro. The most active compound 12 could inhibit not only the secretions of HBsAg and HBeAg, but also HBV DNA replication with IC50 values of 15.02 µM (SI=111.3), 9.00 µM (SI=185.9) and 12.01 µM (SI=139.2).

Lignans from the heartwood of Streblus asper and their inhibiting activities to hepatitis B virus.

Three new lignans, erythro-strebluslignanol (1), threo-7'-methoxyl strebluslignanol (2) and erythro-7'-methoxyl strebluslignanol (3), together with twelve known compounds were isolated from the n-butanol and chloroform fractions of the heartwood of Streblus asper. Their structures were elucidated through extensive spectroscopic methods, including MS and 2D NMR experiments (HMQC and HMBC). The stereochemistry at the chiral center was determined using CD spectra, as well as analysis of coupling constants and optical rotation data, respectively. Primary bioassays showed that 6-hydroxyl-7-methoxyl-coumarin (5) and ursolic acid (10) showed anti-HBV activities, with IC(50) values of 29.60 µM and 89.91 µM for HBsAg at no cytotoxicity, and IC(50) values of 46.41 µM and 97.61 µM for HBeAg at no cytotoxicity, respectively.

[In vitro study on the anti-HSV-1 and HBV activities of extracts from the fruit of Eucalyptus maidenii].

OBJECTIVE: To investigate the antiviral activities of three kinds of extracts from the fruit of Eucalyptus maidenii against herpes simplex virus typel and Hepatitis B Virus. METHODS: Cytotoxicity of extracts on Vero cell lines were estimated using MTT method and anti-HSV-1 activity was observed and determined with CPE and plaque reduction assay. The inhibitory effects of extracts on HBsAg and HBeAg secretion in HepG2.2.15 cell culture were detected using ELISA. RESULTS: Aqueous extract (pl8-E3) had conspicuous anti-HSV-1 activity, the IC50 was 126.77 microg/mL,but the EtOAc extracts( pl8-E1 )and MeOH extracts (pl8-E2) showed little anti-HSV-1 activity. None of these extracts had significant inhibitory eflect on HBsAg and HBeAg secretion in HepG2.2.15 cell culture. CONCLUSION: Aqueous extract(p18-E3) from the fruit of Eucalyptus maidenii has conspicuous anti-HSV-1 activity. It could inactivate virus directly,and inhibit virus attachment,but had no influence on virus penetration. The mechanism that p18-E3 inactivates virus might involve in viral envelope alteration.

Swerilactones L-O, secoiridoids with C12 and C13 skeletons from Swertia mileensis.

Swerilactones L-O (1-4), four unusual secoiridoids with unprecedented C12 and C13 skeletons, were isolated from the traditional Chinese herb Swertia mileensis. Compounds 1 and 2 had moderate inhibitory activities against the secretion of hepatitis B virus surface antigen (IC50 = 1.47 and 1.20 mM, with SI < 1 and 1.53, respectively) and hepatitis B virus e antigen (IC50 = 0.88 and >2.69 mM, with SI 1.62 and <1, respectively) in an antihepatitis B virus assay on the Hep G 2.2.15 cell line in vitro.

Anti-hepatitis B virus active lactones from the traditional Chinese herb: Swertia mileensis.

Swerilactones H-K (1-4), which are four novel lactones with an unprecedented C29 skeleton, were isolated from Swertia mileensis (Qing-Ye-Dan), an endemic Chinese herb used for treating viral hepatitis. Their structures were determined by extensive spectroscopic and X-ray crystallographic diffraction analyses. Swerilactones H-K exhibit potent anti-hepatitis B virus activity against HBV DNA replication with IC(50) values ranging from 1.53 to 5.34 µM. For the first time, a plausible biogenetic pathway for swerilactones H-K, together with the previously reported swerilactones A-D is proposed. From a biogenetic point of view, swerilactones A-D are ascribed as secoiridoid dimers, and swerilactones H-K as secoiridoid trimers.

Homoflavonoid glucosides from Ophioglossum pedunculosum and their anti-HBV activity.

Chemical investigation of the ethanolic extracts of the whole plant of Ophioglossum pedunculosum afforded seven new homoflavonoid glucosides, pedunculosumosides A-G (1-7). Pedunculosumosides A and C exhibit modest activity of blocking HBsAg secretion with IC(50) values of 238.0 and 70.5 µM, respectively.

Schisanwilsonenes A-C, anti-HBV Carotane sesquiterpenoids from the fruits of Schisandra wilsoniana.

Three carotane-type sesquiterpenoids, schisanwilsonenes A (1), B (2), and C (3), were isolated from the fruits of Schisandra wilsoniana. Their structures and relative configurations were elucidated on the basis of spectroscopic methods including 2D-NMR techniques, and the structure of 1 was confirmed by a single-crystal X-ray diffraction experiment. Schisanwilsonene A, at 50 microg/mL, exhibited antiviral activity, inhibiting HBsAg and HBeAg secretion by 76.5% and 28.9%.

In vivo and in vitro anti-hepatitis B virus activity of total phenolics from Oenanthe javanica.

The traditional Chinese medicine Oenanthe javanica (OJ) has been used for many years, mainly for the treatment of inflammatory conditions including hepatitis. In this study, human hepatoma Hep G2.2.15 cells culture system and duck hepatitis B virus (DHBV) infection model were used as in vivo and in vitro models to evaluate the anti-HBV effects of total phenolics from Oenanthe javanica (OJTP). The HBeAg and HBsAg concentrations in cell culture medium were determined by using the enzyme immunoassay kit after Hep G2.2.15 cells were treated with OJTP for 9 d. DHBV-DNA in duck serum was analyzed by dot blot hybridization assay. In the cell model, OJTP could dose-dependently inhibit the production of the HBeAg and HBsAg, and the inhibition rates of OJTP on HBeAg and HBsAg in the Hep G2.2.15 cells were 70.12% and 72.61% on day 9, respectively. In the DHBV infection model, OJTP also reduced HBV DNA level in a dose-dependent manner. The DHBV-DNA levels decreased significantly after the treatment with 0.10 g kg(-1)d(-1) and 0.20 g kg(-1)d(-1) OJTP. The inhibition of the peak of viremia was at the maximum at the dose of 0.20 g kg(-1)d(-1) and reached 64.10% on day 5 and 66.48% on day 10, respectively. Histopathological evaluation of the liver revealed significant improvement by OJTP. In conclusion, our results demonstrate that OJTP can efficiently inhibit HBV replication in Hep G2.2.15 cells line in vitro and inhibit DHBV replication in ducks in vivo. OJTP therefore warrants further investigation as a potential therapeutic agent for HBV infections.

Green tea extract and its major component epigallocatechin gallate inhibits hepatitis B virus in vitro.

Hepatitis B virus (HBV) infection is endemic in Asia and causes major public health problems worldwide. Present treatment strategies for HBV infections are not satisfactory and the clinical limitation of current antiviral drugs for HBV, such as lamivudine, is causing rapid emergence of drug-resistant viral strains during the prolonged therapeutic treatment. In this research, the efficacy of a natural green tea extract (GTE) against HBV in a stably expressed HBV cell line HepG2-N10 is examined. The expression of viral antigens, HBsAg and HBeAg, were determined by using enzyme linked immuno-absorbent assay (ELISA). Quantitative real-time-PCR (Q-PCR) was used for the determination of extracellular HBV DNA and intracellular replicative intermediates and nuclear covalent closed circular DNA (cccDNA). HBV mRNAs were also analyzed by reverse transcription PCR (RT-PCR). Results showed that the 50% effective concentration (EC50) of GTE on HBsAg, HBeAg, extracellular HBV DNA and intracellular HBV DNA were 5.02, 5.681, 19.81, and 10.76 microg/ml, respectively. While the concentration of GTE with the inhibition percentage of 50% on proliferating cells (CC50) was 171.8 microg/ml. Similar analysis of the principal component of GTE, epigallocatechin gallate (EGCG), revealed it has relative weaker efficacy compared to GTE.

Anti-hepatitis B virus activity of wogonin in vitro and in vivo.

The traditional Chinese medicine Scutellaria radix has been used for thousands of years, mainly for the treatment of inflammatory conditions including hepatitis. The major active constituent, wogonin (WG), isolated from S. radix has attracted increasing scientific attention in recent years due to its potent biological activities. However, pharmacologic studies have primarily been focused on wogonin's anti-inflammatory and anti-cancer activities. In this study, we have investigated wogonin's anti-hepatitis B virus (HBV) activity both in vitro and in vivo. In the human HBV-transfected liver cell line HepG2.2.15, wogonin effectively suppressed the secretion of the HBV antigens with an IC(50) of 4 microg/ml at day 9 for both HBsAg and HBeAg. Consistent with the HBV antigen reduction, wogonin also reduced HBV DNA level in a dose-dependent manner. Duck hepatitis B virus (DHBV) DNA polymerase was dramatically inhibited by wogonin with an IC(50) of 0.57 microg/ml. In DHBV-infected ducks wogonin dosed i.v. once a day for 10 days reduced plasma DHBV DNA level with an ED(50) of 5mg/kg. The in vivo anti-HBV effect of wogonin in ducks was confirmed by Southern blotting of DHBV DNA in the liver. Histopathological evaluation of the liver revealed significant improvement by wogonin. In addition, in human HBV-transgenic mice, wogonin dosed i.v. once a day for 10 days significantly reduced plasma HBsAg level. Immunohistological staining of the liver confirmed the HBsAg reduction by wogonin. In conclusion, our results demonstrate that wogonin possesses potent anti-HBV activity both in vitro and in vivo. Currently, wogonin is under early development as an anti-HBV drug candidate.

Antihepatitis activity (anti-HBsAg and anti-HBeAg) of C19 homolignans and six novel C18 dibenzocyclooctadiene lignans from Kadsura japonica.

Bioassay-directed fractionation of the EtOAc extract of Kadsura japonica has led to the isolation of six new C18 dibenzocyclooctadiene lignans, schizanrins I, J, K, L, M, N, along with four known C19 homolignans, taiwanschirins A, B, C, and heteroclitin F. The elucidations of the new structures were based on spectral analysis. Bioassay evaluation against human type B hepatitis revealed that taiwanschirins A and B showed strong activity for anti-HBsAg and a medium effect for anti-HBeAg at 25 microg/mL (12.9 and 11.9 microM for taiwanschirins A and B, respectively).

Inhibition of hepatitis B virus by an aqueous extract of Agrimonia eupatoria L.

Inhibition of HBsAg release against hepatitis B virus (HBV) was investigated in an aqueous extract prepared from the aerial parts (stems and leaves) of Agrimonia eupatoria. The inhibitory effect on HBsAg secretion was footed using aqueous extracts of Agrimonia eupatoria at four different temperatures (37 degrees C 45 degrees C, 55 degrees C and 60 degrees C), and the extract prepared at 60 degrees C was found to have the greatest effect. The inhibitory activity of Agrimonia eupatoria extracts on HBsAg secretion varied over the growing season and was the highest at mid-July. This inhibitory activity was also shown with the aqueous extracts of two other species of the genus Agrimonia: A. pilosa and A. coreana pilosella. These results suggest that some plants of the genus Agrimonia contain potential antiviral activity against HBV.

Homoflavonoids from Ophioglossum petiolatum.

Six homoflavonoids, ophioglonin (1), ophioglonin 7-O-beta-D-glucopyranoside (2), ophioglonol (3), ophioglonol prenyl ether (4), ophioglonol 4'-O-beta-D-glucopyranoside (5), and isoophioglonin 7-O-beta-D-glucopyranoside (6), together with five known compounds, quercetin, luteolin, kaempferol, 3,5,7,3',4'-pentahydroxy-8-prenylflavone, and quercetin 3-O-methyl ether, were isolated from Ophioglossum petiolatum. Their structures were elucidated by analysis of spectroscopic data and chemical evidence. Compounds 1 and quercetin 3-O-methyl ether showed slight anti-HBV surface antigen activity at 25 microM.

Screening of 25 compounds isolated from Phyllanthus species for anti-human hepatitis B virus in vitro.

Using an HBV-producing cell line and inhibition of the expression of the HBsAg and HBeAg as antiviral indicators, a study was conducted on 25 compounds isolated from four Phyllanthus (Euphorbiaceae) plants, including P. amarus Schum. & Thonn., P. multi florus Willd., P. tenellus Roxb. and P. virgatus Forst. f. It was found that niranthin (1), nirtetralin (3), hinokinin (5) and geraniin (13) at the non-cytotoxic concentration of 50 micro m, suppressed effectively both HBsAg and HBeAg expression, with the highest inhibition at 74.3%, 45.3%; 69.6%, 33.9%; 68.1%, 52.3%; 32.1%, 46.6%, respectively. Of these, niranthin (1) showed the best anti-HBsAg activity, while the most potent anti-HBeAg activity was observed with hinokinin (5).

Antiviral activities of extracts isolated from Terminalis chebula Retz., Sanguisorba officinalis L., Rubus coreanus Miq. and Rheum palmatum L. against hepatitis B virus.

The antiviral effects of aqueous extracts of Terminalis chebula Retz., Sanguisorba officinalis L., Rubus coreanus Miq. and Rheum palmatum L. were examined by a cell culture system using a hepatitis B virus (HBV) producing cell line, HepG2 2.2.15. The extracts were assayed for the inhibition of HBV multiplication by measurement of HBV DNA and surface antigen (HBsAg) levels in the extracellular medium of HepG2 2.2.15 cells after an 8-day treatment. All extracts decreased the levels of extracellular HBV virion DNA at concentrations ranging from 64 to 128 microg/mL and inhibited the secretion of HBsAg dose dependently. Of the four tested plants, Terminalis chebula exhibited the most prominent anti-HBV activities.

[Study on the Rheum palmatum volatile oil against HBV in cell culture in vitro].

The antiHBV effect of Rheum palmatum Volatile oil was studied by using 2215 cell line transfected with HBV DNA. At the same time MTT method was applied for the detection of cytoxicity of drugs, selecting acyclovir(ACV) as control medicine. It turns out that the toxic concentration of Rheum palmatum Volatile oil for 50% cells was (CD50) > 1.25 x 10(-1) g/L. When concentration was below 0.625 x 10(-1) g/L, the survival rate of cells was over 90%. The maximum inhibitory rates for HBsAg and HBeAg were 70.71 +/- 5.4% and 30.99 +/- 5.3% respectively. This shows Rheum palmatum Volatile oil possesses the effect of antiHBV in vitro.

Inhibition of HBsAg secretion from Alexander cell line by Phyllanthus amarus.

Alexander cell line, an human hepatocellular carcinoma derived cell line which has the property of secreting HBsAg in the supernatant was used to study the antiviral property of phyllanthus amarus. Aquous extract of Phyllanthus amarus was evaluated for its in vitro ability to inhibit HBsAg secretion on a dose dependent manner. It was seen that P. amarus at 1mg/ml concentration on a single dose inhibited the secretion of HBsAg for a period of 48 hours. This experiment proved the anti hepatitis B virus property of P. amarus at cellular level and further confirmed its beneficial use in the treatment of acute and chronic hepatitis B and healthy carriers of HBV.