Tuan-Wu Cao

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-β-D-glucopyranoside (3), and one new glucosyl caffeoate, 1-O-ethyl-6-O-caffeoyl-β-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).

Chemical constituents of Swertia yunnanensis and their anti-hepatitis B virus activity

Four new triterpenoids, sweriyunnangenin A (1), sweriyunnanosides A (2), B (3) and C (4), along with nineteen known compounds (5-23) were isolated from Swertia yunnanensis. Based on extensive spectroscopic analyses (1D- and 2D-NMR, HRESIMS, UV, IR, [α]D), the structures of sweriyunnangenin A (1), sweriyunnanosides A (2), B (3) and C (4) were elucidated as taraxer-14-ene-3α,6β-diol, oleanolic acid 28-O-β-D-glucopyranosyl-(1→2)-O-β-D-glucopyranoside, 2α,3β-di-hydroxyolean-12-en-28-oic acid 28-O-β-D-glucopyranosyl(1→6)-β-D-glucopyranosyl (1→6)-β-D-glucopyranosyl(1→2)-β-D-glucopyranoside and hederagenin 28-O-β-D-glucopyranosyl(1→6)-β-D-glucopyranosyl(1→6)-β-D-glucopyranosyl(1→2)-β-D-glucopyranoside, respectively. Twenty-two compounds were evaluated for their anti-HBV activities on the HepG 2.2.15 cell line in vitro, of which nine compounds showed potent anti-HBV activities. Compounds 1, 5-6, 14-16 and 19 showed activities against the secretion of HBsAg (IC50 values from 0.10 to 1.76 mM) and HBeAg (IC50 values from 0.04 to 1.41 mM), and compounds 11 and 13-16 exhibited significant inhibition on HBV DNA replication (IC50 values from 0.01 to 0.09 mM).

Seven New Secoiridoids with Anti-Hepatitis B Virus Activity from Swertia angustifolia

Seven new secoiridoids, swertianglide (1) and swertianosides A-F (2-7), together with fifteen known compounds, were isolated from the whole plants of Swertia angustifolia. Their structures were elucidated on the basis of extensive spectroscopic analyses ([α](D), UV, IR, MS, 1D- and 2D-NMR). Fourteen compounds were evaluated for their anti-hepatitis B virus (HBV) activities on the Hep G 2.2.15 cell line in vitro. Compound 2, an unusual secoiridoid glycoside dimer, showed significant activities inhibiting the secretion of HBsAg (IC₅₀ 0.18 mM, SI 3.11) and HBeAg (IC₅₀ 0.12 mM, SI 4.67).

Xanthones with Anti-Hepatitis B Virus Activity from Swertia mussotii

Two new xanthones, 8-O-β-D-glucopyranosyl-1-hydroxy-2,3,5-trimethoxyxanthone (1) and 8-O-[β-D-xylopyranosyl-(1 → 6)-β-D-glucopyranosyl]-1-hydroxy-2,3,5-trimethoxyxanthone (2), along with eighteen known xanthones (3-20) were isolated from Swertia mussotii. Their structures were elucidated on the basis of extensive spectroscopic analyses (1D- and 2D-NMR, HRESIMS, UV, IR, [α]D). All compounds were evaluated for their anti-hepatitis B virus activities on HepG 2.2.15 cells line in vitro, and compounds 3-10 exhibited significant activity inhibiting hepatitis B virus DNA replication with IC50 values from 0.01 mM to 0.13 mM. Compounds 3-5 showed remarkable activity with IC50 values of 0.77, > 0.98, and 0.21 mM for HBsAg, and < 0.62, 0.35, and 0.04 mM for HBeAg, respectively. Meanwhile, the effects of different substitutions on the anti-hepatitis B virus activity of xanthones from S. mussotii were discussed.

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.

Anti-hepatitis B virus active secoiridoids from Swertia kouitchensis

Three new secoiridoids, swertiakoulactone (1) and swertiakosides A and B (2 and 3), were isolated from Swertia kouitchensis. Their structures were elucidated by comprehensive spectroscopic analyses including MS, IR, 1D and 2D NMR data. By the anti-hepatitis B virus (HBV) assay on Hep G 2.2.15 cells line in vitro, compound 1 showed moderate activities inhibiting the HBsAg secretion (IC50 = 1.10 mM, SI = 4.39) and HBV DNA replication (IC50 = 1.16 mM, SI = 4.12).

Two new secoiridoids and other anti-hepatitis B virus active constituents from Swertia patens

Abstract Two new secoiridoids, swerpatic acid (1) with an unusual C8 skeleton and swerpalactone (2), were isolated along with ten known compounds (3–12) from the whole plants of Swertia patens. Their structures were elucidated by comprehensive spectroscopic analyses. Eight compounds were evaluated for their anti-hepatitis B virus (HBV) activities on Hep G 2.2.15 cell line in vitro. Compounds 4 and 10 showed moderate inhibitory activities on the secretion of HBsAg with IC50 values of 1.96 and 0.50 mM.