In Vitro Study on Anti-Hepatitis C Virus Activity of Spatholobus suberectus Dunn

Abstract

Hepatitis C virus (HCV) infects 200 million people worldwide, and 75% of HCV cases progress into chronic infections, which consequently cause cirrhosis and hepatocellular carcinoma. HCV infection is treated with currently considered standard drugs, including direct anti-viral agents (DAAs), alone or in combination with peginterferon-α plus ribavirin. However, sustained viral responses vary in different cohorts, and high costs limit the broad use of DAAs. In this study, the ethanol and water extracts of 12 herbs from Lingnan in China were examined in terms of their inhibitory effect on HCV replication. Among the examined extracts, Spatholobus suberectus ethanol extracts suppressed HCV replication. By comparison, Extracts from Fructus lycii, Radix astragali (root), Rubus chingii Hu (fruit), Flos chrysanthemi Indici (flower), Cassia obtusifolia (seed), Lonicera japonica Thunb (flower), Forsythia suspense Thunb (fruit), Poria cocos (sclerotia), Carthamus tinctorius L. (flower), Crataegus pinnatifida Bge. (fruit), and Leonurus japonicas Houtt. (leaf) extracts failed to show a similar activity. Active S. suberectus fractions containing tannins as the major component also inhibited the in vitro translation of HCV RNA. The combination treatments of single compounds, such as epigallocatechin gallate and epicatechin gallate, were not as potent as crude S. suberectus fractions; therefore, crude S. suberectus extract may be a potential alternative treatment against HCV either alone or in combination with other agents.

Keywords: HCV; S. suberectus extraction; anti-HCV therapy; tannin.

Figure 1

Extraction scheme of S. suberectus. Ethanol fractions were labeled as E followed by the percentage of ethanol used; for example, JXT-E25 means 25% ethanol eluted fraction of S. suberectus (JXT).

Figure 2

Expression levels of HCV NS proteins with the treatment of (A) JXT; (B) JXT-E0; (C) JXT-E25; (D) JXT-E50, and (E) JXT-E75 extracts. Huh-luc/neo-ET cells were seeded in a 6-well plate, and treated with different concentrations of JXT fractions for 72 h. The cells were collected and lysed, and 30 ng of the total protein was subjected to SDS PAGE and western blot. The expression levels of NS3, NS5A, and NS5B were determined. The expression level of GAPHD was used as the housekeeping control. The results are representative of three independent experiments.

Figure 3

Quantitative results of protein expression levels of NS3, NS5A, and NS5B with treatments of (A) JXT; (B) JXT-E0; (C) JXT-E25; (D) JXT-E50; and (E) JXT-E75 extracts. The expression levels of NS3, NS5A, and NS5B were normalized to that of GAPDH.

Figure 4

Effect of (A) JXT; (B) JXT-E0; (C) JXT-E25; (D) JXT-E50; and (E) JXT-E75 extracts on HCV RNA levels. Huh-luc/neo-ET cells were seeded in a 6-well plate and treated with 5 μg/mL of JXT fractions for different time periods. At the end of the treatment, RNA was extracted with Trizol and converted to cDNA. The level of HCV RNA was determined through real-time PCR. The expression level of 18S rRNA was used as a control. The absolute copy number of HCV RNA was determined with the standard curve generated by using the linearized pFK-I389/NS3-3′ plasmid. The results were obtained from three independent experiments and presented as mean ± SD (* p < 0.05, ** p < 0.01, *** p < 0.001 compared with DMSO control). Some of the error bars were too small to be seen.

Figure 5

Effects of whole water and different ethanol extracts of JXT, and CHX on the translation of HCV RNA in a rabbit lysis cell-free lysate system. The results were obtained from three independent experiments and presented as mean ± SD (* p < 0.05, ** p < 0.01, *** p < 0.001 compared with DMSO control).

Figure 6

Chromatogram of the representative constituents in the water extract of S. suberectus and sub-fractions obtained from macroporous resin with gradient ethanol elution. (A) Mixed standard solution of gallic acid (1); epigallocatechin (2, EGC); catechin (3); epicatechin (4, EC); epigallocatechin gallate (5, EGCG), and epicatechin gallate (6, ECG) at 2.50, 21.92, 10.67, 14.25, 7.64 and 11.75 μg/mL, respectively; (B) Prepared samples of crude water extracts of S. suberectus, and sub-fractions eluted from macroporous resin by (C) 0%; (D) 25%; (E) 50%; and (F) 75% ethanol solutions at 41.28, 23.72, 33.46, 33.70, and 31.75 mg/mL, respectively.

Figure 7

Chemical structures of (A) ECG; (B) EGCG; (C) EC; (D) EGC; (E) gallic acid; (F) (+)-catechin.

Figure 8

Anti-HCV replication effects under the combined treatment with EGCG and ECG in HCV replicon cells. Huh-luc/neo-ET replicon cells were incubated with different concentrations of EGCG and ECG for 72 h alone or in combination. The ratios of the detected EC₅₀ of EGCG and ECG alone or in combination at EC₅₀ of single compound were plotted against each other in an isobologram. Each point on the isobologram represents a combined treatment that inhibited 50% of HCV replication (mean ± SD from three separate experiments).