Inhibitory efficiency of Andrographis paniculata extract on viral multiplication and nitric oxide production

Abstract

Andrographis paniculata (Burm. F.) Nees is a medicinal plant previously reported with broad-spectrum antivirals but the mode of inhibition remains elusive. The objective of this study was to identify the most active fraction from A. paniculata ethanol extract (APE, APE-2A, APE-2B and APE-2C) and dry powder extract (APSP) against influenza A (H3N2), representing RNA viruses, and herpes simplex virus-1 (HSV-1), representing DNA viruses. The results showed that the fractions APSP, APE, APE-2B, and APE-2C directly neutralized the HSV-1 and influenza A (H3N2) when incubated at room temperature for 60 min before infecting the cells. The results also showed that the additional APE-2A fraction also directly neutralized the influenza A (H3N2), but not the HSV-1. The APE, APE-2B and APE-2C inhibited the HSV-1 by more than 0.5 log when the fractions were introduced after infection. Similarly, the APSP and APE inhibited the influenza A (H3N2) more than 0.5 log after infection. Only 50 μg/mL APE-2C inhibited the viruses greater than 0.5 log. In addition, A. paniculata extracts were also evaluated for their interfering capacities against nitric oxide (NO) production in LPS-activated RAW 264.7 macrophages. As well, APE-2C potently inhibited NO production at the IC₅₀ of 6.08 μg/mL. HPLC and LC-MS analysis indicated that the most actively antiviral fractions did not contain any andrographolide derivatives, whereas the andrographolide-rich fractions showed moderate activity.

Figure 1

Cytotoxicity assay by MTS for 4 h of Vero and MDCK cells at 24 and 48 h reaction with various concentrations of A. paniculata extracts as follows: APSP, APE, APE-2A, APE-2B, and APE-2C.

Figure 2

Cytotoxicity concentration (CC₅₀) of A. paniculata extracts as follows; APSP, APE, APE-2A, APE-2B, and APE-2C in Vero cells and MDCK cells. Data represented Mean ± standard error of mean (SEM) of three independent experiments.

Figure 3

Pre-exposure experiment of A. paniculata extracts as follows; APSP, APE, APE-2A, APE-2B, and APE-2C against HSV-1 in Vero cell (A) and Influenza A (H3N2) in MDCK cell (B). Data represented means ± standard error of mean (SEM) of three independent experiments. p-value indicated significant difference between treated and control groups (unpaired t-test).

Figure 4

Post-exposure experiment of A. paniculata extracts as follows; APSP, APE, APE-2A, APE-2B, and APE-2C against HSV-1 in Vero cell (A) and Influenza A (H3N2) in MDCK cell (B). Data represented means ± standard error of mean (SEM) of three independent experiments. p-value indicated significant difference between treated and control groups (unpaired t-test).

Figure 5

Log10 reduction of HSV-1 (A) and Influenza A (H3N2) (B) after post-exposure with various concentrations of A. paniculata extracts. Error bar represented (SEM).

Figure 6

Nitric oxide inhibitory activity (A) and cell viability (B) at the effective doses.

Figure 7

HPLC-VWD chromatogram of APSP (A), APE (B), APE-2B (C) and APE-2C (D).

Figure 8

Total ions chromatogram (TIC) of fraction APE-2B in positive mode (A), fraction APE-2B in negative mode (B), fraction APE-2C in positive mode (C) and fraction APE-2C in negative mode (D). Peak numbers of compounds correspond to those in Tables 5 and 6.