Study on Phaleria macrocarpa fruit protein aqueous extract for anti-human herpesvirus type-1 and its mode of action

Abstract

Aqueous extract of Phaleria macrocarpa fruit (AEPMF) has previously exhibited in vitro anti-HHV-1 with a selective index (SI) of 17.9. This study aims to further evaluate the human herpesvirus type-1 (HHV-1) inhibitory action of P. macrocarpa fruit protein aqueous extract (PMFPAE). PMFPAE contains 16 ± 1% protein, and further fractionation by gel filtration chromatography (GFC) produces six protein fractions (P1-P6). The freeze-dried extract was analysed for the associated compounds by liquid chromatography-tandem mass spectrometry (LC/MS-MS). At least three important antiviral compounds were determined, including quercetin, mangiferin and gluconic acid. PMFPAE and its fractions are non-cytotoxic to Vero cells with 50% cytotoxic concentration (CC50) values ranging between 96 ± 1.3 and 1450 ± 2 μg/mL. Antiviral activity determined by post-treatment assay showed that PMFPAE is effective against HHV-1 clinical strain, KOS-1 strain and acyclovir (ACV) resistant strain (UKM-1) with 50% effective concentrations (EC50) values of 18 ± 1.2, 29 ± 2.3 and 41 ± 2.5 μg/mL, respectively. The SI of PMFPAE against HHV-1 clinical, KOS-1 and UKM-1 strains were 80, 50 and 35, respectively, and protein fractions P1-P6 ranged between 67.9 and 4.2. It is worth noting that the SI values for PMFPAE and some of the fractions were higher than the SI value of AEPMF. PMFPAE mode of action on virus replication was evaluated on the three HHV-1 strains. PMFPAE has the ability to inhibit the early stages of the HHV-1 replication cycle during viral attachment and at 50-60 μg/mL was more than 80% at 1.5 and 2 hours post-infection (hpi) during penetration. Treatment of the host cells with PMFPAE prior to virus infection has approximately 30-42% inhibitory effect on HHV-1 replication. Incubation of different strains of HHV-1 infected cells with PMFPAE for 24 h reduced progeny release, and the effect was dose-dependent. Treatment with PMFPAE decreases plaque formation when added 2 to 12 hpi against different HHV-1 strains as proven in the time-addition assay. The effect on the early HHV-1 replication stage was supported in the time-removal assay when treatments were removed at 2 to 30 hpi. PMFPAE showed virucidal activity when treated at 1×103 pfu/mL. Observation by transmission electron microscopy (TEM) supported the virucidal activity whereby PMFPAE treated at 60 μg/mL caused damage or loss of the virus envelope. Further confirmation of the anti-HHV-1 mechanism was done on the viral gene by quantitative reverse transcription-polymerase chain reaction (qRT-PCR). Treatment with PMFPAE affected the immediate-early (IE) genes (ICP0 and ICP4), early (E) gene (UL30) and late (L) genes (UL27 and UL44) of HHV-1 clinical strain. As for the viral protein, when analysed by in situ ELISA, PMFPAE highly increases IE proteins (ICP0 and ICP4) at different time points on clinical HHV-1 strain. In conclusion, PMFPAE contains antiviral proteins and compounds that support the antiviral activity of P. macrocarpa. PMFPAE displayed virucidal activity, affects virus attachment, penetration and viral progeny release against different HHV-1 strains, including an ACV-resistant strain. The effect on viral immediate-early, early and late replication phases is supported by inhibiting the selected viral genes and protein.