Malik et al. BMC Complementary and Alternative Medicine (2016) 16:139 DOI 10.1186/s12906-016-1120-2 RESEARCH ARTICLE Open Access Antiviral and virucidal activities of Duabanga grandiflora leaf extract against Pseudorabies virus in vitro Farhah Zaidar Abdul Malik1, Zeenathul Nazariah Allaudin1*, Hwei San Loh2,3, Ting Kang Nee4, Homayoun Hani1 and Rasedee Abdullah5 Abstract Background: Duabanga grandiflora or known in Malaysia as Berembang Bukit, Megawasih,orPedada Bukit, is a native plant of the Southeast Asian countries. In this study, the anti-viral properties of D. grandiflora were investigated. Methods: The D. grandiflora leaf extracts were obtained with ethyl acetate, hexane, and ethanol as solvents and labelled 37 leaf ethyl acetate (37 L EA), 37 leaf hexane (37 L H), 37 leaf ethanol (37 L ET), respectively. The cytotoxicity of the extracts on Vero cells were determined by the 3-(4,5-Diamethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide (MTT) assay. Results: Among extracts, 37 L EA was most cytotoxic to Vero cells, followed by 37 L H and 37 L ET, with CC50 of 218, 833, and >1000 μg/mL, respectively. The cytopathic effect (CPE) and plaque reduction, inhibition, and virucidal assays and the selective index (SI) were employed to determine the effect of the extracts on infectivity and replication of pseudorabies virus (PrV) in Vero cells. The D. grandiflora leaf extracts showed dose-dependent antiviral activities, with higher activities at high doses. The 37 L ET and 37 L EA showed anti-viral effects through plaque formation and viral replication inhibitions, and virucidal property. The SI of the 37 L ET and 37 L EA by the viral replication inhibition assay was 8.3 and 1.9, respectively, and by the CPE reduction assay, 6.7 and 2.9, respectively. Conclusion: Ethanol is the best solvent for the preparation of D. grandiflora leaf extract as an antiviral agent. Keywords: Antiviral assay, Duabanga grandiflora, Plaque reduction assay, Inhibition assay, Virucidal assay Background through the stimulation of type II collagen production Duabanga, a genus originally included in the Sonnera- [2]. The D. grandiflora extracts and partial purified frac- tiaceae, is now classified under the subfamily Duaban- tions exhibited bacterial growth inhibition and re-sensitized goideae of the family Lythraceae. Duabanga, native to methicillin-resistant Staphylococcus aureus (MRSA) to- Southeast Asia, consists of three species, Duabanga wards ampicillin treatment by inhibiting MRSA biofilm for- grandiflora, D. moluccana, and D. taylorii Jayaweera [1]. mation and the penicillin-binding protein, PBP2a [3–6]. In Malaysia, Duabanga is known as Berembang Bukit, The plant extracts also possess insecticidal activity [7]. Megawasih and Pedada Bukit. Duabanga grandiflora The antimicrobial and phytochemical properties of D. contains eugeniin, comprising of gallic acid, ellagic acid, grandiflora was first described in 2011 [3] in a report and sugar [2]. that described the use of higher plant species for com- Traditionally, the Duabanga grandiflora tree has sev- bating newly emerging and drug resistant pathogens. eral medicinal properties. The leaf extract is used topic- The ethanol D. grandiflora leaf extract appears to con- ally to whiten skin, retard aging, and heal inflammation tain a high concentration of phytochemicals including tannins, phenolic compounds, flavonoids and steroids. On the other hand, the ethyl acetate D. grandiflora ex- * Correspondence: [email protected] 1Department of Veterinary Pathology and Microbiology, Faculty of Veterinary tract contain high level of tannins and moderate levels Medicine, Universiti Putra Malaysia, Serdang 43400, Selangor, Malaysia of alkaloids and phenols [3]. Previously, phytochemicals Full list of author information is available at the end of the article © 2016 Malik et al. Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Malik et al. BMC Complementary and Alternative Medicine (2016) 16:139 Page 2 of 10 especially tannins, phenolic compounds, flavonoids were RPMI maintenance media supplemented with 1 % FBS, reported to possess antiviral activities against human seeded into sterile 96-well flat bottom and 24-well plates 4 and animal viruses [8–12]. at 1 × 10 cells/well, and incubated under 5 % CO2 Herpesviruses are highly prevalent in humans and humidified atmosphere at 37 °C. most animals [13]. Members of Herpesviridae are host- specific agents that share properties, which allow them Plant extract to cross species barriers [14, 15]. This group of viruses All plant extracts were dissolved, with frequent shaking has relatively short reproduction cycle, can efficiently for 48 h in dimethyl sulfoxide (DMSO) and then diluted destroy infected cells, spread rapidly, and establish latent with sterile deionized water to obtain stock concentra- infection in the host. tions of 10 mg/mL in 10 % DMSO and 50 mg/mL in In this study, the antiviral properties of D. grandiflora 50 % DMSO. The 37 L ET extract was the most soluble plant extracts against PrV was determined. The cellular followed in order by 37 L EA, and 37 L H. The solutions toxicity and the most effective solvent to be used in the were stored as stock at −20 °C. preparation of D. grandiflora leaf extracts were also determined. Cytotoxicity assay The stock solutions were diluted serially with RPMI Methods media without FBS to obtain extract concentrations of Crude plant extract 62.5, 125, 250, 500, and 1000 μg/mL. In general, the final D. grandiflora was collected from the Semenyih Dam concentration of DMSO in the working concentration area (3.079066°N 101.886091°E), Selangor, Malaysia, was below 0.3 %. 100 μL of each concentration was used identified by Dr. Christopher Wiart of the University of to treat confluent monolayer Vero cells in a 96-well flat- Nottingham, Malaysia Campus, and the herbarium vou- bottom plate. For reference the Vero cells were treated cher specimens (UNMC 37) deposited at the Herbarium with 100 μL of either 0.05, 1.0, 10, or 12 % DMSO in of Faculty of Science of the University. Crude plant ex- PBS. Non-treated Vero cells served as the negative con- tracts, harvested from the leaves, extracted with hexane, trol. Positive controls were treated with DMSO at the ethanol, and ethyl acetate, and labelled 37 L H, 37 L ET, same concentrations as the plant extracts. All experi- and 37 L EA, respectively were prepared by Dr. Kang ments were performed in triplicates. The Vero cells were Nee Ting and Prof. Dr. Hwei San Loh of the University then incubated for 48 h under 5 % CO2, humidified of Nottingham, Malaysia. atmosphere at 37 °C and visualized under light micros- The leaves of the D. grandiflora were cleaned with copy. The cytotoxic effect of treatments was determined sterile deionized water, cut into small fragments, and by MTT assay. Cytotoxicity scoring was according a 5- dried in a closed room at 25 to 28 °C for 2 weeks. The point scale: 5 = confluent well-defined monolayer with dried leave fragments were grounded, weighed and cell-to-cell contact and the cell morphology and density soaked in 95 % ethanol at a proportion of 1:8 (fragment: intact; 4 = occasional cell lysis with ≤20 % of the cells ethanol) at room temperature for 24 h. The plant mate- round in shape, loosely attached, devoid of intracytoplas- rials were then subjected to sequential fractionation mic granule; 3 = prevalent cell lysis with <50 % of cells using hexane, ethanol, and ethyl acetate. Each plant ma- round in shape and devoid of intracytoplasmic granule; terial was soaked and fractionated trice before subjecting 2 = the majority of the cells are affected but <70 % of to rotary evaporation at 40 °C under reduced pressure. cells are round in shape or lysed; 1 = almost total lysis The concentrated crude extracts were stored at −20 °C. and destruction of cells and presence of spaces between cells [16]. The cytotoxic concentrations (CC50) of crude Pseudorabies Virus (PrV) propagation plant extracts, which are the concentrations causing The PrV strain AIP used in this study is from the working 50 % cell cytotoxicity of Vero cells were determined stock virus (1 × 108 pfu/mL) prepared and stored at - 80 °C (Additional file 1). in the Virology Laboratory, Faculty of Veterinary Medicine, Universiti Putra Malaysia. Quantitation of stock virus was Antiviral assays by plaque titration assay using Vero cells. Cytopathic Effect (CPE) reduction assay The CPE reduction assay was conducted according to Vero cell culture method described previously [17]. The Vero cells were Vero cells (ATCC No. CCL-81) were grown in RPMI seeded in a 96-well flat-bottom plate for 24 h to obtain a 1640 supplemented with 10 % fetal bovine serum (FBS) monolayer. 50 μL of 50 TCID50 PrV with 50 μL of either and antibiotic-antimycotic combination [1 % Penicillin 18.75, 37.5, 75, 150, or 300 μg/mL extracts was added to (100 U/mL), 1 % Streptomycin (100 mg/mL) and 1 % the cells and incubated under 5 % CO2 at 37 °C for 72 h. Fungi zone (2.5 mg/mL)]. The cells were maintained in The CPE was determined under light microscopy and Malik et al. BMC Complementary and Alternative Medicine (2016) 16:139 Page 3 of 10 graded as follows: 0 = 0 %, 1 = 0 to 25 %, 2 = 26 to 50 %, methylcellulose in 2 % FBS/DMEM was added to the cells 3 = 51 to 75 %, and 4 = 76 to 100 % CPE.