INTRODUCTION:

Dengue virus (DENV) is a member of the Flavivirus (genus) of the Flaviviridae (family). It is a significant human pathogen that causes a wide spectrum of clinical diseases ranging from dengue fever (DF) to severe dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS)¹.

In Indonesia, dengue occurred for the first time as an outbreak in Jakarta and Surabaya in 1968². Up until today, there is no effective antiviral or vaccine available for dengue³. Besides Indonesia, the other ASEAN countries where outbreaks of DENV have been substantial were Malaysia, Philippines, Thailand, and Vietnam⁴. Recently, there are four dengue virus genotypes; DENV-1, DENV-2, DENV-3, and DENV-4⁵. The rates of transmission and spread vary greatly among the four dengue serotypes⁶. In addition, Mustafa et al. led the discovery of the new serotype of the dengue virus, DENV-5⁷. World Health Organization stated that dengue viruses are spread in metropolitan cities mainly by two species of mosquitoes, Aedes agypti and Aedes albopictus⁸.

This condition happens especially in tropical and subtropical areas. Indonesia is stated to possess the second largest biodiversity in the world, with around 40,000 endemic plant species including 6,000 medicinal plants^9,10. For this reason, natural products have become the main source of test material in the development of antiviral drugs based on traditional medicine¹¹. P. merkusii or Sumatran pine is a native pine of the Malesia region of Southeast Asia, mainly in Indonesia. P. merkusii belongs to the family of Pinaceae and genus Pinus. It is one of the most important tropical softwood tree species in Indonesia. In Java Island, P. merkusii is the most important producer of pine resin. However, Indonesia is a major producer of turpentine distilled from this resin¹².

Many studies reported that the medicinal properties of P. merkusii are due to the phytochemicals possessed, including saponins, flavonoids, lignans, polyphenols, triterpenes, sterols, triterpenoids, glycosides, and alkaloids. P. merkusii is an important source of pycnogenol that contains proanthocyanidins (procyanidins). Proanthocyanidins are potent, free radical scavengers, antibacterial, exhibit vasodilatory, anticancer, antiallergic, anti-inflammatory, cardioprotective, immune-stimulating, antidiabetes, anti-atherosclerosis, estrogenic activities^13,14,15. In the present study, we investigated the antiviral activity of P. merkusii stem bark and cone against DENV-2 isolated from Surabaya, Indonesia.

MATERIALS AND METHODS:

Chemicals:

The chemical reagents used in this study were the dimethyl sulfoxide (Merck, Germany), Minimum Essential Medium Eagle (MEM) (Sigma-Aldrich, USA), ethanol (≥99.8%, Sigma-Aldrich, USA), DENV-2 originally from Surabaya, Indonesia (NCBI accession number: KT012513) obtained from Dengue Study Group, Institute of Tropical Disease, Universitas Airlangga, Vero cell (African green monkey kidney), penicillin-streptomycin (Gibco, USA), fetal bovine serum (FBS), fungizone (Sigma-Aldrich, USA), Viral ToxGlo™ Assay reagents (Promega, USA), and CellTiter-Glo^® Luminescent Cell Viability Assay (Promega, USA).

Preparation of P. merkusii Stem Bark and Cone Extract:

P. merkusii were collected from Malang, Indonesia. Taxonomic identification of P. merkusii was carried out by Purwodadi Botanical Garden, Indonesian Institute of Sciences, Purwodadi, Indonesia. The dry of P. merkusii barks and cones were cut into pieces and ground into powder. 350 g of the powdered barks and cones were soaked in ethanol (1.75 L) for 3 days, and then the macerate was separated and concentrated using Rotavapor^®R-300 (250 rpm, 60°C) according to Proboningrat et al.¹⁵.

Cell Culture:

Vero cells were cultured in Minimum Essential Medium Eagle (MEM) containing 10% fetal bovine serum (FBS), 0.5% fungizone (Sigma-Aldrich, USA), and 2% penicillin-streptomycin (Gibco, USA) at 37°C in a 5% CO₂ incubator. The confluent monolayer of Vero cells were detached with trypsin-EDTA and incubate cells at 37 °C for 5 minutes. The medium was gently added through pipetting and the cells were counted using a haemacytometer (Paul Marienfeld, Germany). The mixture was transferred into a 96-well plate with 1×10⁶ cells/10 mL and incubated in 37 °C with 5% CO₂³.

Antiviral Activity Assay:

Confluent monolayers of Vero cells were prepared on a 96-well plate (1×10⁶ cells/10mL), and the titer of DENV-2 (2×10⁴ FFU/well). We used Viral ToxGlo™ Assay reagents (Promega, USA) following the manufacturer’s guidelines. In addition, the Viral ToxGlo™ Assay is a method intended for use in research studies to identify cytopathic effect (CPE) induced by viral infection. The assay measures cellular ATP, a stable and tightly regulated surrogate of cell viability.

Cytotoxicity Assay:

We used CellTiter-Glo^® Luminescent Cell Viability Assay (Promega, USA) for cytotoxicity assay following the manufacturer’s guidelines. the CellTiter-Glo^® Luminescent Cell Viability Assay is a homogeneous method to determine the number of viable cells in culture based on the quantitation of the ATP present, which signals the presence of metabolically active cells. It is important to determine the cytotoxic potential of the formulation on the cell line used in the antiviral assays. This allows the calculation of the selectivity index defined as the ratio of the 50% cytotoxic concentration (CC₅₀) to the 50% antiviral concentration (IC₅₀) as previously described by Zandi et al.¹⁶. The ideal drug would be cytotoxic only at very high concentrations and have antiviral activity at very low concentrations, thus yielding a high SI value and thereby able to eliminate the target virus at concentrations well below its cytotoxic concentration. The selectivity index of a compound is a widely accepted parameter used to express a compound’s in vitro efficacy in the inhibition of virus replication.

RESULTS AND DISCUSSION:

Here, an initial report of P. merkusii stem bark and cone against DENV-2 isolated from Surabaya, Indonesia. In this study, we revealed that P. merkusii stem bark and cone inhibited DENV-2 in Vero cells with IC₅₀= 140.63 μg/mL and 73.78 μg/mL, CC₅₀= 89.65 μg/mL and 249.5 μg/mL, SI= 0.64 and 3.38, respectively (see Figure 1).

Figure 1. IC₅₀ and CC₅₀ of P. merkusii stem bark (SB) and cone (c) against DENV-2 in Vero cells.

To date, the use of traditional medicinal plants to treat many diseases is growing worldwide as they have few or no adverse effects^17,18. Traditional medicinal plants have been reported to have antiviral activity and some have been used to treat viral infections in animals and humans. Besides Pinaceae, some members of the family of Acanthaceae, Amaranthaceae, Caricaceae, Cucurbitaceae, Elaeagnaceae, Euphorbiaceae, Fabaceae, Fagaceae, Flagellariaceae, Halymeniaceae, Labiatae, Meliaceae, Myrtaceae, Piperaceae, Phyllophoraceae, Poaceae, Rhizophoraceae, Rubiaceae, Saururaceae, Solieriaceae, Verbenaceae, Zingiberaceae, and Zosteraceae have been reported as anti-dengue¹⁹.

The phytochemicals of the plants serve as compounds that have biological action^10,20. Tannins, flavonoids, saponins, and alkaloids are known to possess medicinal properties⁹. The genus of Pinus (Pinaceae) contains more than 110 species. Previous studies reported that more than 280 compounds, including phenols, flavonoids, terpenoids, and many other compounds, have been isolated from the genus of Pinus. Previous studies also revealed that the chemical constituents of species of Pinus exhibited antioxidant, anti-inflammatory, antiasthmatic, anticancer, and antivirus activities²¹.

In addition, proanthocyanidins are natural compounds that are ubiquitously found in Pinus plants. Proanthocyanidins are present in flowers, bark, fruits, and seeds of various plants as a defense against biotic and abiotic stressors²². Chemically, they are oligomeric and polymeric products of the flavonoid biosynthetic pathway²³. Flavonoids are a class of polyphenolic compounds having significant human health benefits²² and described as less toxic in comparison to other plant compounds¹⁶. Moreover, several studies have shown that flavonoids exerted significant antiviral activities against a number of common viruses including Aichi virus, dengue virus, canine distemper virus, and few others^16,24,25. There are also reports of inhibition of virus replication cycle by flavones, a subgroup of flavonoids that include compounds such as baicalein¹⁶.

P. merkusii was a good source of traditional medicine and it provides a noteworthy basis in pharmaceutical biology for the development or formulation of new drugs and future clinical uses to combat DENV infection. Moreover, the establishment of new anti-dengue products from bioactive compounds is needed in order to find more effective and less toxic anti-dengue drugs. Therefore, any comprehensive study on the potential of medicinal plants with isolated active compounds that have shown anti-dengue activity should undergo additional in vitro and in vivo animal testing followed by toxicity and clinical tests. This may result to the discovery of a promising compound to be optimized and thus be considered suitable for application in the production of new anti-dengue compounds.

CONCLUSION:

In summary, P. merkusii stem bark and cone are potent to inhibit DENV-2. Therefore, it should be considered for in vivo evaluation in the development of an effective antiviral compound against DENV-2.

ACKNOWLEDGEMENT:

This study was supported by the Mandat Research Grant Universitas Airlangga (HRMUA) 2019, Institute of Tropical Disease (ITD) the Center of Excellence (COE) program by the Ministry of Research and Technology (KEMENRISTEK) of the Republic of Indonesia, and PMDSU Scholarship - Batch III which was awarded to Arif Nur Muhammad Ansori, Amaq Fadholly, Annise Proboningrat, Suhailah Hayaza, and Raden Joko Kuncoroningrat Susilo. We thank EJA Translation for editing the manuscript.

CONFLICT OF INTEREST:

The authors declare no conflict of interest.

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Received on 19.12.2019 Modified on 21.04.2020

Research J. Pharm. and Tech. 2021; 14(7):3705-3708.

DOI: 10.52711/0974-360X.2021.00641