105 Vol. 7 No. 5 May-August 2019 ANTI-DENGUE TYPE 2 VIRUS ACTIVITIES OF ZINC (II) COMPLEX C O M P O U N D S W I T H 2 - ( 2 , 4 - D I H Y D R O X Y P H E N Y L ) - 3 , 5 , 7 - TRIHYDROXYCROMEN-4-ONE LIGANDS IN VERO CELLS Teguh Hari Sucipto1,α, Harsasi Setyawati2, Siti Churrotin1, Ilham Harlan Amarullah1, Sri Sumarsih2, Puspa Wardhani1, Aryati1, Soegeng Soegijanto1 1 Dengue Study Group, Institute of Tropical Disease, Universitas Airlangga, Indonesia 2 Department of Chemistry, Faculty of Science and Technology, Universitas Airlangga, Indonesia α Corresponding author: teguhharisucipto@staf.unair.ac.id ABSTRACT Dengue virus (DENV) is a disease that is transmitted through Aedes aegypti and Aedes albopictus mosquitoes, and is spread in tropical and sub-tropical regions. Now, dengue or antiviral vaccines for humans do not yet exist, but there are great efforts to achieve this goal. Complex compounds are reported to fungicidal, bactericidal and antiviral activity. Antiviral activity against DENV is an important alternative to the characterization and development of drugs candidate. The purpose of this study was to study zinc(II) compounds with 2-(2,4-dihydroxyphenyl)-3,5,7-trihydroxycromen-4-one ligand on DENV-2 replication in Vero cells. Vero cell lines (African green monkey kidney) was used in this study, maintained and propagated in Minimum Essential Eagle Medium containing 10% fetal bovine serum at 37°C in 5% CO2. The activity of dengue virus was carried out by enzyme-immunosorbent assay (ELISA) method and CellTiter96® Non-Radioactive Proliferation. The value of activity inhibition (IC50) of complex compounds with variations of mol metal: ligand 1:2, 1:3, and 1:4 against dengue virus type 2 (DENV2) was 2.44 μg/ml, 2.75 μg/ml, respectively and 2.00 μg/ml, also the toxicity value (CC50) of complex compounds with variation mol metal: ligand 1:4 for Vero cells is 3.59 μg/ml. The results of this study were indicate that these properties have been shown to inhibit anti-dengue type 2 virus (DENV-2), but are also toxic in Vero cells. Including previous study about complex compound interaction with dengue virus type 2 activity, Zn(II) more reactive compound then Cu(II), and Co(II). The comparison with Cu(II) complex compound, it has been revealed that Co(II) and Zn(II) is more toxic, was found to be nontoxic to human erythrocyte cells even at a concentration of 500 μg/ml. Keywords: Anti-DENV2, Complex Compounds, Zinc(II), 2-(2,4-dihydroxyphenyl)-3,5,7-trihydroxycromen-4-one ABSTRAK Virus Dengue (DENV) adalah penyakit yang ditularkan melalui nyamuk Aedes aegypti dan Aedes albopictus, serta didistribusikan di daerah tropis dan sub-tropis. Kini, vaksin dengue atau antivirus untuk manusia belum disetujui secara klinis, meski telah ada upaya besar untuk mencapai tujuan ini. Senyawa kompleks dilaporkan menunjukkan aktivitas fungisida, bakterisida, dan antivirus. Aktivitas antivirus melawan DENV merupakan alternatif penting untuk karakterisasi dan pengembangan obat-obatan. Tujuan dalam penelitian ini adalah untuk investigasi aktivitas senyawa kompleks logam seng(II) dengan ligan 2-(2,4-dihidroksifenil)-3,5,7-trihidroksikromen-4-on terhadap replikasi DENV-2 pada sel Vero. Sel Vero (African green monkey kidney) yang digunakan dalam penelitian ini, dipelihara dan diperbanyak dalam Medium Essential Eagle yang mengandung 10% serum janin sapi pada 37°C dalam 5% CO2. Aktivitas senyawa kompleks virus dengue dilakukan dengan metode enzyme-immunosorbent assay (ELISA) dan toksititas dengan metode CellTiter96® Non-Radioactive Proliferation. Nilai penghambatan aktivitas (IC50) senyawa kompleks dengan perbandingan mol logam:ligan 1:2, 1:3, dan 1:4 terhadap virus dengue tipe 2 (DENV2) masing-masing adalah 2.44 μg/ml, 2.75 μg/ml, dan 2.00 μg/ml, serta nilai toksisitas (CC50) senyawa kompleks dengan perbandingan mol logam:ligan 1:4 untuk sel Vero adalah 3,59 μg/ml. Hasil penelitian ini menunjukkan bahwa senyawa kompleks tersebut menunjukkan aktivitas penghambatan anti-dengue virus tipe 2 (DENV-2), tetapi juga bersifat toksik pada sel Vero. Termasuk penelitian sebelumnya tentang interaksi senyawa kompleks dengan aktivitas virus dengue tipe 2, Zn (II) lebih Research Report 106 Indonesian Journal of Tropical and Infectious Disease, Vol. 7 No. 5 May-August 2019: 105–108 reaktif senyawa kemudian Cu (II), dan Co (II). Perbandingan dengan kompleks Cu (II), telah diketahui bahwa Co (II) dan Zn (II) lebih toksik, ditemukan tidak beracun pada sel eritrosit manusia bahkan pada konsentrasi 500 μg / ml. Kata kunci: Anti-DENV2, Senyawa Kompleks, Seng(II), 2-(2,4-dihidroksifenil)-3,5,7-trihidroksikromen-4-on INTRODUCTION A major public health concern worldwide in recent years is a most prevalent mosquito-borne viral pathogen dengue virus (DENV), was transmitted through Aedes aegypti and Aedes albopictus mosquitoes, and is spread in tropical and sub-tropical regions.1,2,3 Presently around the world dengue is endemic in 112 countries.4 The incidence of DENV has increased approximately 30-fold over the past 50 years.5 Dengue virus, the causal agent of dengue, has been shown to induce apoptosis in vitro and in vivo.6,7 The mechanisms that trigger the apoptotic cellular responses, however, have not been thoroughly investigated.8 Micronutrient homeostasis is a key factor in maintaining a healthy immune system. Trace element zinc is a critical cofactor for many proteins involved in cellular processes like differentiation, proliferation and apoptosis that zinc is a nutritionally fundamental trace element and is second most abundant trace metal in the human body after iron.9 In previous study, Zn2+ was suggested that DENV-2 infection of Vero cells and Human breast adenocarcinoma cell line resulted in the induction of apoptosis.8,10 The complex compound from metal and organic compound reaction can be used an anti-DENV-2, especially Cu(II) with 2,4,5-triphenylimidazole exhibited adsorption inhibitory activity at IC50 = 2.3 μg/ml. 11 A significant inhibitory activity to that of the complex Co(II) with 2-(2,4- dihydroxyphenyl)-3,5,7-trihydroxycromen -4-one ligand was reported against the tested pathogenic DENV-2 in Vero cells 3.08 μg/ml.12 In the present study, the inhibitory activity of Zinc(II) with 2-(2,4-dihydroxyphenyl)-3,5,7-trihydroxycromen-4- one ligand against the replication of DENV-2 in Vero cells was investigated. MATERIAL AND METHODS Chemicals and Medium The chemical reagents used in this research were the Zinc(II)–2-(2,4-dihydroxyphenyl)-3,5,7-trihydroxycromen -4-one complex compound, dimethyl sulfoxide by Merck 99.98%, Germany, Minimum Essential Eagle Medium by Sigma-Aldrich (Germany), dengue virus type 2 Surabaya Isolate (KT012513), Vero cell by African green monkey kidney, CellTiter96® Non-Radioactive Proliferation reagent by Promega (USA), and DENV antibody (4G2) for enzyme-linked immunosorbent assay (ELISA). Vero Cells Preparation Vero cell lines (African green monkey kidney) was used in this study, maintained and propagated in Minimum Essential Eagle Medium containing 10% fetal bovine serum. Cultured Vero cell lines were incubated at 37 °C, respectively in 5% CO2. Confluent monolayer of Vero cells were detached with trypsin-EDTA and incubate cells at 37 °C for 5 minutes. Add Minimum Essential Eagle Medium containing 10% fetal bovine serum, pipetting gently to break up any clumps of cells and counted using a Hemocytometer. Add cells in 96-well plate with 1 × 106 cells/10 ml and incubated in 37°C incubator with 5 CO2. Monitor cells daily or every other day, cells reach a >90 % confluent monolayer.13,14 Anti-dengue Type 2 Virus Assay Confluent monolayers of Vero cells were prepared on a 96-well plate (1 × 106 cells/10 ml), and the titer of DENV-2 (2 × 104 FFU/well). The 50% inhibitory concentration (IC50) was calculated as follows: IC50 = (NC − AC) 100/ NC after incubating at 37°C for 2 days in 5% CO2, where NC is the mean of the absorbance of negative controls and AC is the absorbance of the compound tested. The DENV-2 inhibition of replication by compound was further investigated by using quantitative ELISA at 415 nm.15 Cytotoxicity assay The dye of CellTiter96® Non-Radioactive Proliferation reagent by Promega is a modification of MTT assay method by Mosmann. The assay is suitable to use in adherent and suspension cells. Assay is very sensitive, it can detect 1,000 cells/well of a plate reader. Vero cells (1×105 cells/ml) were seeded in plate at 37°C in 5% CO2 overnight. A total of 100 μl of serial delusion compound were incubated with Vero cells for 24 h. A total of 100 μl of Cell Proliferation Reagent was added into each well, incubated for 4 hour at 37°C. The plate was read at 570 nm using ELISA reader (iMarkTM Microplate Absorbance Reader). RESULT AND DISCUSSION Anti-dengue Type 2 Virus Activity Metal complex compounds are a promising class of drug leads, and the associated studies have attracted more attention. However, the systematic basic research of metal complex compounds is lagging behind, partly because very few efforts have been made to establish a set activity screening and subsequent evaluation system for the comprehensive investigations on the structure and activity relationship of metal complex compounds. The in vitro anti-dengue virus activity of the Zinc(II)–2- (2,4-dihydroxyphenyl)-3,5,7-trihydroxycromen-4-one complex compound was tested against one type dengue virus (dengue virus type 2) by enzyme-linked immunosorbent 107Sucipto, et al.: Anti-Dengue Type 2 Virus Activities of Zinc(II) Complex Compounds assay (ELISA).15 The susceptibility of the strains toward the present compounds was judged measuring the size of inhibition. Zinc(II)– 2-(2,4-dihydroxyphenyl)-3,5,7- trihydroxycromen-4-one complex compound was further studied for their inhibitory effect on replication of the dengue virus type 2 in Vero cells. The IC50 (inhibitory concentration 50) was determined from the dose response curve with variations of mol metal:ligand 1:2 (Figure 1), 1:3 (Figure 2), and 1:4 (Figure 3). Figure 1. Inhibition curve of dengue virus type 2 at several concentrations of Zn(II) complex with variations of mol metal:ligand 1:2 Figure 2. Inhibition curve of dengue virus type 2 at several concentrations of Zn(II) complex with variations of mol metal:ligand 1:3 Figure 3. Inhibition curve of dengue virus type 2 at several concentrations of Zn(II) complex with variations of mol metal:ligand 1:4 The IC50 value with variations of mol metal:ligand 1:2, 1:3, and 1:4 against dengue virus type 2 was 2.44 μg/ml, 2.75 μg/ml, respectively and 2.00 μg/ml. The comparison of the complex compounds and the known anti-dengue virus type 2 activity showed that the 1:4 variation mol metal:ligand was more effective than 1:2 and 1:3. The bulky 2-(2,4-dihydroxyphenyl)-3,5,7-trihydroxycromen -4-one ligand on binding to the metal cation reduces the polarity of the metal ion due to the ligand orbital overlap with the metal orbitals, resulting in a delocalization of positive charge.16 This increases the lipophilic character of the metal favors its permeation through the lipoid layer of the virus membranes.17 The effects of other compounds against cellular RNA polymerases and formation of the complex with RNA have reported suggesting that compound could also affect the similar replication enzymes.18 Previous research was reported anti-dengue type 2 activity, especially Cu(II) with 2,4,5-triphenylimidazole exhibited adsorption inhibitory activity at IC50 = 2.3 μg/ml.11 A significant inhibitory activity to that of the complex Co(II) with 2-(2,4-dihydroxyphenyl)-3,5,7- trihydroxycromen -4-one ligand was reported against the tested pathogenic dengue virus type 2 in Vero cells 3.08 μg/ ml. The comparison of the other complex, Zn(II) complex compound more significant activity for inhibit dengue virus type 2 replication then Cu(II) complex, and Co(II) complex. As for the central ion (M2+), when chelated with ligand to form the complex, it has the following order in stability: Zn2+ > Cu2+ > Co2+. Besides that, Cu(II) free ligand more reactive to dengue virus type 2 up to 0.13 μg/ ml because Cu2+ has stronger oxidative activity19 and react with cysteine residues on the surface of the protease.18 At molecular scale such complex compound interact directly with proteins and DNA, leading to dysfunction and cleavage of the structure of macromolecular.20 Cytotoxicity Activity This compound was tested for cytotoxicity by modification of MTT assay method by Mosmann assay on Vero cell lines, Zinc(II)–2-(2,4-dihydroxyphenyl)- 3,5,7-trihydroxycromen-4-one complex compound showed cytotoxicity with CC50 at 3.59 μg/ml. The CC50 value was found to increase with an increasing concentration of the test compound, as shown in Figure 4. Figure 4. Cytotoxicity of Zn(II) complex curve for Vero cell lines at several concentrations 108 Indonesian Journal of Tropical and Infectious Disease, Vol. 7 No. 5 May-August 2019: 105–108 Previous research was reported cytotoxicity other complex compounds to Vero cell lines, Cu(II) with 2,4,5-triphenylimidazole at CC50 = 44.74 μg/ml11 and the complex Co(II) with 2-(2,4-dihydroxyphenyl)-3,5,7- trihydroxycromen-4-one ligand at 3.36 μg/ml.12 The comparison with Cu(II) complex compound, it has been revealed that Co(II) and Zn(II) is more toxic, was found to be lower toxic to human MCF7 cell proliferation.21 CONCLUSION Metal complex compounds are a promising class of drug leads, and the associated studies have attracted more attention. Including previous study about complex compound interaction with dengue virus type 2 activity, Zn(II) more reactive compound then Cu(II), and Co(II). ACKNOWLEDGEMENT This work was supported by the Institute of Tropical Disease (ITD) the Center of Excellence (COE) program by the Ministry of Research and Technology (RISTEK) Indonesia. REFERENCES 1. Carrington LB, Simmons CP, 2014. Human to mosquito transmission of dengue viruses. Front. Immunol. 5(290): 1-8 2. Pongsiri A, Ponlawat A, Thaisomboonsuk B, Jarman RG, Scott TW, Lambrechts L, 2014. Differential susceptibility of two field Aedes aegypti populations to a low infectious dose of dengue virus. Plos One. 9(3): 1-6 3. Paixão ES, Teixeira MG, Rodrigues LC, 2017. Zika, chikungunya and dengue: the causes and threats of new and re-emerging arboviral diseases. BMJ Glob. Health. 3: 1-6 4. Gurugama P, Garg P, Perera J, Wijewickrama A, Seneviratne SL, 2010. Dengue viral infections. Indian J. Dermatol., 55(1): 68-78 5. Oliveira AFCS, Teixeira RR, Oliveira AS, Souzza APM, Silva ML, Paula SO, 2017. Potential Antivirals: Natural Products Targeting Replication Enzymes of Dengue and Chikungunya Viruses. Molecules, 22(505): 1-20 6. Ho MR, Tsai TT, Chen CL, Jhan MK, Tsai CC, Lee YC, Chen CH, Lin CF, 2017. Blockade of dengue virus infection and viral cytotoxicity in neuronal cells in vitro and in vivo by targeting endocytic pathways. Sci. Rep. 7(6910): 1-11 7. Sun P, Kochel TJ, 2013. The battle between infection and host immune responses of dengue virus and its implication in dengue disease pathogenesis. Sci. World J. 2013: 1-11 8. Shafee N, AbuBakar S, 2011. Characterization of dengue type 2 NGC virus infection in C6/36, Vero and MRC-5 cells. Intl. J. Virol., 7(1): 24-32 9. Gammoh NZ, Rink L, 2017. Zinc in infection and inflammation. Nutrients, 9(624): 1-25 10. Lee SY, Ching YW, Shafee N, 2017. Zinc induces normoxic accumulation of transcriptionally active hypoxia-inducible factor 1-Alpha in mammary epithelial cells. Molecular Biology, 51(1): 89-95 11. Sucipto TH, Churrotin S, Setyawati H, Martak, F, Mulyatno KC, Amarullah IH, Kotaki T, Kameoka M, Yotopranoto S, Soegijanto S, 2018. A new copper(II)-imidazole derivative effectively inhibits replication of DENV-2 in Vero cell. Afr. J. Infect. Dis., 12(S): 116-119 12. Sucipto TH, Churrotin S, Setyawti H, Mulyatno KC, Amarullah IH, Ueda S, Kotaki T, Sumarsih S, Wardhani P, Bendryman SS, Aryati, Soegijanto S, Kameoka M, 2017. Inhibitory activity of cobalt(II)- morin complex against the replication of dengue virus type 2. Indones J. Trop. Infect. Dis. 6(6): 141-144 13. Ammerman NC, Beier-Sexton M, Azad AF, 2009. Growth and maintenance of vero cell lines. Curr. Protoc. Microbiol. 11(1): 1-10 14. Plotkin BJ, Sigar IM, Swartzendruber JA, Kaminski A, 2018. Anaerobic growth and maintenance of mammalian cell lines. J. Vis. Exp. 137: 1-6 15. Koishi AC, Zanello PR, Bianco EM, Bordingon J, dos Santos CND, 2012. Screening of dengue virus antiviral activity of marine seaweeds by an in situ enzyme-linked immunosorbent assay. Plos One. 7(12): 1-11 16. Kirubavathy SJ, Velmurugan R, Parameswari K, Chitra S, 2014. Synthesis, characterization, single crystal XRD, in vitro antimicrobial and cytotoxicity study of tris(ethylenediamine)cobalt(III)chloride oxalate trihydrate. Arab. J. Chem. xxx: 1-6 17. Inba PJK, Annarej B, Thalamuthu S, Neelakantan MA, 2013. Cu(II), Ni(II), and Zn(II) complexes of salan-type ligand containing ester groups: synthesis, characterization, electrochemical properties, and in vitro biological activities. Bioinorg. Chem. Appl. 2013: 1-12 18. Sucipto TH, Churrotin S, Setyawati H, Kotaki T, Martak, F, Soegijanto S, 2017. Antiviral activity of copper(II)chloride dihydrate against dengue virus type-2 in Vero cell. Indones J. Trop. Infect. Dis. 6(4): 84-87 19. Liu Y., Guo M., 2015, Studies on transition metal-quercetin complexes using electrospray ionization tandem mass spectrometry, Molecules, No. 20, 8583-8594 20. Ejelonu BC, 2016. Potential of copper and its complexes of therapeutic agents. World J. Res. Rev. 3(3): 47-52 21. Rosu T, Pahontu E, Illies DC, Georgescu R, Mocanu M, Leabu M, Shova S, Gulea A, 2012. Synthesis and characterization of some new complexes of Cu(II), Ni(II), and V(IV) with Schiff base derived from indole-3-carboxaldehyde. Biological activity prokaryote and eukaryotes. Eur. J. Med. Chem. 53: 380-389