Antiviral activity of four types of bioflavonoid against dengue virus type-2

Zandi Keivan, Teoh Boon-Teong, Sam Sing-Sin, Wong Pooi-Fong, Mustafa Mohd, Abubakar, Abubakar Sazaly

Description

Dengue is a major mosquito-borne disease currently with no effective antiviral or vaccine available. Effort to find antivirals for it has focused on bioflavonoids, a plant-derived polyphenolic compounds with many potential health benefits. In the present study, antiviral activity of four types of bioflavonoid against dengue virus type -2 (DENV-2) in Vero cell was evaluated. Anti-dengue activity of these compounds was determined at different stages of DENV-2 infection and replication cycle. DENV replication was measured by Foci Forming Unit Reduction Assay (FFURA) and quantitative RT-PCR. Selectivity Index value (SI) was determined as the ratio of cytotoxic concentration 50 (CC 50 ) to inhibitory concentration 50 (IC 50 ) for each compound. Results The half maximal inhibitory concentration (IC 50 ) of quercetin against dengue virus was 35.7 μg mL -1 when it was used after virus adsorption to the cells. The IC 50 decreased to 28.9 μg mL -1 when the cells were treated continuously for 5 h before virus infection and up to 4 days post-infection. The SI values for quercetin were 7.07 and 8.74 μg mL -1 , respectively, the highest compared to all bioflavonoids studied. Naringin only exhibited anti-adsorption effects against DENV-2 with IC 50 = 168.2 μg mL -1 and its related SI was 1.3. Daidzein showed a weak anti-dengue activity with IC 50 = 142.6 μg mL -1 when the DENV-2 infected cells were treated after virus adsorption. The SI value for this compound was 1.03. Hesperetin did not exhibit any antiviral activity against DENV-2. The findings obtained from Foci Forming Unit Reduction Assay (FFURA) were corroborated by findings of the qRT-PCR assays. Quercetin and daidzein (50 μg mL -1 ) reduced DENV-2 RNA levels by 67% and 25%, respectively. There was no significant inhibition of DENV-2 RNA levels with naringin and hesperetin. Conclusion Results from the study suggest that only quercetin demonstrated significant anti-DENV-2 inhibitory activities. Other bioflavonoids, including daidzein, naringin and hesperetin showed minimal to no significant inhibition of DENV-2 virus replication. These findings, together with those previously reported suggest that select group of bioflavonoids including quercetin and fisetin, exhibited significant inhibitory activities against dengue virus. This group of flavonoids, flavonol, could be investigated further to discover the common mechanisms of inhibition of dengue virus replication.

Sujets

• Antiviral
• Dengue virus
• Flavonoid
• Quercetin
• Naringin
• Daidzein
• Hesperetin

Informations

• Publié par: biomed
• Publié le: 01 janvier 2011
• Nombre de lectures: 35
• Langue: English

Extrait

Zandi et al. Virology Journal 2011, 8:560
http://www.virologyj.com/content/8/1/560
RESEARCH Open Access
Antiviral activity of four types of bioflavonoid
against dengue virus type-2
1 1 1 2 2 1*Keivan Zandi , Boon-Teong Teoh , Sing-Sin Sam , Pooi-Fong Wong , Mohd Rais Mustafa and Sazaly AbuBakar
Abstract
Background: Dengue is a major mosquito-borne disease currently with no effective antiviral or vaccine available.
Effort to find antivirals for it has focused on bioflavonoids, a plant-derived polyphenolic compounds with many
potential health benefits. In the present study, antiviral activity of four types of bioflavonoid against dengue virus
type -2 (DENV-2) in Vero cell was evaluated. Anti-dengue activity of these compounds was determined at different
stages of DENV-2 infection and replication cycle. DENV replication was measured by Foci Forming Unit Reduction
Assay (FFURA) and quantitative RT-PCR. Selectivity Index value (SI) was determined as the ratio of cytotoxic
concentration 50 (CC ) to inhibitory concentration 50 (IC ) for each compound.50 50
-1Results: The half maximal inhibitory concentration (IC ) of quercetin against dengue virus was 35.7 μgmL when50
-1it was used after virus adsorption to the cells. The IC decreased to 28.9 μgmL when the cells were treated50
continuously for 5 h before virus infection and up to 4 days post-infection. The SI values for quercetin were 7.07
-1and 8.74 μgmL , respectively, the highest compared to all bioflavonoids studied. Naringin only exhibited
anti-1
adsorption effects against DENV-2 with IC = 168.2 μgmL and its related SI was 1.3. Daidzein showed a weak50
-1
anti-dengue activity with IC = 142.6 μgmL when the DENV-2 infected cells were treated after virus adsorption.50
The SI value for this compound was 1.03. Hesperetin did not exhibit any antiviral activity against DENV-2. The
findings obtained from Foci Forming Unit Reduction Assay (FFURA) were corroborated by findings of the qRT-PCR
-1
assays. Quercetin and daidzein (50 μgmL ) reduced DENV-2 RNA levels by 67% and 25%, respectively. There was
no significant inhibition of DENV-2 RNA levels with naringin and hesperetin.
Conclusion: Results from the study suggest that only quercetin demonstrated significant anti-DENV-2 inhibitory
activities. Other bioflavonoids, including daidzein, naringin and hesperetin showed minimal to no significant
inhibition of DENV-2 virus replication. These findings, together with those previously reported suggest that select
group of bioflavonoids including quercetin and fisetin, exhibited significant inhibitory activities against dengue
virus. This group of flavonoids, flavonol, could be investigated further to discover the common mechanisms of
inhibition of dengue virus replication.
Keywords: Antiviral, Dengue virus, Flavonoid, Quercetin, Naringin, Daidzein, Hesperetin
Background (DSS). There are four dengue virus genotypes, DENV-1,
Denguevirus(DENV)isamemberofthegenusflavi- DENV-2, DENV-3 and DENV-4 which are transmitted
virus of the Flaviviridae family. It is a significant human to humans mainly by two species of mosquitoes, Aedes
pathogen which causes a wide spectrum of clinical ill- agypti and Aedes Albopictus [1]. All four DENV can
nesses ranging from a silent or mild febrile infection, cause dengue. To date there are no effective vaccine or
self-limited dengue fever (DF) to the severe dengue antiviral treatment for dengue. Dengue patients are
hemorrhagic fever (DHF) and dengue shock syndrome usually supportively-treated until they recover without
any specific treatment measures. Several studies have
shown that the level of viremia correlates with the* Correspondence: [e-mail]
1Tropical Infectious Disease Research and Education Center (TIDREC), severity of disease with high viremia often seen in severe
Department of Medical Microbiology, Faculty of Medicine, University of dengue. Hence, antivirals that can reduce the level of
Malaya, Kuala Lumpur, Malaysia
Full list of author information is available at the end of the article
© 2011 Zandi et al; licensee BioMed Central Ltd This is an Open Access article distributed under the terms of the Creative Commons
Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in
any medium, provided the original work is properly cited.Zandi et al. Virology Journal 2011, 8:560 Page 2 of 11
http://www.virologyj.com/content/8/1/560
-1viremia or the viremic phase could possibly reduce the stock solutions (20 mg mL )werestoredat-20C.
severity of dengue. Stock solution was diluted using cell culture medium
Plants and plant’s derived compounds remain an and sterilized by a syringe filter with 0.2 μmporesize
important source for the discovery and the development (Millipore, MA, USA) right before each experiment.
of new antiviral drugs because of their expected low
side effects and their high accessibility in the nature Cells and virus
[2-4]. There have been numerous reports on the anti- C6/36 mosquito cell line derived from Aedes albopictus
viral activity of various phytochemicals against dengue and Vero (African green monkey kidney) cell line were
viruses and these include various flavonoids [5-8]. Flavo- used in this study. Both cell lines were maintained and
noids are basically low molecular weight phenolic com- propagated in Eagle’s Minimum Essential Medium
pounds found widely in different kinds of plants. (EMEM) (Gibco, NY, USA) containing 10% fetal bovine
Different types of flavonoids can be found in fruits, serum (Gibco, NY, USA). Cultured C6/36 and Vero
roots, nuts, seeds, bark, steams and flowers of plants. cells were incubated at 28 C and 37 C, respectively in
These include quercetin which can be found in some 5% CO humidified chamber. At the time of virus pro-2
foods and fruits such as green and black tea, apple, pagation, serum concentration was reduced to 2%.
Denonion, citrus, tomato and some other plants [9,10]. Anti- gue virus type-2 (DENV-2) New Guinea C strain (NGC)
viral activities of various other flavonoids have also been was propagated using C6/36 cell line and harvested after
reported against some viruses including human cytome- CPEpresentationondaysevenpost-infection.After
galovirus (HCMV), HSV-1, HSV-2 and some types of titration, viral stock was stored at -70 C. Cell lines and
human adenoviruses [11-13]. virus were provided by Virology laboratory of the
TropiIn the present study, we are interested to examine the cal Infectious Disease Research and Education Center,
anti-dengue virus properties of quercetin, hesperetin, Faculty of Medicine, University of Malaya (Kuala
Lumnaringin and daidzein. Hesperetin is a flavonone and its pur, Malaysia).
glycoside form, hesperidin is water soluble and it could
be found in various citrus fruits. After ingestion of In vitro cytotoxicity assay
hesperidin, its sugar moiety is released from the back- MTT assay was performed following the manufacturer’s
bone of this compound and the aglycone form known instructions(Promega,WI,USA).Briefly,confluent
as hesperetin can be released. In vitro antiviral activities Vero cells in 96-well cell culture microplates were
treaof hesperetin have been reported against some RNA ted with different concentrations of each compound in
viruses [14-16]. Naringin on the other hand, is a flavo- triplicate. The treated cells were incubated for four days
none glycoside found abundantly in grapefruit juice. at 37 C followed by the addition of 15
μlofMTTsoluAntiviral activity of naringin were reported against tion to each well. The microplate was incubated at 37 C
HSV-1 and HSV-2 but this finding remains controver- for 4 h. Then, 100 μl of the solubilisation/stopping
solusial [12,17]. Daidzein is an isoflavone found in soybeans tion was added to each well. The optical density (OD)
and its antiviral activity against influenza viruses has of wells was measured at 570 using 96-well plate reader
been reported [18]. Currently, there is no published data (TECAN, Mannendorf, Switzerland). Dose-response
on the possible anti-dengue virus activities of quercetin, curves were plotted using Graph Pad Prism 5 (Graph
hesperetin, naringin and daidzein. Therefore, in this Pad Software Inc., San Diego, CA).
study we evaluated these compounds activities on
DENV-2 (NGC strain) replication in cell culture system. Pretreatment of cells with bioflavonoids
The effects of each compound were evaluated against In order to determine the prophylactic anti-dengue
different stages of dengue virus replication including activity of compounds, different concentrations of
comvirus adsorption, intracellular replication and direct vir- pounds were added to the Vero monolayer cells in
triucidal activities. plicate at different times, 5 h before virus infection.
After 5 h of pre-infection treatment, the cells were
Methods washed twice with sterile PBS and then 200 FFU of
Bioflavonoids DENV-2 was inoculated to the cells and incubated at 37
Four different types of bioflavonoid, quercetin, naringin, C for 1 h. To determine the effects of continuous
treathesperetin (Sigma-Aldrich, St. Louis, MO, USA) and ment, different concentrations of each compound were
daidzein (Indofine Chemical Co. Inc., Hillsborough, NJ, added to the Vero cells, 5 h pre-infection and
continuUSA) were evaluated for their potential activities against ously for 4 days post-infection.
dengu