Antiviral Activity of Cynometra ramiflora Linn Leaves Extract

Against Replication of Dengue Virus Serotype 2

on Huh 7.5 Cell In Vitro

Amry Irsyada Yusuf

, Beti Ernawati Dewi

and Fithriyah Sjatha

Undergraduate Program, Faculty of Medicine, Universitas Indonesia

Department of Microbiology, Fculty of Medcicine, Universitas Indonesia,Jl. Pegangsaan Timur no. 16 Jakarta 10320

Keywords: Antiviral, Cynometra ramiflora Linn, Dengue virus, Huh7.5 cells

Abstract: Dengue hemorrhagic fever (DHF) remains a major world health problem particularly in Indonesia caused by

dengue virus (DENV) infection. Until now, there is no specific antiviral therapy for DENV and the

treatment for its infection is still supportive. The extract of Cynometra ramiflora Linn leaves known for

having potencies such as bactericide, analgesic, antiviral, anti-inflamation and anti-allergy. The objective of

this study is to assessed antiviral activity of C. ramiflora Linn leaves extract against DENV-2 in-vitro. The

potency of C. ramiflora Linn leaves extract at concentration of 1.25; 2.5; 5; 10 and 20 μg/ml towards DENV

was performed on Huh 7.5 cell infected by DENV-2 with moi of 0.5. Each treatment was repeated 6 times

compared with DMSO treated and infected DENV-2 cells as control. Inhibition rate of the extract against

DENV replication was measured using foci-forming immunoassay. Statistically, administration of C.

ramiflora Linn leaves extract at 1,25 ; 2,5 ;5 ; 10 and 20 μg/ml resulting 36,06 %, 45,96 %, 47,35%,

55,94%, 62,70% inhibition towards DENV-2 respectively, with significant value (p < 0,05). This result

showed that the extract of C. ramiflora Linn leaves has potency as anti-dengue.

1 INTRODUCTION

Dengue virus (DENV) infection as formed in

Dengue Fever (DF), Dengue Hemorrhagic Fever

(DHF) and Dengue Shock Syndrome (DSS) are the

common infectious diseases mainly on tropical and

subtropical area, Pacific Islands, South and Central

America, North Australia nor Africa (Chan et al,

2009). Moektiwardoyo (2014) reported theres an

intention the raised of dengue cases in Indonesia

each year as the consequences of global warming.

Since 1968 until 2009, WHO recorded Indonesia as

a country with the highest dengue cases in South

East Asia region (Indonesia Minstry of Health,

2010). In 2009 it self in Indonesia there are 77.489

cases of dengue infection with 585 fatality

(Moektiwardoyo et al, 2014; Indonesia Ministry of

Health, 2010).

DENV belong to Flaviviridae family, divided in to 4

serotypes; DENV-1, DENV-2, DENV-3 and DENV-

4 which each serotype was grouped into several

genotypes. DENV genome composed of 11 kbp

nucleotides, encoding 3 structural (prM, C and E)

and 7 nonstructural (NS1, 2A, 2B, 3, 4A, 4B and 5)

proteins. DENV is transmitted through Aedes

albopictus and Aedes aegypti mosquito as vector

(Castro et al, 2015).

Several efforts are conducted against DENV

infection, such as vaccine development and vector

control. Although dengue vaccine is now limitedly

available, long term effiacy of this vaccine is remain

unclear. Until now, vector control was proven can

not be pefomed effectively nor giving a direct

impact to reduce DENV infection. Not like other

viral infection such as Human Immunodeficiency

Virus (HIV), influenza or Hepatitis viruses which

have anti-viral therapy, exact anti-DENV treatment

is not avalilable until now and refered to high

burden of DENV infection this anti-DENV is urgely

needed. Balancing haemostatic status is the only

cure available to overcome DENV infection in

patients.

Despite all of those efforts against DENV infection,

several research to find a novel anti dengue is also

progressing. Several plants as natural resources

found to have activity against DENV. Tang et al

198

Yusuf, A., Dewi, B. and Sjatha, F.

Antiviral Activity of Cynometra ramiﬂora Linn Leaves Extract Against Replication of Dengue Virus Serotype 2 on Huh 7.5 Cell In Vitro.

DOI: 10.5220/0008359901980201

In Proceedings of BROMO Conference (BROMO 2018), pages 198-201

ISBN: 978-989-758-347-6

(2012) reported a methanol extract from Momordica

charantiia leaves and leaves extract of Ocimum

sanctum was able to inhibit DENV-1.

Other plant that also potential to be an antiviral is

Cynometra ramiflora Linn. Leaves of C.ramiflora

Linn was proven to have an antiviral capability and

antiseptic properties and widely applied for skin

diseases, such as scabies nor leprae infection

(Quattrocchi U et al, 2012). Several chemical

compounds were found in C. ramiflora Linn such as

flavonoids, tannin, alkaloid, phenolic and saponin

(Paguigan et al, 2014). Saponin was reported for

having a pharmachology activity such as

bactericidal, antiviral, cytotoxic, analgetic, anti-

inflamatory, anti-cancer and anti-allergy. Meanwhile

flavonoid was found to hegative effect against

prostaglandin as anti-analgetic agent (Afialus et al,

2013). Nevertheless, Cynometra ramiflora Linn is

widely distributed in India, Southeast Asia and

Australia (Globinmed, 2015, Hafidh et al., 2009).

With its promising properties as anti-DENV and its

abundancy worldwide, we assessed several

concentration of C.ramiflora Linn leaves estract

against DENV-2 in-vitro using Huh7.5 cell line. As

our result show that this extract have a good potency

as anti-DENV infection.

2 MATERIAL AND METHODS

2.1 Cell and Viruses

Huh7.5 hepatocarcinoma cell line was grown in

MEM containing 10% FBS and 1% of antimycotic-

antibiotic on 5% CO

at 37

C. DENV-2 NGC strain

was propagated in Huh7.5 cell line and viral titer

was measured with immunostaining formed as

FFU/mL (focus forming unit).

C.ramiflora Linn leaves extract was diluted in

Dimethyl sulfoxide (DMSO) as prepared from

Indonesian Institute of Sciences (LIPI).

2.2 Antiviral Assay

A mixture of 1.25; 2.5; 5; 10 and 20 μg/ml

C.ramiflora Linn leaves extract was prepared using

2% of FBS MEM. Thus, extract mixture was applied

to a confluent Huh7.5 culture cell in 96 well plate

followed by addition of DENV-2 with m.o.i

(multiplicity of infection) value 0.5. Cells was then

incubate on 5% CO

at 37

C for 1 hour.

After 1 hour of incubation a semi-overlayed media

(MEM contain of 2% FBS and 0.5%

methylcellulose) was applied. Cells were then

incubated on 5% CO

at 37

C for 48 hours.

Groups of Huh7.5 cells with DMSO and DENV-2

infected were included on each experiment as

negative and positive control. Each group was

performed in 6 repetitive well.

2.3 Focus Forming Immunoassay

After incubation, supernatant was discarded and

cells were washed with excess PBS. A solution of

3.7% formaldehyde was added in to the cells and

incubated for 15 minutes. After incubation,

formaldehyde solution was discarded and cells were

washed vigorously with excess PBS.

Cells were then permeabilized with 0.5% Triton-x

solution for 15 minutes followed by 1:500 dengue

patient sera (confirmed with RT-PCR) in 1% skim

milk-PBS solution. This mixture was then incubate

for 1 h RT.

After incubation, antibody was discarded and cells

were then washed 3 times with excess PBS. A

solution of 1:500 rabbit anti-human IgG peroxidase

conjugated antibody was added in 1% skim milk-

PBS followed further incubation fr 1 h RT.

After incubation, antibody was discarded and cells

were then washed 3 times with excess PBS. A

solution of Diaminobenzidine (DAB) substrate was

then added in to the cells followed by incubation for

15 minutes at RT.

Stained group of cell will appear as brown color

under microscope represent as one viral foci.

Number of foci was calculated and statistical

analysis were performed compared to positive

control.

3 RESULT AND DISCUSSION

Our research show the potency of C.ramiflora Linn

leaves extract against DENV-2 in-vitro using culture

of Huh7.5 cell line. As we know DENV is an

arbovirus with cell tropism thus the use of Huh7.5

hepatocarcinoma cell line is a suitable cell line to be

used in dengue study (Blight et al, 2002). Antiviral

potency from C.ramiflora Linn leaves extract

against DENV-2 was ranged from 36.06%, 45.96%,

47.35%, 55.94% and 62.7% for each concentration

of 1.25; 2.5; 5; 10 and 20 µg/ml respectively. As

depicted from figure 1, significant DENV-2

inhibition compare to positive control was obtained

in p value < 0.05 for all concentration tested.

Antiviral Activity of Cynometra ramiﬂora Linn Leaves Extract Against Replication of Dengue Virus Serotype 2 on Huh 7.5 Cell In Vitro

199

Figure 1. Number of average FFU for each concentration of C.ramiflora Linn leaves extract against DENV-2 represent

with standard error of mean (SEM) and ** P < 0,001.

Based on phytochemical analysis, there are several

chemical compounds can be found in C.ramiflora

Linn leaves extract such as flavonoid, tannin,

alkaloids phenolic and saponin (Paguigan et al,

2014).

Lee et al (2012) reported that saponin has the ability

to inhibit Hepatitis-C virus replication through up-

regulation of cytokine signal-2. We are assuming

that saponin compound can also inhibit DENV

replication through similar mechanism towards Hep-

C virus, since both viruses belong to Flaviviridae

genera. Meanwhile triterpenoid saponin, derivate of

saponin, which also found in C.ramiflora Linn could

also inhibit Herpes simplex virus (Simos et al,

1999).

Flavonoid compound from Carica papaya can

inhibit DENV enzyme of NS2B-NS3 protease whic

required in viral assembly which those proteins also

a good target for viral inhibition (Sethivel et al,

2013). Further bioinformatic analysis shown that

flavonoid has high energitical bind against receptor

binding site of NS2B-NS3 DENV protein. Other

flavonoid deivate, quarcetin and fisetin also show

DENV-2 inhibition (Zandi et al, 2011).

Other compound like tannin was proved to be able to

inhibit HIV-1 and influenza virus through

decreasing viral peptide synthesis. Alkaloids

compound such as 33 isoquinoline have

antibacterial, antifungal and antiviral such as herpes

simplex virus (HSV) and parainfluenza virus on

varied concentration tested (Orhana et al, 2007). As

for phenolic compound, Ara et al (2011) shown that

this compund has anti replication DENV-2 activity

with IC

of 362,68+ 0,04 µg/ ml and SI of 2,75.

Our result show there are DENV inhibition in the

addition of C.ramiflora Linn leaves extract on cell

culture, although on which DENV replication steps

does the inhibition appear is still unknown.

Comparing similar study was performed by Meutia

et al, 2017 which also testing the C.ramiflora Linn

leaves extract against DENV2 in different mtehod

shows that direct and indirect measurement DENV

titer may give different result of inhibition value. On

the other hand, the existance of the extract during

cell incubation and extract removal just after viral

infection can produce different value of DENV

inhibition. Moreover form this both studies, in line

result was shown which C.ramiflora Linn leaves

extract have consistent ability to inhibit DENV

replication in vitro.

4 CONCLUSION

Our study shows that C.ramiflora Linn leaves

extract is having significant anti DENV activity.

ACKNOWLEDGEMENTS

Publication of this was study supported by Hibah

PITTA UI 2018/2019.

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