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

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: 10.5220/0009844500002406

In Proceedings of BROMO Conference (BROMO 2018) - Symposium on Natural Product and Biodiversity, page 1

ISBN: 978-989-758-347-6

et al (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.

BROMO 2018 - Bromo Conference, Symposium on Natural Products and Biodiversity

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.

REFERENCES

Afjalus, S.M., Malik, S., Emrul, H., Sanjana, S., Farjana,

Y. (2013). Evaluation of neuropharmacological,

antibacterial and antinociceptive activity of methanolic

extract of the bark of cynometra ramiflora

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

linn.(Leguminose). Int J Res Ayurveda Pharm, 2, (4),

192–7.

Ara, S., Morais, S.M., Marques, M.M.M., Lima, D.M.,

Santos, S.C.C., Almeida, R.R. et al. (2011). Antiviral

activities of extracts and phenolic components of two

spondias species against dengue virus.J. Venom. Anim.

Toxins incl. Trop. Dis, 17(4).

Blight, K.J., McKeating, J.A., Rice, C.M. (2002). Highly

permissive cell lines for subgenomic and genomic

hepatitis C virus RNA replication. J Virol, 24(76),

13001–14.

Castro, C., Castillo, F., Gutierrez, MM. (2015). Ethanol

extracts of Cassia grandis and Tabernaemontana

cymosa inhibit the in vitro replication of dengue virus

serotype 2. Asian Pac J Trop Dis, 5, (2), 98–106.

Chan, K.S., Chang, K., Lin, C.C., Huang, J.H., Lin, W.R.,

Chen, T.C., et al.(2009). Effect of serotypes on clinical

manifestations of dengue fever in adults. J Microbiol

Immunol Infect, 42,471–8.

Cynometra ramiflora L. Accessed on January 24, 2018

fromhttp://www.globinmed.com/index.php?option=co

m_content&view=article&id=79525:cynometra-

ramiflora-l&catid=367:c

Hafidh, R.R., Abdulamir, A.S., Jahanshiri, F., Abas, F.,

Abu Bakar, F., Sekawi, Z. (2009). Asia is the mine of

natural antiviral products for public health. Open

Complement Med J, (1), 58-68.

Kementrian Kesehatan RI. (2010). Demam berdarah

dengue. 2nd ed. Jakarta: Kementrian Kesehatan RI.

Lee, J., Lim, S., Kang, SM., Min, S., Son, K., et al. (2012).

Saponin Inhibits Hepatitis C Virus Propagation by Up-

regulating Suppressor of Cytokine Signaling 2. PLoS

ONE, 7(6), e39366. doi:10.1371/journal.pone.0039366

Meutia, ZZ., Dewi, BE., (2017) Effects of C.ramiflora

Linn leaves extract against dengue virus replication in

vitro on Huh7it-1 cell. Advanced Science Letters.

olume 23, Number 7, July 2017, pp. 6834-6837(4)

Moektiwardoyo, M., Tjitraresmi, A., Susilawati, Y.,

Yoppi, I., Halimah, E., Zahryanti, D.(2014).The

potential of dewa leaves (Gynura pseudochina (L)

D.C) and temu ireng rhizomes (Curcuma aeruginosa

Roxb.) as medicinal herbs for dengue fever treatment.

Procedia Chemistry,13, 134–41.

Muhtadi, Haryoto, Azizah, T., Indrayudha P., Suhendi, A.,

Yen, K.H. (2014). Antioxidant, cytotoxic activities

and chemical constituents of cynometra ramiflora

Linn. J Med Plants Res, 47(1), 1578–82.

Orhana, I., Ozcelik, B., Karaoglu, T., Sener, B. (2007).

Antiviral and antimicrobial profiles of selected

isoquinoline alkaloids from fumaria and corydalis

species. Z Naturforsch C. 62(1-2), 19-26.

Paguigan, N.D., Castillo, D.H., Hernandez, C.L. (2014).

Anti-ulcer activity of leguminose plants [Internet].

Anti-ulcer activity of leguminose plants. Accessed on

January 24, 2015 from

http://www.scielo.br/scielo.php?pid=S0004-

28032014000100064&script=sci_arttext

Quattrocchi, U. (2012). CRC World Dictionary of

Medicinal and Poisonous Plants: Common Names,

Scientific Names, Eponyms, Synonyms, and

Etymology. United States of America: Taylor &

Francis Group, LLC, 1287.

Senthilvel, P., Lavanya, P., Kumar, K.M., Swetha, R.,

Anitha P., Bag, S. et al. (2013). Flavonoid from carica

papaya inhibits NS2B-NS3 protease and prevents

dengue 2 viral assembly. Bioinformation, 9(18), 889–

895.doi: 10.6026/97320630009889

Simoes, C.M.O., Amoros, M., Girre, L. (1999).

Mechanism of antiviral activity of triterpenoid

saponins.Phytother.Res,13, 323–28.

Tang, L.I., Ling, A.P., Koh, R.Y., Chye, S.M., Voon,

K.G.(2012).Screening of antidengue activity in

methanolic extracts of medicinal plants. BMC

Complement Altern Med, 12, 3.

Yacoub, S., Farrar, J. (2014). Dengue (3rd ed). China:

Elsevier Saunders, 162-170.

Zandi, K., Teoh, B.T., Sam, S.S., Wong, P.F., Mustafa,

M.R., Abubakar, S. (2011). Antiviral activity of four

types of bioflavonoid against dengue virus type-2.

Virol J, 28,8, 560, doi: 10.1186/1743-422X-8-560.

BROMO 2018 - Bromo Conference, Symposium on Natural Products and Biodiversity