Advance Publication by J-STAGE     

Japanese Journal of Infectious Diseases      

Anti-hepatitis C virus activity of a crude extract from longan (Dimocarpus longan Lour.) leaves   

Dadan Ramadhan Apriyanto, Chie Aoki, Sri Hartati, Muhammad Hanafi, Leonardus Broto Sugeng Kardono, Ade Arsianti, Melva Louisa, Tjahjani Mirawati Sudiro, Beti Ernawati Dewi, Pratiwi Sudarmono, Amin Soebandrio, and Hak Hotta

Received: March 6, 2015. Accepted: June 22, 2015 Published online: August 7, 2015

DOI: 10.7883/yoken.JJID.2015.107             

Advance Publication articles have been accepted by JJID but have not been copyedited

or formatted for publication.

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Anti-hepatitis C virus activity of a crude extract from longan (Dimocarpus longan

Lour.) leaves

Dadan Ramadhan Apriyanto1, Chie Aoki2,3*, Sri Hartati4, Muhammad Hanafi4,

Leonardus Broto Sugeng Kardono4, Ade Arsianti5, Melva Louisa6, Tjahjani Mirawati

Sudiro1, Beti Ernawati Dewi1, Pratiwi Sudarmono1, Amin Soebandrio1, Hak Hotta3

1Department of Microbiology, Faculty of Medicine, University of Indonesia, JL.

Salmeba 4, Jakarta 10430, Indonesia; 2JST/JICA SATREPS of Kobe University

Graduate School of Medicine, JL. Salemba 4, Jakarta 10430, Indonesia; 3Division of

Microbiology, Kobe University Graduate School of Medicine, 7-5-1 Kusunoki-cho,

Chuo-ku, Kobe 650-0017, Japan; 4Research Center for Chemistry, Indonesian Institute

of Sciences (LIPI), Kawasan Puspiptek, Serpong 15310, Indonesia; and 5Department of

Chemistry and 6Department of Pharmacology, Faculty of Medicine, University of

Indonesia, JL. Salmeba 4, Jakarta 10430, Indonesia.

*Corresponding author: Chie Aoki

E-mail address: nu_chie@msn.com

Address: Division of Microbiology, Kobe University Graduate School of Medicine,

7-5-1 Kusunoki-cho, Chuo-ku, Kobe 650-0017, Japan.

Telephone number: +81-78-382-5502, Fax number: +81-78-382-5519

Keywords: Hepatitis C Virus, Antiviral, crude extract of Dimocarpus longan leaves

Running title: Anti-HCV activity of D. longan crude extract

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青木千恵 1,2、堀田博 2

1. 神戸大学大学院医学研究科 JST/JICA SATREPS

〒10430, インドネシアジャカルタ Salemba 4

2. 神戸大学大学院医学研究科 微生物学分野

〒650-0017, 兵庫県神戸市中央区楠町 7-5-1, TEL: 078-382-5502

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SUMMARY:

Hepatitis C is a disease caused by hepatitis C virus (HCV) that causes chronic infection,

cirrhosis, and hepatocellular carcinoma. The current standard therapy is a combination

of pegylated interferon-α plus ribavirin with NS3 protease inhibitors. Addition of NS3

protease inhibitors increases response rates; however, this addition is associated with

significant side effects and an increase in the overall cost of the treatment. Therefore,

there remains a need to develop safe and inexpensive drugs for the treatment of HCV

infections. In this study, we examined the antiviral activity of a crude extract from

Dimocarpus longan leaves against HCV (genotype 2a strain JFH1). The D. longan

crude extract exhibited anti-HCV activity with a 50% effective concentration (EC50) of

19.4 μg/ml without cytotoxicity. A time-of-addition study demonstrated that the crude

extract exerts anti-HCV activity at both the entry and post-entry steps. The crude extract

markedly blocked viral entry step through a direct virucidal effect with a marginal

inhibition of virion assembly. The co-treatment of the crude extract with cyclosporine A

or telaprevir, an NS3 protease inhibitor, had additive and synergistic antiviral effects,

respectively. Our findings suggest that the D. longan crude extract may be a candidate

of the add-on therapy for HCV infection.

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INTRODUCTION:

Hepatitis C virus (HCV) is a small, enveloped virus belonging to the Hepacivirus

genus of the Flaviviridae family. The HCV genome is a single-stranded, positive-sense

RNA of 9.6 kb in length and encodes for a single open reading frame (1). The single

open reading frame is translated by an internal ribosome entry site located in the

5′-untranslated region (5′UTR) and processed by host peptidases and viral-encoding

proteases into ten polypeptides: three structural (core, E1, E2) and seven nonstructural

(p7, NS2, NS3, NS4A, NS4B, NS5A, NS5B) proteins (2). The life cycle of HCV can be

divided into three major steps: entry of the virus into its target cells by

receptor-mediated endocytosis, cytoplasmic and membrane-associated replication of the

RNA genome, and assembly and release of the progeny virions.

HCV is an important human pathogen infecting approximately 170–200

million people worldwide (3) and contributing to 3–4 million new infections each year

(4). HCV infection can lead to chronic hepatitis, liver cirrhosis, and hepatocellular

carcinoma. These chronic liver diseases are a major cause of mortality. HCV is

commonly transmitted from infected blood and organ transplants (5). HCV isolates are

classified into seven major genotypes and more than 100 subtypes (6). The distribution

of HCV genotypes varies geographically and HCV genotypes 1 to 3 are distributed

worldwide. Genotypes 1b and 2a are most common in Asia, including Japan and

Indonesia (7-9).

Diversity of HCV isolates hampers the development of vaccine. Combination

therapy with a pegylated interferon-α (PEG-IFNα) and ribavirin have been used as the

initial treatment of choice. This therapy improved the sustained virological response

(SVR); however, the SVR rates are no better than 50% in patients infected with

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genotype 1 infections (1). Till date, many anti-HCV compounds have been investigated

and those are classified into two main classes: direct-acting antivirals (DAAs) and host

targeting antivirals. The DAAs target the NS3 protease, NS5A protein, and NS5B RNA

polymerase, while host–targeting antivirals target host proteins critical for virus

replication, such as cyclophilin A and micro RNA122 (10). Recently, the US Food and

Drug Administration approved some NS3 protease inhibitors (telaprevir, boceprevir,

etc.) for therapy of patients infected with HCV genotype 1. The addition of the

telaprevir to PEG-IFNα plus ribavirin-based therapy has a higher barrier to drug

resistance and increases SVR rates up to 75% in patients with HCV genotype 1

infections (11-13). However, this combination therapy is very expensive and sometimes

causes significant adverse effects (14, 15). Thus, there remains a need to develop safe,

inexpensive, and well-tolerated drugs for the treatment of HCV infections.

Medicinal plants are attractive resources for the discovery of new

biologically–active natural products for the treatment of many diseases, including

infectious diseases (16, 17). Because natural products from medicinal plants have

advantages of lower cost and fewer side effects as well as characteristics of high

chemical diversity, researchers have explored active molecules from medicinal plants

during the last several decades. Phytochemical contents of plants, such as flavonoids,

terpenoids, lignin, alkaloids, tannins, polyphenolics, coumarins, saponins and

chlorophyllins, were reported to have anti-HCV activity (18-26). Thus, natural products

from medicinal plants are alternative approach to control HCV infection.

In the present study, we found that the crude extract from Dimocarpus

longan leaves has a potential anti-HCV activity. We investigated the mechanism of

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action of the inhibition and also antiviral effect of the crude extract in combination with

cyclosporine A and an NS3 protease inhibitor, telaprevir.

MATERIALS AND METHODS

Cells and viruses: A clone from a human hepatoma-derived cell line, Huh7it-1

(19), were grown in Dulbecco's modified Eagle's medium (Gibco-Invitrogen, Carlsbad,

CA, USA) supplemented with 10% fetal bovine serum (Biowest, Nuaille, France),

non-essential amino acids (Gibco-Invitrogen) and kanamycin (Sigma-Aldrich, St. Louis,

MO, USA). Cultured cells were incubated at 37°C in 5% CO2 humidified chamber. A

cell culture-adapted HCV variant (JFH1 strain of genotype 2a) was propagated as

described previously (27, 28). An Indonesian strain of dengue virus type 2 (DENV,

DS-18/09 strain) was propagated in mosquito-derived C6/36 cells and African green

monkey kidney-derived Vero cells as described previously (29, 30).

Virus titration: Virus titration was performed as described previously (19, 27).

Culture supernatants were serially diluted in culture medium and inoculated to the cells in

96-well plates. After virus adsorption for 4 h at 37°C, the cells were incubated in medium

containing 0.4% methylcellulose (Sigma-Aldrich) for 40 h. Virus titers were determined

by a focus-forming assay as described previously (27, 28). Virus-infected cells were

stained with anti-HCV or anti-DENV human patient’s serum followed by horseradish

peroxidase-conjugated goat anti-human IgG (MBL, Nagoya, Japan). Infectious foci were

visualized with Metal Enhanced DAB Substrate kit (Thermo Fisher Scientific, Rockford,

IL, USA). Foci image was captured using Olympus digital camera DP21 attached to

Olympus CKX41 microscope (Olympus, Tokyo, Japan) and counted using katikati

counter (http://www.vector.co.jp/soft/win95/art/se347447.html).

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To determine intracellular HCV infectivity, freeze-and-thaw experiments were

performed. In brief, virus-infected cells were washed with phosphate-buffered saline

(PBS) and suspended in the fresh culture medium. The samples were subjected to

3 cycles of freezing and thawing at −80°C and 37°C, respectively, and were centrifuged

at 12,000 × g for 5 min at 4°C to remove cell debris. HCV titers were measured as

described above.

Antiviral activity assay: Antiviral activity assay was performed as described

previously (19). In brief, Huh7it-1 cells were inoculated with HCV or DENV at

multiplicity of infection (MOI) of 1 in the presence of D. longan crude extract (100, 50,

25, 12.5, 6.25 and 3.125 μg/ml) for 2 h at 37°C. After removing residual virus by

washing, the cells were incubated for 46 h with the same extract samples. Cell culture

supernatants were collected for virus titration. In time-of-addition studies, cells were

treated with 50 μg/ml D. longan crude extract only during viral inoculation or only after

inoculation for the remaining culture period until virus harvest.

To evaluate the possible effect of co-treatment effect of the D. longan crude extract

with cycolosporin A or telaprevir (AdooQ BioScience, CA, USA), the classical

isobologram analysis was performed (31).

Quantitative real time-polymerase chain reaction (qRT-PCR): Total RNA

was extracted using Trizol reagent (Invitrogen, Carlsbad, CA, USA) according to the

manufacturer’s instructions. One μg of total RNA was transcribed using a ReverTra

Ace® qPCR RT Kit (Toyobo Co. Ltd., Osaka, Japan) with random primers and cDNA

was amplified using SYBR Premix Ex Taq (Takara, Kyoto, Japan). PCR was performed

using Roche 480 LightCycler II system using specific primers to amplify an NS3 region

of the HCV genome; 5'-CTTTGACTCCGTGATCGACT-3' (sense) and

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5'-CCCTGTCTTCCTCTACCTG-3' (antisense), and human -actin (ACTB) mRNA;

5'-TGGCACCCAGCACAATGAA-3' (sense) and

5'-CTAAGTCATAGTCCGCCTAGAAGCA-3' (antisense), as described previously

(32).

Western blot analysis: Immunoblotting was performed as described

previously with a slight modification (33, 34). Cells were lysed in sodium dodecyl

sulfate (SDS) sample buffer. Equal amounts of protein were separated by

SDS-polyacrylamide gel electrophoresis and transferred onto a polyvinylidene

difluoride membrane (Amersham Pharmacia, Piscataway, NJ, USA). Membranes were

incubated with an HCV NS3-specific mouse monoclonal antibody (clone H23, Abcam,

Cambridge, MA, USA), or glyceraldehyde-3-phosphate dehydrogenase (GAPDH)

(MBL, Nagoya, Japan) followed by horseradish peroxidase-conjugated goat anti-mouse

immunoglobulin (MBL, Nagoya, Japan) as the secondary antibody. Bound antibody

complexes were detected with immobilon Western (Millipore, Bedford, MA, USA).

Viral adsorption assay: Cells were inoculated with HCV (MOI=3) in the

presence or absence of the D. longan crude extract (50 μg/ml) at 4°C for 1 h. The cells

were extensively washed with cold PBS and suspended in Trizol reagent for total RNA

extraction.

Virucidal activity assay: The HCV suspension (106 ffu/ml, 75 µl) mixed with

an equal volume of D. longan crude extract or heparin (Sigma-Aldrich) was incubated

for 2 h at 37°C. Cells were inoculated with a dilution (1250 times) of the treated virus

suspension for 4 h at 37°C. After removing viral inoculum, the cells were overlaid with

0.4% methylcellulose-containing medium and incubated for 40 h.

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Plasmid construction: pFK-SGR-GLuc/JFH1 was generated from subgenomic

replicon pSGR-JFH1, in which neo gene was replaced with Gaussia luciferase gene

(GLuc). In brief, an HCV replicon expressing GLuc was constructed by replacing Neo

gene of plasmid pSGR-JFH1 with the luciferase gene from Gaussia princeps. The T7

promoter and the HCV 5′UTR were amplified from pSGR-JFH1 by PCR using primers

JFH1-EcoRI (5′-GGAATTCTAATACGACTCACTATAG-3′) and JFH1-GLuc

(5′-ACTTTGACTCCCATTTTGGTTTTTCTTTGAGG-3′). GLuc gene was amplified

from pRNAi-hGL (TAKARA Bio Inc., JAPAN) by PCR using primers GLuc-ATG

(5′-ATGGGAGTCAAAGTTCTGTTTGC-3′) and GLuc-T (5′-

TTAGTCACCACCGGCCCCCT-3′). The two PCR DNA fragments were used as

templates for the second round of PCR to combine the 5′UTR with GLuc gene. The

second PCR product was digested with EcoRI and ligated with the pSGR-JFH1 vector

by digesting with EcoRI and PmeI. After the correct DNA sequences were verified, the

generated pSGR-GLuc/JFH1 was double digested with Age I and EcoRV, respectively,

and put into linearized pFK with same restriction enzyme sites.

Replication inhibition assay using subgenomic replicon: Methods for in

vitro transcription of HCV RNA and its electroporation into cells have been reported

previously (32). SGR-GLuc/JFH1 RNA-transfected cells were seeded into a 12-well

plate. At 4 h post-electroporation, D. longan crude extract or cyclosporin A

(Sigma-Aldrich, St. Louis, MO, USA) was added to the medium, and the cells were

incubated for 48 h. Portions of the culture medium were collected for luciferase assay

and luciferase activities were measured with a Gaussia luciferase assay kit (New

England Biolabs, Ipswich, MA, USA) using a GloMax-96 Microplate Luminometer

(Promega, Madison, WI, USA). The luminometer was set to automatically inject 50 µl

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of the Gluc assay solution with the following parameters, 5 seconds of delay and 5

seconds of integration.

Cytotoxicity assay: Cytotoxicity of D. longan crude extract against Huh7it-1

cells was determined by MTT [3-(4,5-dimethylthiazol-2-

yl)-5(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium] method. Briefly,

cells seeded in 96-well plates were treated with various concentrations of the crude

extract for 48 h. The MTT assay was performed as described previously (19). The

percentage of viable cells was plotted versus the concentration of crude extract. The

concentration by which to mediate 50% cytotoxicity (CC50) was determined by

non-linear regression analysis using GraphPad Prism graphing software.

Plant material: D. longan leaves were obtained from the Research Center for

Chemistry, Indonesian Institute of Sciences (LIPI), Serpong, Indonesia. The species

were determined by botanists at the Botanical Research Center for Biology, LIPI,

Chibinong, Indonesia. A herbarium specimen was deposited in the Research Center for

Chemistry, LIPI.

Preparation of a crude extract from D. longan leaves: Dried D. longan leaves

(500 g) were ground to powder and extracted (3-times) using methanol (2 L) under

reflux conditions. The extracts were combined and concentrated under vaccum at 40°C

using rotavapor to obtain the crude extract. The crude extract was dissolved in dimethyl

sulfoxide (DMSO) at a concentration of 100 mg/ml, and stored at −30°C.

Data analysis: Results were expressed as the mean ± SEM. Differences

between two data sets was evaluated by Student’s two-tailed t- test. A p-value of 0.05

was considered as statistically significant.

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RESULTS:

Crude extract from D. longan inhibits HCV infection:

We collected approximately 250 kinds of Indonesian medicinal plants from a

botanical garden in LIPI, Serpong, Indonesia and evaluated crude extracts from those

plants for anti-HCV activity. We found that a crude extract from D. longan leaves

inhibited HCV infection in a dose-dependent manner, with a 50% effective

concentration (EC50) of 19.4 µg/ml (Fig. 1A). The cytotoxicity of the crude extract

against Huh7it-1 cells was examined by an MTT assay. No apparent cytotoxicity was

observed when Huh7it-1 cells were treated with D. longan crude extract up to 400

µg/ml (CC50=681.9 µg/ml) (Fig. 1B). The selectivity index (SI: CC50/EC50) was 35.1.

To determine the possible inhibition step(s) in the viral life cycle, we carried out

a time-of-drug addition study. Cells were treated with D. longan crude extract only

during 2 h of viral inoculation (entry step) or only after viral inoculation for the

remaining culture period until virus harvest (post-entry step). As a positive control,

HCV mixed with the crude extract was inoculated to the cells, and after viral

inoculation for 2 h, cells were re-fed with fresh medium containing the crude extract for

46 h (whole step). D. longan crude extract showed anti-HCV activity at both the entry

and the post-entry steps (Fig. 2B). The treatment at the entry and the post-entry steps

exhibited 75.2% and 90.8% inhibition, respectively, at 50 µg/ml. The levels of

intracellular HCV NS3 protein accumulation were examined by western blotting

analysis. In contrast to the results of infectivity in supernatants, the reduction in the

amounts of the HCV NS3 protein accumulation by the treatment was not clearly

observed (Fig. 2C). In addition, cells were pretreated with the crude extract for 2 h

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before viral inoculation. The result showed that the pretreatment of the cells with the

crude extract did not affect HCV infection into the cells (Fig. 2D).

D. longan crude extract exerts anti-HCV activity through a direct virucidal effect

and by a partial inhibition of the virion assembly: The time-of-drug addition analysis

showed that D. longan crude extract exerts anti-HCV activity at both the entry and the

post-entry steps. Thus, we proceeded to determine the mechanism(s) of action of the

crude extract by conducting three categorized experiments: (i) a virucidal activity assay,

(ii) a viral adsorption assay, and (iii) tests for the post-entry event (virus replication,

virus translation, and virion assembly). Firstly, the virucidal activity was measured.

HCV premixed with the crude extract (50 g/ml) was incubated for 2 h at 37°C before

viral inoculation to the cells at 37°C. The result showed that the crude extract could

reduce HCV infectivity by 99.7% (Fig. 3A). Next, we investigated the effect of the

crude extract on virus adsorption. Cells were preincubated at 4°C and inoculated with

HCV at 4°C for 1 h in the presence of the crude extract. The treatment of cells at 4°C

can minimize virus internalization into the cells occurring after virus binding to the

specific receptor(s). Subsequently, total RNAs from the cells after viral adsorption were

extracted and subjected to qRT-PCR to quantify the amount of HCV bound to the cell

surface. The result showed that the crude extract exhibited weak anti-adsorption activity

and the HCV copy number decreased by 33.8% (Fig. 3B). As a positive control,

treatment with heparin (400 g/ml) blocked the virus adsorption by 53.8%.

Next, to explore antiviral effect(s) on the post-entry event, we first evaluated the

effect of the crude extract on HCV replication. Cells were infected with HCV and then

treated with the crude extract at 50 µg/ml for 46 h, followed by quantifying intracellular

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HCV RNA levels using qRT-PCR. Treatment with the crude extract at the post-entry

step slightly reduced intracellular HCV RNA levels compared with the untreated control

(15% reduction) (Fig. 4A). A similar result was observed in HCV subgenomic replicon

system, which revealed that a treatment with the crude extract causes a 30% decrease of

luciferase activity (Fig. 4B). Next, HCV protein accumulation was analyzed by western

blotting. In agreement with the effect on the virus RNA replication, treatment with the

crude extract hardly affected the levels of HCV NS3 protein accumulation (Fig. 4C); on

the other hand, cyclosporin A (2 μg/ml), a positive control inhibitor, reduced HCV NS3

accumulation. Furthermore, to consider the possibilities of defective virion assembly

and impaired release of virion, we quantified the amount of intracellular and

extracellular infectious viruses. The crude extract at 50 µg/ml reduced the extracellular

virus titer from 1.1 × 106 ffu/ml to 2.2 × 105 ffu/ml (79.8% reduction) (P < 0.01), while

the intracellular virus titer from 6.1 × 105 ffu/ml to 3.3 × 105 ffu/ml (48.6% reduction)

(P < 0.01) (Fig. 4D). Taken together, our data suggested that D. longan crude extract

exhibits anti-HCV activity through a direct virucidal effect and by a partial inhibition of

the virion assembly.

Co-treatment of a D. longan crude extract with cyclosporine A or telaprevir has

additive and synergistic anti-HCV effects, respectively: Cyclosporine A, a

well-known immunosuppressive drug, inhibits HCV replication by mediating the

blockage of cyclophilins (35). Telaprevir, an inhibitor of HCV NS3 protease, is used to

treat HCV genotype 1 infection in combination with PEG-IFNα plus ribavirin in routine

clinical practice. Because we found that the D. longan crude extract inhibits HCV

infection, we examined the inhibition effect of the D. longan crude extract in

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combination with cyclosporine A or telaprevir. The percent infectivity by co-treatment

with cyclosporinA or telaprevir was plotted for fixed concentration of the D. longan

crude extract. The addition of cyclosporine A or telaprevir resulted in a slight increase

of the anti-HCV effect (Fig. 5A). The co-treatment did not increase cytotoxicity (data

not shown). Moreover, isobologram analysis demonstrated that inhibitory effect of

co-treatment with cyclosporine A or telaprevir was additive and synergistic,

respectively (Fig. 5B).

D. longan crude extract inhibits DENV infection: We further examined the effect of

D. longan crude extract against DENV, another RNA virus belonging to the same

Flaviviridae family. The D. longan crude extract exhibited anti-DENV activity with an

EC50 of 40.9 μg/ml, at day 2 post-infection (Fig. 6).

DISCUSSION:

    D. longan, a member of the Sapindaceae family, is commonly found in tropical

and subtropical areas, such as China, Thailand, Malaysia, Philippines, and Indonesia. It

has been used as a traditional medicinal herb and possesses anti-inflammatory,

antibacterial, antifungal, antiviral, antioxidant, and anticancer properties (36-39). Crude

extract from D. longan aerial parts was reported to exhibit antiviral activity against HIV

(39); however, till date there has been no reports that the crude extract from D. longan

leaves exhibits antiviral activity against HCV and other viruses.

In the present study, we have not yet isolated a compound responsible for the

anti-HCV activity of D. longan crude extract. Phytochemical analysis showed that the

crude extract contains high amounts of triterpenes, tannins, flavonoids, and

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carbohydrates (data not shown). D. longan leaves were reported to contain eight

polyphenolic compounds: ellagic acid, 3,4-O-dimethyl ellagic acid, (+)-catechin, ethyl

gallate, gallic acid, kaempferol, quercetin and kaempferol-3-O-α-L-rhamnoside (40).

Recently, Xue et al. reported 12 compounds isolated from D. longan leaves; quercetin

3-O-(3″-O-2‴-methyl-2‴-hydroxylethyl)-β-D-xyloside, quercetin

3-O-(3″-O-2‴-methyl-2‴-hydroxylethyl)-α-L-rhamnopyranoside, afzelin,

kaempferol-3-O-α-L-rhamnopyranoside, (-)-epicatechin and proanthocyanidin A-2,

friedelin, epifriedelanol, β-amyrin, N-benzoylphenylalanyl-N-benzoylphenylalaninate,

β-sitosterol, and daucosterol (41). Ellagic acid and gallic acid, ubiquitous tannins, were

reported to exhibit anti-HCV activity. Ellagic acid was reported to suppress HCV

replication of genotype 2a replicon by targeting the HCV NS3 protease (42), while

gallic acid inhibits HCV at the early viral entry step (43). Catechin and epicatechin were

reported to exhibit anti-HCV activity. Catechin inhibits virus assembly (44), and

epicatechin inhibits virus replication by attenuating the COX-2-dependent signal

pathway (45). The flavonoid quercetin was reported to inhibit HCV replication and

HCV virion production (19, 44). Quercetin inhibits HCV replication by targeting the

cellular heat shock protein 40 and 70 (46). It also suppresses HCV NS3/NS4A protease

activity (47). The anti-HCV activity of the D. longan crude extract in the present study

may reflect either the effect(s) of the principal constituent or of multiple bioactive

compounds described above. Further analysis is required to identify the active

molecule(s) responsible for the anti-HCV activity of D. longan crude extract.

The result of time-of-drug addition study demonstrated that the D. longan crude

extract exerts the anti-HCV activity both at the entry and the post-entry steps. The crude

extract blocked HCV entry through a direct virucidal effect (Fig. 3A). There was only

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marginal inhibition of HCV RNA replication and HCV protein accumulation by the

crude extract (Fig. 4A, B and C). The crude extract exhibited marginal antiviral effect

on the virus assembly (Fig. 4D). These data suggested that the crude extract exhibits

anti-HCV activity primarily through a direct virucidal effect. In addition, the observed

reduction in the HCV infectivity at the post-entry step is thought to result from a direct

inactivation of virion released from the infected cells and a partial inhibition of the

virion assembly.

The current standard treatment for chronic hepatitis patients with HCV genotype

1 infection is a triple combination regimen using PEG-IFNα plus ribavirin with NS3

protease inhibitors (telaprevir, simeprevir, etc.). Moreover, all oral, IFN-free regimen

consisting of an NS3 protease inhibitor (asunaprevir) and an NS5A replication-complex

inhibitor (daclatasvir) was recently approved for clinical practice. Clinical guidelines

discuss optimizing the use of several DAAs with a different mechanism of action in the

HCV life cycle to maximize SVR rates and minimize the risk of selection of drug

resistance. We demonstrated that a co-treatment of D. longan crude extract with

cyclosporine A or telaprevir had additive and synergistic antiviral effects, respectively

(Fig. 5B). There are some advantages of medicinal use of the crude extract such as less

cost, fewer side effects and easier access compared to isolated medical constituents or

synthetic medicines. Although the use of D. longan crude extract alone has relatively

lower anti-HCV effect, coadministration of the crude extract as a supplement might

increase therapeutic outcome in combination therapy with IFN-free regimens.

In the present report, we also demonstrated that D. longan crude extract exhibits

anti-DENV activity (Fig. 6). Dengue is a mosquito-borne virus disease and found in

tropical and subtropical regions. There is currently no effective vaccine or antiviral drug

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available; thus, there is a strong need to develop cheap and effective antivirals. Our data

revealed that D. longan crude extract inhibits DENV infection with EC50 being 40.9

μg/ml, although the effectiveness of the extract against DENV is weaker than the

effectiveness against HCV. Because both HCV and DENV are members of the

Flaviviridae family, an intensive work on anti-HCV antivirals can also benefit the

search for antivirals against DENV.

In conclusion, the present study demonstrates that a D. longan crude extract

exhibits anti-HCV activity primarily by inhibiting the entry step through a direct

virucidal effect. The crude extract may be useful to develop a candidate(s) of the add-on

therapy for HCV infection.

Acknowledgments:

We thank Dr. Takaji Wakita (National Institute of Infectious Diseases, Tokyo, Japan)

for providing pJFH-1 and pSGR-JFH1. This work was supported in part by Science and

Technology Research Partnerships for Sustainable Development (SATREPS) from

Japan Science and Technology Agency (JST) and Japan International Cooperation

Agency (JICA).

Conflict of interest: The authors declare no conflict of interests.

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REFERENCES:

1. Lauer GM, Walker BD. Hepatitis C virus infection. N Engl J Med. 2001;345:41-52. 2. Lohmann V, Korner F, Koch J, et al. Replication of subgenomic hepatitis C virus RNAs in a hepatoma cell line. Science. 1999;285:110-3. 3. Wendt A, Adhoute X, Castellani P, et al. Chronic hepatitis C: future treatment. Clin Pharmacol. 2014;6:1-17. 4. Shepard CW, Finelli L, Alter MJ. Global epidemiology of hepatitis C virus infection. Lancet Infect Dis. 2005;5:558-67. 5. Brown RS. Hepatitis C and liver transplantation. Nature. 2005;436:973-8. 6. Messina JP, Humphreys I, Flaxman A, et al. Global distribution and prevalence of hepatitis C virus genotypes. Hepatology. 2015;61:77-87. 7. Dusheiko G, Schmilovitz-Weiss H, Brown D, et al. Hepatitis C virus genotypes: an investigation of type-specific differences in geographic origin and disease. Hepatology. 1994;19:13-8. 8. Takada N, Takase S, Takada A, et al. Differences in the hepatitis C virus genotypes in different countries. J Hepatol. 1993;17:277-83. 9. Utama A, Tania NP, Dhenni R, et al. Genotype diversity of hepatitis C virus (HCV) in HCV-associated liver disease patients in Indonesia. Liver Int. 2010;30:1152-60. 10. Pybus OG, Cochrane A, Holmes EC, et al. The hepatitis C virus epidemic among injecting drug users. Infect Genet Evol. 2005;5:131-9. 11. Kwo PY. Boceprevir: a novel nonstructural 3 (NS3) protease inhibitor for the treatment of chronic hepatitis C infection. Therap Adv Gastroenterol. 2012;5:179-88. 12. Poordad F, McCone J, Jr., Bacon BR, et al. Boceprevir for untreated chronic HCV genotype 1 infection. N Engl J Med. 2011;364:1195-206. 13. Zeuzem S, Andreone P, Pol S, et al. Telaprevir for retreatment of HCV infection. N Engl J Med. 2011;364:2417-28. 14. Hynicka LM, Heil EL. Anemia management in patients with chronic viral hepatitis C. Ann Pharmacother. 2013;47:228-36. 15. McHutchison JG, Everson GT, Gordon SC, et al. Telaprevir with peginterferon and ribavirin for chronic HCV genotype 1 infection. N Engl J Med. 2009;360:1827-38. 16. Kitazato K, Wang Y, Kobayashi N. Viral infectious disease and natural products with antiviral activity. Drug Discov Ther. 2007;1:14-22. 17. Yang SS, Cragg GM, Newman DJ, et al. Natural product-based anti-HIV drug discovery and development facilitated by the NCI developmental therapeutics program. J Nat Prod. 2001;64:265-77. 18. Adianti M, Aoki C, Komoto M, et al. Anti-hepatitis C virus compounds obtained from Glycyrrhiza uralensis and other Glycyrrhiza species. Microbiol Immunol. 2014;58:180-7. 19. Aoki C, Hartati S, Santi MR, et al. Isolation and identification of substances with anti-hepatitis c virus activities from kalanchoe pinnata. Int J Pharm Pharm Sci. 2014;6:211-5. 20. Calland N, Dubuisson J, Rouille Y, et al. Hepatitis C virus and natural compounds: a new antiviral approach? Viruses. 2012;4:2197-217.

Accep

ted M

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  19

21. Jardim AC, Igloi Z, Shimizu JF, et al. Natural compounds isolated from Brazilian plants are potent inhibitors of hepatitis C virus replication in vitro. Antiviral Res. 2015;115:39-47. 22. Kong L, Li S, Liao Q, et al. Oleanolic acid and ursolic acid: novel hepatitis C virus antivirals that inhibit NS5B activity. Antiviral Res. 2013;98:44-53. 23. Lee J, Lim S, Kang SM, et al. Saponin inhibits hepatitis C virus propagation by up-regulating suppressor of cytokine signaling 2. PLoS One. 2012;7:e39366. 24. Lee JC, Tseng CK, Young KC, et al. Andrographolide exerts anti-hepatitis C virus activity by up-regulating haeme oxygenase-1 via the p38 MAPK/Nrf2 pathway in human hepatoma cells. Br J Pharmacol. 2014;171:237-52. 25. Patrick L. Hepatitis C: epidemiology and review of complementary/alternative medicine treatments. Altern Med Rev. 1999;4:220-38. 26. Ratnoglik SL, Aoki C, Sudarmono P, et al. Antiviral activity of extracts from Morinda citrifolia leaves and chlorophyll catabolites, pheophorbide a and pyropheophorbide a, against hepatitis C virus. Microbiol Immunol. 2014;58:188-94. 27. Aoki C, Hidari KI, Itonori S, et al. Identification and characterization of carbohydrate molecules in mammalian cells recognized by dengue virus type 2. J Biochem. 2006;139:607-14. 28. Yu L, Aoki C, Shimizu Y, et al. Development of a simple system for screening anti-hepatitis C virus drugs utilizing mutants capable of vigorous replication. J Virol Methods. 2010;169:380-4. 29. Ito M, Takasaki T, Yamada K, et al. Development and evaluation of fluorogenic TaqMan reverse transcriptase PCR assays for detection of dengue virus types 1 to 4. J Clin Microbiol. 2004;42:5935-7. 30. Putri DH, Sudiro TM, Yunita R, et al. Immunogenicity of a candidate DNA vaccine based on the prM/E genes of a dengue type 2 virus Cosmopolitan genotype. Jpn J Infect Dis. 2015. 31. Tanabe Y, Sakamoto N, Enomoto N, et al. Synergistic inhibition of intracellular hepatitis C virus replication by combination of ribavirin and interferon- alpha. J Infect Dis. 2004;189:1129-39. 32. Hou W, Aoki C, Yu L, et al. A recombinant replication-competent hepatitis C virus expressing Azami-Green, a bright green-emitting fluorescent protein, suitable for visualization of infected cells. Biochem Biophys Res Commun. 2008;377:7-11. 33. Deng L, Adachi T, Kitayama K, et al. Hepatitis C virus infection induces apoptosis through a Bax-triggered, mitochondrion-mediated, caspase 3-dependent pathway. J Virol. 2008;82:10375-85. 34. Wahyuni TS, Widyawaruyanti A, Lusida MI, et al. Inhibition of hepatitis C virus replication by chalepin and pseudane IX isolated from Ruta angustifolia leaves. Fitoterapia. 2014;99:276-83. 35. Nakagawa M, Sakamoto N, Tanabe Y, et al. Suppression of hepatitis C virus replication by cyclosporin a is mediated by blockade of cyclophilins. Gastroenterology. 2005;129:1031-41. 36. Meng FY, Ning YL, Qi J, et al. Structure and antitumor and immunomodulatory activities of a water-soluble polysaccharide from Dimocarpus longan pulp. Int J Mol Sci. 2014;15:5140-62. 37. Pan Y, Wang K, Huang S, et al. Antioxidant activity of microwave-assisted extract of longan (Dimocarpus Longan Lour.) peel. Food Chem. 2008;106:1264-70.

Accep

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anus

cript

  20

38. Rasched KN, Fouche G. Anticancer Activity of Dimocarpus longan Lour. Leaf Extracts in vitro and Phytochemical Profile. Greener J Med Plant Res. 2013;1:1-5. 39. Rashed K, Luo MT, Zhang LT, et al. Anti-HIV-1 Activity of of Dimocarpus longan Lour. Extracts and the Main Chemical Content. Global J Microbiol Biochem. 2013;1:1-7. 40. Wu Q, Wang L, Yu X, et al. Polyphenols from longan leaf and their radical-scavenging activity. Int Proc Chem Biol Environ. 2013;50:180-5. 41. Xue Y, Wang W, Liu Y, et al. Two new flavonol glycosides from Dimocarpus longan leaves. Nat Prod Res. 2015;29:163-8. 42. Reddy BU, Mullick R, Kumar A, et al. Small molecule inhibitors of HCV replication from pomegranate. Sci Rep. 2014;4:5411. 43. Hsu WC, Chang SP, Lin LC, et al. Limonium sinense and gallic acid suppress hepatitis C virus infection by blocking early viral entry. Antiviral Res. 2015;118:139-47. 44. Khachatoorian R, Arumugaswami V, Raychaudhuri S, et al. Divergent antiviral effects of bioflavonoids on the hepatitis C virus life cycle. Virology. 2012;433:346-55. 45. Lin YT, Wu YH, Tseng CK, et al. Green tea phenolic epicatechins inhibit hepatitis C virus replication via cycloxygenase-2 and attenuate virus-induced inflammation. PLoS One. 2013;8:e54466. 46. Gonzalez O, Fontanes V, Raychaudhuri S, et al. The heat shock protein inhibitor Quercetin attenuates hepatitis C virus production. Hepatology. 2009;50:1756-64. 47. Bachmetov L, Gal-Tanamy M, Shapira A, et al. Suppression of hepatitis C virus by the flavonoid quercetin is mediated by inhibition of NS3 protease activity. J Viral Hepat. 2012;19:e81-8.

 

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FIGURE LEGENDS:

Fig. 1: Anti-HCV activity of D. longan crude extract. (A) HCV was mixed with

serial dilutions of D. longan crude extract (12.5 to 100 μg/ml) and inoculated to

Huh7it-1 cells at an MOI of 1. After virus adsorption, the cells were incubated with the

same concentrations of the crude extract for 48 h. Virus titers from the culture

supernatants determined. The percentage of HCV infectivity compared to the untreated

control is shown. (B) Huh7it-1 cells were treated with different concentrations of D.

longan crude extract (12.5 to 800 μg/ml) for 48 h and cell viability was evaluated by

MTT assay. The percentage of cell viability compared to the untreated control is shown.

Data represent means ± SD of data from triplicate cultures.

Fig. 2: Mode-of-action of D. longan crude extract. (A) Schematic representation of

the time-of-drug addition experiment. (B) Cells were treated with 50 μg/ml of D. longan

crude extract for 48 h during and after viral inoculation (whole treatment), for 2 h only

during viral inoculation (coaddition), or for 46 h only after viral inoculation

(post-infection). At day 1 and day 2 post-infection, extracellular virus infectivity was

determined by a focus-forming assay. (C) Cells treated with 50 μg/ml of the crude

extract described in (B) were analyzed by western blotting against HCV NS3 and

GAPDH as a loading control. (D) Cells were treated with the crude extract for 2 h only

before viral inoculation (pretreatment), for 2 h only during viral inoculation (coaddition),

or for 46 h only after viral inoculation (post-infection).  The percentage of HCV

infectivity compared to the untreated control is shown. Data represent means ± SD of

data from triplicate cultures. Control: untreated control.

Fig. 3: Effect of D. longan crude extract on virus entry and adsorption steps. (A)

Virucidal activity test. HCV suspension was mixed with D. longan crude extract (50

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μg/ml) or heparin (400 μg/ml), and incubated for 2 h at 37°C. The mixtures were

inoculated to the Huh7it-1 cells to determine the remaining virus infectivity. (B) Virus

adsorption assay. Cells were inoculated with HCV (MOI=3) in the presence or absence

of the D. longan crude extract (50 μg/ml) at 4°C for 1 h. After being washed with PBS

three times, total cellular RNAs were extracted and HCV RNA copy number was

measured by qRT-PCR analysis. -actin mRNA was used as an internal control for

normalization of the HCV RNA amounts. Data represent means ± SD of data from

triplicate cultures. Control: untreated control. ǂ, P < 0.000001; *, P < 0.05; **, P < 0.01,

compared with the control. 

Fig. 4: Effect of D. longan crude extract on the post-entry step. (A) HCV infected

cells (MOI=1) were treated with D. longan crude extract for 46 h. HCV RNA copy

number in the cells was measured by qRT-PCR analysis. -actin mRNA was used as an

internal control for normalization of the HCV RNA amounts. (B) Cells were transfected

with HCV subgenomic replicon RNA. At 4 h post-transfection, culture medium from

the transfected cells was changed to medium containing the crude extract or

cyclosporine A (CycA). Culture supernatants were collected after 48 h of drug treatment

and the luciferase activity was measured as described in the Materials and Methods

section. (C) Lysates from the infected cells described in (A) were subjected to western

blotting analysis to detect HCV NS3 and GAPDH as a loading control. The relative

amount of HCV NS3/GAPDH in each line was shown. (D) Extracellular and

intracellular virus infectivity titers from cells described in (A) were determined. Data

represent means ± SD of data from triplicate cultures. Control: untreated control. *,

P < 0.005; **, P < 0.001; ǂ, P < 0.000001; ♯, P < 0.00001, compared with the control. 

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Fig. 5: Co-treatment effect of a D. longan crude extract in combination with

cyclosporine A or telaprevir. (A) HCV-infected cells were co-treated with indicated

concentrations of D. longan crude extract with cyclosporine A or telaprevir. Virus titers

at 48 h post-infection were determined. (B) Isobologram analysis. The fixed ratios

adjusted by the EC50 (FICs) were calculated and the FICs for D. longan crude extract,

cyclosporine A or telaprevir were plotted. Data represent means ± SD of data from

duplicate cultures.

Fig. 6: Anti-DENV activity of D. longan crude extract. DENV (DS-18/09 strain) was

mixed with serial dilutions of D. longan crude extract and inoculated to Huh7it-1 cells

(MOI=1). After virus adsorption, the cells were incubated with the same concentrations

of the crude extract. Virus titers from the culture supernatants determined. The

percentage of HCV infectivity compared to the untreated control is shown. Data

represent means ± SD of data from triplicate cultures.

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Fig. 1: Anti-HCV activity of D. longan crude extract.

0

25

50

75

100

125

0 12.5 25 50 100

HC

V i

nfe

ctiv

ity

(%

of

con

tro

l)

D. longan (g/ml)

0

25

50

75

100

125

0 12.5 25 50 100 200 400 800C

ell

viab

ility

(%

of

con

tro

l)

D. longan (g/ml)

BA

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Fig. 2: Mode-of-action of D. longan crude extract.

HCV NS3

GAPDH

A

C

post-infection

pretreatment

whole treatment -2 +2 +48

HCV virus titrationB

0 (h)

coaddition

cells+virus+drug

cells+drug

cells+virus+drug

cells+virus cells+drug

cells+virus

HC

V i

nfe

ctiv

ity

(%

of

con

tro

l)

HC

V i

nfe

ctiv

ity

(%

of

con

tro

l)

0

25

50

75

100

pretreatmentcoadditionpost-infection

D. longan (50 g/ml)

D

cells+drug

0

25

50

75

100

Day1 Day2

whole treatment

coaddition

post-infection

+24

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Fig. 3: Effect of D. longan crude extract on virus entry and adsorption steps.

0

25

50

75

100

125

**

B

ǂ

A

HC

V R

NA

co

py

nu

mb

er

(% o

f co

ntr

ol)

HC

V i

nfe

ctiv

ity

(lo

g1

0 f

fu/m

l)

2

3

4

5

6

*

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Fig. 4: Effect of D. longan crude extract on the post-entry step.

GAPDH

NS3/GAPDH 10.240.72 0.68

Extracellular titer

Intracellular titer

D

2

4

6

8

10

12

HC

V in

fect

ivit

y

(×10

5 f

fu/m

l)

ControlCycA

D. longan (g/ml)

50 25

HCV NS3

C

0

0.5

1

1.5

2

0

HC

V R

NA

(a

rbit

ary

un

it)

A B

0

25

50

75

100

125HCV replicon

Rel

ativ

e lu

cife

rase

ac

tivi

ty (

%)

HCV infection

D. longan(g/ml)

25 50

25 50

D. longan (g/ml)

ǂ ♯

*

**

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Fig.

cyclo

5: Co-treat

osporine A

2

4

6

8

10

12

HC

V i

nfe

ctiv

ity  

(% o

f co

ntr

ol)

 

 

Co

mp

ou

nd

B 

A 

B 

tment effec

or telaprev

0

20

40

60

80

00

20

0 0.0

cyclos

 Additive 

Synergism 

 Antagonis

compound

ct of D. long

vir.

06 0.25 1

0 6.25 12

D. longan (

sporin A (g/

0.0

0.2

0.4

0.6

0.8

1.0

1.2

0

FIC

:D.l

on

gan

 

 

sm 

A 

28

gan crude e

4

.5 25

0

20

40

60

80

100

120

(g/ml)

ml)

0.0 0.2 0.4 0.6 0

FIC:cyclosp

cyclospor

extract in c

0 3.33

0.8 1.0 1.2

porin A

rin A

telapre

0.0

0.2

0.4

0.6

0.8

1.0

1.2

combinatio

10 33.3 1

evir (nM)

FIC:telap

0.0 0.2 0.4 0.6

telapre

on with

00

previr 

0.8 1.0 1.2

evir 

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Fig. 6: Anti-DENV activity of D. longan crude extract.

 

DE

NV

in

fect

ivit

y  

(% o

f co

ntr

ol)

 

D. longan (g/ml)

0

25

50

75

100

125

0 25 50 100

Day1

Day2

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