Antiviral activity of extracts from Morinda citrifolia leaves and chlorophyll catabolites,...

ORIGINAL ARTICLE

Antiviral activity of extracts from Morinda citrifolia leavesand chlorophyll catabolites, pheophorbide a andpyropheophorbide a, against hepatitis C virusSuratno Lulut Ratnoglik1, Chie Aoki1,2, Pratiwi Sudarmono4, Mari Komoto1, Lin Deng1, Ikuo Shoji1,Hiroyuki Fuchino, Nobuo Kawahara and Hak Hotta1

1Division of Microbiology, Kobe University Graduate School of Medicine, 7-5-1 Kusunoki-cho, Chuo-ku, Kobe 650 0017, Japan,2JST/JICA within the Science and Technology Research Partnership for Sustainable Development Laboratory, Tokyo, Japan, 3Faculty ofMedicine, University of Indonesia Jl., Salemba 4, Jakarta 10430, Indonesia and 4Research Center for Medicinal Plant Resources, NationalInstitute of Biomedical Innovation, 1-2 Hachimandai, Tsukuba Ibaraki 305 0843, Japan

ABSTRACTThe development of complementary and/or alternative drugs for treatment of hepatitis C virus (HCV)infection is still needed. Antiviral compounds in medicinal plants are potentially good targets tostudy. Morinda citrifolia is a common plant distributed widely in Indo-Pacific region; its fruits andleaves are food sources and are also used as a treatment in traditional medicine. In this study, using aHCV cell culture system, it was demonstrated that a methanol extract, its n-hexane, and ethyl acetatefractions from M. citrifolia leaves possess anti-HCV activities with 50%-inhibitory concentrations(IC50) of 20.6, 6.1, and 6.6mg/mL, respectively. Bioactivity-guided purification and structural analysisled to isolation and identification of pheophorbide a, the major catabolite of chlorophyll a, as an anti-HCV compound present in the extracts (IC50 ¼ 0.3mg/mL). It was also found that pyropheophorbidea possesses anti-HCV activity (IC50¼ 0.2mg/mL). The 50%-cytotoxic concentrations (CC50) ofpheophorbide a and pyropheophorbide a were 10.0 and 7.2mg/mL, respectively, their selectivityindexes being 33 and 36, respectively. On the other hand, chlorophyll a, sodium copper chlorophyllin,and pheophytin a barely, or onlymarginally, exhibited anti-HCV activities. Time-of-addition analysisrevealed that pheophorbide a and pyropheophorbide a act at both entry and the post-entry steps. Thepresent results suggest that pheophorbide a and its related compounds would be good candidates forseed compounds for developing antivirals against HCV.

Key words antiviral, hepatitis C virus, pheophorbide a, pyropheophorbide a.

Hepatitis C virus (HCV) belongs to the Hepacivirusgenus within the Flaviviridae family. The viral genome, asingle-stranded, positive-sense RNA of 9.6 kb, encodes apolyprotein precursor consisting of about 3000 aminoacid residues (1). The polyprotein is cleaved by the hostand viral proteases to generate 10 mature proteins,namely core, envelope (E) 1, E2, a putative ion channelp7, and nonstructural proteins NS2, NS3, NS4A, NS4B,

NS5A and NS5B. Core, E1, and E2 are the structuralproteins and form the infectious virus particle togetherwith the viral genome. The nonstructural proteins playessential roles in viral RNA replication. Based on aconsiderable degree of sequence heterogeneity of itsgenome, HCV is currently classified into seven geno-types (1–7) and more than 70 subtypes (1a, 1b, 2a, 2b,etc.) (2).

CorrespondenceHak Hotta, Division of Microbiology, Kobe University Graduate School of Medicine, 7-5-1 Kusunoki-cho, Chuo-ku, Kobe 650 0017, Japan.Tel:þ81 78 382 5500; fax: þ81 78 382 5519; e-mail: [email protected]

Received 8 January 2014; revised 11 January 2014; accepted 16 January 2014.

List of Abbreviations: CC50, 50%-cytotoxic concentrations; dpi, days post-infection; FR., fraction; GAPDH, glyceraldehyde-3-phosphatedehydrogenase; HCV, hepatitis C virus; IC50, 50%-inhibitory concentration; ID, internal diameter; NMR, nuclear magnetic resonance; ODS, octadecylsilane; SI, selectivity index; TLC, thin layer chromatography.

Microbiol Immunol 2014; 58: 188–194doi: 10.1111/1348-0421.12133

188 © 2014 The Societies and Wiley Publishing Asia Pty Ltd

Hepatitis C virus is a major cause of chronic liverdiseases, such as hepatitis, cirrhosis, and hepatocellularcarcinoma, with substantial morbidity andmortality (3, 4).The prevalence of HCV is about 2%, representing 120million people worldwide. Current standard treatmentusing pegylated interferon and ribavirin is effective in onlyhalf the patients infected with HCV genotype 1, which isthemost resistant of allHCVgenotypes to interferon-basedtherapy. Therefore, development of complementary and/or alternative drugs for treatment of HCV infection is stillneeded from both clinical and economic points of view. Inthis regard, antiviral substances obtained from medicinalplants are potentially good targets to study (5–7), as has alsobeen reported for other viruses (8).Morinda citrifolia belongs to the Rubiaceae family and

is thought to have originated in Indonesia. This commonplant is distributedwidely in the Indo-Pacific region. Thefruits and leaves ofM. citrifolia are food sources for localpeople and are also used as a treatment for infections andinflammatory diseases (9) in traditional medicine.Nowadays, the juice from the ripe fruits, traditionallyknown as “noni,” is sold as a health food even inindustrialized countries. It has been reported thatmethanol or ethanol extracts of M. citrifolia fruits and/or leaves have antibacterial activities against somebacteria, such as Staphylococcus aureus (10) andMycobacterium tuberculosis (11). Using an HCV sub-genomic replicon, anti-HCV activity has also beenreported for both methanol and ethanol extracts ofM. citrifolia fruits (12). In this study, we used an HCVinfection system in cultured cells to explore the anti-HCV activities of methanol extracts of the fruits (ripeand frozen), leaves, roots, and branches of M. citrifolia.We report here that a methanol extract of M. citrifolialeaves and its subfractions, as well as an isolatedcompound, pheophorbide a, and its catabolite, pyro-pheophorbide a, possess antiviral activities against HCV.

MATERIALS AND METHODS

Cells and viruses

Huh7.5 cells and the plasmid pFL-J6/JFH1 to producethe J6/JFH1 strain of HCV genotype 2a (13) were kindlyprovided by Dr. C.M. Rice (Rockefeller University, NewYork, NY, USA). The J6/JFH1-P47 strain of HCV wasprepared as described previously (14). Huh7.5 cellswere cultured in Dulbecco’s modified Eagle’s medium(Wako, Osaka, Japan) supplemented with FBS (Biowest,Nuaillé, France), non-essential amino acids (Invitrogen,Carlsbad, CA, USA), penicillin (100 IU/mL), andstreptomycin (100mg/mL) (Invitrogen) at 37 °C in a5% CO2 incubator.

Extraction of various parts of M. citrifoliaand further fractionation and purificationof the samples

M. citrifolia fruits (ripe and frozen), leaves, roots, andbranches were collected in Okinawa Prefecture, Japan.Methanol extracts of each of these components ofM. citrifolia were prepared and subjected to purificationprocedures, as described previously (15–18). In brief, theplant components were dried at room temperature,pulverized according to their characteristics, and thenextracted with methanol at 50 °C for 6 hr. The extractswere then filtered and the filtrates concentrated by usingan evaporator at temperatures not exceeding 40 °C. Theresidues thus obtained were resuspended in water andsuccessively partitioned between n-hexane, ethyl acetate,and 1-butanol. Next, the n-hexane extractswere subjectedto recycling preparative HPLC (solvent system, 100%methanol; column, GS-320; ID, 21.5mm� 500mm; flowrate, 5.0mL/min; detection, UV 210 nm) to yield fivefractions (Fr. 1–5). Fr. 5 was subjected to HPLCseparation (solvent system, 100% methanol; column,TSK-gel GOLIGOPW [Tosoh Bioscience Diagnostics,Tessenderlo, Belgium]; ID, 4.6mm � 250mm; flow rate,1.0mL/min; detection, UV 210nm) to yield threefractions (Fr. 5-1 to 5-3). Fr. 5-1 was rechromatographedby ODS column chromatography (Varian Mega Bond-Elut C18; Agilent Technologies Japan, Tokyo, Japan) with100%methanol as an eluent to yield three fractions (Fr. 5-1-1 to 5-1-3). Fr. 5-1-3 was subjected to HPLC (solventsystem, methanol–acetone [9:1]; column, CosmosilCholester [Nacalai Tesque, Kyoto, Japan]; ID, 4.6mm� 450mm;flowrate, 2mL/min; detection,UV400 nm) toobtain two fractions (Fr. 5-1-3-1 and 5-1-3-2).

The 1H-NMR spectra weremeasuredwith a JEOLECA500 spectrometer (500MHz; Tokyo, Japan). HPLC wasperformed on a JASCO LC-2000 plus system (JASCO,Tokyo, Japan). A Merck TLC plate (Art. 5715; Merck,Darmstadt, Germany) was used for TLC comparisons.

Chemicals

Chlorophyll a (from spinach) and sodium copperchlorophyllin were purchased from Sigma–Aldrich (StLouis, MO, USA) and pheophytin a from Wako PureChemical Industries (Osaka, Japan). Pheophorbide aand pyropheophorbide a were purchased from FrontierScientific (Logan, UT, USA).

Analysis of anti-HCV activities of plantextracts and purified compounds

For anti-HCV activity assay, test samples were weighedand dissolved in DMSO to obtain stock solutions, whichwere stored at –20 °C until used. Huh7.5 cells were

Anti-HCV activity of pheophorbide a

© 2014 The Societies and Wiley Publishing Asia Pty Ltd 189

seeded in 24-well plates (1.9� 105 cells/well). A fixedamount of HCV was mixed with serial dilutions of theplant extracts (100, 30, 10, 3, and 1mg/mL) andinoculated into the cells. After 2 hr, the cells werewashed with medium to remove the residual virus andfurther incubated in medium containing the sameconcentrations of the plant extracts as those used duringvirus inoculation. In order to assess themode of action ofthe samples examined, in some experiments treatmentwith the plant extracts was performed only during virusinoculation or only after virus inoculation until virusharvest. Culture supernatants were obtained at 1 and2 days post-infection and titrated for virus infectivity, asdescribed previously (19). Virus and cells treated withmedium containing 0.1% DMSO served as controls.Percent inhibition of virus infectivity by the samples wascalculated by comparing with the controls; IC50 weredetermined.

Time-of-addition experiments

To determine whether anti-HCV activities of the testsamples occurred at the entry or the post-entry step,time-of-addition experiments were performed as de-scribed previously (6, 7). In brief:1 HCV was mixed with each of the compounds and

the mixture was inoculated into the cells. After virusadsorption for 2 hr, the residual virus and test samplewere removed and the cells refed with fresh mediumwithout the test sample for 46 hr. This experimentexamines antiviral effect during the entry step.2 HCV was inoculated into the cells in the absence of

test samples. After virus adsorption for 2 hr, the residualvirus was removed and the cells refed with fresh mediumcontaining the test samples for 46 hr. This experimentexamines the antiviral effect during the post-entry step.3 As a positive control, HCV mixed with the test

sample was inoculated into the cells. After virusadsorption for 2 hr, the residual virus and test samplewere removed and the cells refed with fresh mediumcontaining the test samples for 46 hr.

WST-1 assay for cytotoxicity

WST-1 assay was performed as described previously witha slightmodification (19). In brief, Huh7.5 cells in 96-wellplates were treated with serial dilutions of the plantextracts or 0.1% DMSO as a control for 48 hr. After thistreatment, 10mL of WST-1 reagent (Roche, Mannheim,Germany) was added to each well and the cells culturedfor 4 hr. The WST-1 reagent is absorbed by the cells andconverted to formazanbymitochondrial dehydrogenases.The amount of formazan, which correlates with thenumber of living cells, was determined by measuring the

absorbance of each well using a microplate reader at 450and 630 nm. Percent cell viability compared to the controlwas calculated for each dilution of the plant extracts andCC50 were determined.

Immunoblotting

Immunoblotting analysis was performed as describedpreviously (6, 7, 20). In brief, cells lysed with an SDSsample buffer were subjected to SDS-PAGE andtransferred onto polyvinylidene difluoride membranes(Millipore, Bedford, MA, USA). The membranes wereincubated with the respective primary antibody, such asmouse monoclonal antibodies against HCV NS3 andGAPDH (Millipore). Horseradish peroxidase-conjugat-ed goat anti-mouse immunoglobulin (Invitrogen) wasused to visualize the respective proteins by means of anenhanced chemiluminescence detection system (GEHealthcare, Buckinghamshire, UK).

Real-time quantitative RT-PCR

Real-time quantitative RT-PCR was performed asdescribed previously (6, 7, 20). In brief, 1mg of totalRNA extracted from the cells using a ReliaPrep RNA cellminiprep system (Promega, Madison, WI, USA) wasreverse transcribed using a GoScript Reverse Transcrip-tion system (Promega) with random primers. Theresultant cDNA was subjected to real-time quantitativePCR analysis using SYBR Premix Ex Taq (TaKaRa,Kyoto, Japan) in a MicroAmp 96-well reaction plate andan ABI PRISM 7500 system (Applied Biosystems, FosterCity, CA, USA). The HCV NS5A-specific primers usedwere 50-AGACGTATTGAGGTCCATGC-30 (sense)and 50-CCGCAGCGACGGTGCTGATAG-30 (antisense).Human GAPDH gene expression measured by usingprimers 50-GCCATCAATGACCCCTTCATT-30 (sense)and 50-TCTCGCTCCTGGAAGATGG-30 (antisense)served as internal controls.

Statistical analysis

Results are expressed as mean� SEM. Statistical signifi-cance was evaluated by Student’s t-test. P< 0.05 wasconsidered statistically significant.

RESULTS

Anti-HCV activities of methanol extracts ofM. citrifolia fruits, leaves, roots, andbranches

Methanol extracts of the fruits (ripe and frozen), leaves,roots, and branches of M. citrifolia were examined forantiviral activities against the HCV J6/JFH1-P47 strain.


S.L. Ratnoglik et al.

It was found that amethanol extract ofM. citrifolia leavesat a concentration of 30mg/mL inhibits HCV infectionby 98.3%, whereas extracts of ripe fruits, frozen fruits,roots and branches at the same concentration inhibitHCV infection by 23.4%, 34.0%, 59.6%, and 27.7%,respectively. The following analyses, therefore, focus onthe extract from M. citrifolia leaves.

Anti-HCV activities of further purifiedsamples of M. citrifolia leaves

The methanol extract of M. citrifolia leaves wasfurther partitioned with different solvents, comprisingn-hexane, ethyl acetate, 1-butanol and water, and theirIC50, CC50, and SIs (SI: CC50/IC50) determined. The IC50

values of the partitions with n-hexane and ethyl acetatewere 6.1 and 6.6mg/mL, respectively; both of theseshowed stronger anti-HCV activities than did themethanol extract, which had an IC50 of 20.6mg/mL(Table 1). The n-hexane-partitioned sample was frac-tionated into a further five fractions by a recyclingpreparative HPLC method; of these fractions, only Fr. 5showed anti-HCV activity, the IC50 being 7.8mg/mL(Table 2). Fr. 5 was therefore further purified by HPLC,ODS column chromatography and another HPLC andFr. 5-1-3-2 identified as the most potent and purifiedfraction, having an IC50 of 4.6mg/mL.On TLC analysis (detection: UV irradiation 366 nm)

of Fr. 5-1-3-2, a red-fluorescent spot was detected,suggesting that Fr. 5-1-3-2 consists almost solely ofchlorophyll a and its degraded products. The presence ofpheophorbide a, which is reportedly the major cataboliteof chlorophyll a (21, 22), was confirmed by directcomparison with a standard sample by TLC analysis.Structural analyses using HPLC and NMR identified thepurified compound as pheophorbide a (23).

Anti-HCV activities of pheophorbide a andpyropheophorbide a

Pheophorbide a is a breakdown product of chlorophyll.In its breakdown process, chlorophyll loses theMg2þ ion

through demetallation to generate pheophytin a insenescent leaves (21, 22). Pheophytin a is catabolized topheophorbide a through dephytylation. In fruits,dephytylation of chlorophyll takes place first to generatechlorophyllide, which is then catabolized to pheophor-bide a through demetallation. Pheophorbide a is furthercatabolized to generate pyropheophorbide a. On theother hand, sodium copper chlorophyllin is a semi-synthetic compound in which the Cu2þ ion replaces theMg2þ ion.

Anti-HCV activities of commercially available, re-agent-grade chlorophyll a and its-related compoundswere examined. It was found that chlorophyll a barely,and sodium copper chlorophyllin and pheophytin a onlyweakly, exhibit anti-HCV activities (IC50¼ 220, 32.0,and 54.5mg/mL, respectively). On the other hand,pheophorbide a and pyropheophorbide a showed potentanti-HCV activities with IC50 of 0.3 and 0.2mg/mL,respectively (Table 3).

Table 1. Anti-HCV activity (IC50), cytotoxicity (CC50) and selectivity

index (SI) of a methanol extract and solvent partitions obtained from

of M. citrifolia leaves

Sample IC50 (mg/mL) CC50 (mg/mL) SI

Methanol extract 20.6 >30‡ >1.5n-Hexane partition 6.1 >30‡ >4.9Ethyl acetate partition 6.6 >30‡ >4.5n-Butanol partition 20.8 >30‡ >1.5Water partition >30† >30‡ na

†no detectable HCV inhibition at 30mg/mL. ‡no detectable cytotoxicityat 30mg/mL; na, not applicable.

Table 2. Anti-HCV activity (IC50), cytotoxicity (CC50), and selectivity

index (SI) of fractions from M. citrifolia leaves obtained by recycling

preparative HPLC and ODS column chromatography


Fr. 1 >30 >30‡ naFr. 2 >30 >30‡ naFr. 3 >30† >30‡ naFr. 4 >30† >30‡ naFr. 5 7.8 >30‡ >3.8ODS column chromatography

Fr. 5-1-1 8.6 >30‡ >3.5Fr. 5-1-2 5.2 >30‡ >5.8Fr. 5-1-3 4.6 >30‡ >6.5

HPLCFr. 5-1-3-1 >30 >30 naFr. 5-1-3-2 4.6 >30 >6.5

†no detectable HCV inhibition at 30mg/mL. ‡no detectable cytotoxicityat 30mg/mL; na, not applicable.

Table 3. Anti-HCV activity (IC50), cytotoxicity (CC50), and selectivity

index (SI) of commercially available chlorophyll a, sodium copper

chlorophyllin, pheophytin a, pheophorbide a, and pyropheophorbide a


Chlorophyll a 220 >300 naSodium copper chlorophyllin 32.0 158 4.9Pheophytin a 54.5 328 6.0Pheophorbide a 0.3 10.0 33Pyropheoborbide a 0.2 7.2 36

na, not applicable.



Mode-of-action of pheophorbide a andpyropheophorbide a

To determine whether the anti-HCV effects of pheo-phorbide a and pyropheophorbide a are exerted duringthe entry or post-entry step, time-of-addition experi-ments were performed. It was found that, when added tothe culture only during virus adsorption followed byvirus entry, pheophorbide a (1.0mg/mL) and pyropheo-phorbide a (0.5mg/mL) inhibit HCV infection by 64.0%and 53.0%, respectively (Table 4). On the other hand,when added to the culture only after virus inoculation,they inhibited HCV replication by 95.3% and 98.1%,respectively. These results suggest that pheophorbide aand pyropheophorbide a act during both the entry andpost-entry steps.

Inhibition of HCV RNA replication and HCVprotein synthesis by pheophorbide a andpyropheophorbide a

To further confirm that pheophorbide a and pyropheo-phorbide a exert their anti-HCV activities not onlyduring the virus entry step but also during the post-entrystep (after the virus has entered the cells), Huh7.5 cellswere inoculated with HCV for 2 hr in the absence of thetest samples, and then treated with either one of thecompounds for 1–2 days. Real-time quantitative RT-PCR and immunoblotting analyses demonstrated thatboth pheophorbide a and pyropheophorbide a inhibitHCV RNA replication in a dose-dependent manner(Fig. 1a) and, consequently, inhibit HCV proteinsynthesis in the cells (Fig. 1b).

DISCUSSION

It has been reported that methanol and ethanol extractsofM. citrifolia fruits show anti-HCV activities in a HCVsubgenomic replicon system (12). In the present study,we found that a methanol extract of M. citrifolia leavesinhibits HCV replication in an HCV cell culture systemmore efficiently than doM. citrifolia fruits extracts (98%vs. ca. 30% inhibition at 30mg/mL), the IC50 of the

M. citrifolia leaves extract being 20.6mg/mL (Table 1).Subsequent bioactivity-guided purification and struc-tural analysis demonstrated that pheophorbide a, knownto be the major catabolite of chlorophyll a (21, 22), and

Table 4. Time-of-addition analysis of pheophorbide a and

pyropheophorbide a against HCV

Anti-HCV activity†

Compound (mg/mL)During virusinoculation

After virusinoculation

During & aftervirus inoculation

Pheophorbide a (1.0) 64.0 95.3 98.7Pyropheophorbide a (0.5) 53.0 98.1 99.7

†% inhibition when tested by time-of-addition analysis.

Fig. 1. Effects of pheophorbide a and pyropheophorbide a onHCV RNA replication and protein synthesis. (a) Huh 7.5 cells wereinoculated with HCV J6/JFH1 at an MOI of 2.0. After 2 hr, the cellswere washed with medium to remove residual virus and treated witheither pheophorbide a or pyropheophorbide a (both at 0.5, 1.0, 2.0,and 4mg/mL) or left untreated, and then subjected to real-timequantitative RT-PCR analysis 1 and 2 dpi. The HCV RNA amounts inthe cells are normalized to degree of GAPDH mRNA expression. Datarepresent means� SEMs of data from two independent experiments.The value for the untreated control at 1 dpi is arbitrarily expressed as1.0. *, P< 0.0001. (b) Amounts of the HCV NS3 protein in thepyropheophorbide a-treated cells described in (a) were measured bywestern blot analysis using monoclonal antibody against the NS3protein. GAPDH served as an internal control to verify equal amountsof sample loading.



its related catabolite, pyropheophorbide a, have potentanti-HCV activities (Table 3). A time-of-addition studysuggested that pheophorbide a and pyropheophorbide aact during both the entry and post-entry steps (Table 4).It should be noted that, although Fr. 5-1-3-2 purifiedfrom the M. citrifolia leaves extract consists almostentirely of pheophorbide a, its anti-HCV activity is muchweaker than that of reagent-grade pheophorbide a(Tables 2, 3). One possible explanation for this apparentdiscrepancy is that a copurified small molecule(s) in thefraction interfered with the anti-HCV activity ofpheophorbide a. Further studies are needed to clarifythis issue.Pheophorbide a reportedly inhibits influenza A virus

infection (24). Pheophorbide a and pyropheophorbide aalso reportedly show antiviral activities against herpessimplex virus type 2 and influenza A virus, but notpoliovirus (25). Given that herpes simplex virus type 2and influenza A virus are envelope viruses whereaspoliovirus is a non-envelope virus, Bouslama et al.speculate that pheophorbide a and pyropheophorbide ainhibit envelope viruses, targeting specific envelopeproteins and thereby interfering with viral binding to thehost cell receptors (25). On the other hand, our presentresults suggest that pheophorbide a and pyropheophor-bide a inhibit HCV infection not only during the viralbinding/entry step but also during the post-entry step(Table 4, Fig. 1). The post-entry step can be furtherdivided into the following stages of the HCV lifecycle: (i)uncoating of the viral particles and capsid; (ii) synthesisand processing of the viral proteins and replication of theviral genome; and (iii) assembly, intracellular transport,and release of the viral particles (1, 26, 27). Now that wehave shown that pheophorbide a and pyropheophorbidea inhibit HCV infection during the post-entry step,it is important to elucidate the specific molecularmechanism(s) in the viral lifecycle targeted by thosecompounds.Pheophorbide a and pyropheophorbide a are known to

induce photosensitivity: the resultant photo-activatedcharacteristics play important anti-tumor roles inphotodynamic therapy using pheophorbide a and itsderivatives (28–30). Pheophorbide a also reportedlyinduces apoptosis of cancer cells and potentiatesimmunostimulating functions of macrophages (31, 32).However, photosensitivity can cause serious adverseeffects when these agents are used to treat cancer and viralinfections. Importantly, the photosensitizing effect can beseparated from anti-tumor effect (28). It is thereforetempting to speculate that a new derivative(s) with morepotent anti-HCV activities and less capacity to inducephotosensitivity could be synthesized from the seedcompounds of pheophorbide a and pyropheophorbide a.

It has previously been reported that pheophytin ashows anti-HCV activities with IC50 of 4.97mM(equivalent to 4.3mg/mL) (33). Also chlorophyllin, asemi-synthetic derivative of chlorophyll, reportedly hasantiviral activities against poliovirus and bovine herpes-virus, with IC50 of 19.8 and 8.6mg/mL, respectively (34).However, in our study, compared with the more potentanti-HCV activities of pheophorbide a and pyropheo-phorbide a, pheophytin a and sodium copper chlor-ophyllin exhibited only marginal anti-HCV activity, theIC50 being 54.5 and 32.0mg/mL, respectively (Table 3).

In this study, we demonstrated anti-HCV activities ofpheophorbide a and pyropheophorbide a using the J6/JFH1 strain of HCV genotype 2a (13, 14). Whether thesecompounds inhibit replication of other HCV strains ofdifferent genotypes is an important question to answer.Currently, some other HCV genotypes, such asgenotypes 1a, 1b and 3a to 7a, are available for drugscreening tests (2, 35). Such in vitro cell culture systemswould help in determining the possible anti-HCVactivities of pheophorbide a and pyropheophorbide aagainst different genotypes of HCV.

In conclusion, we have demonstrated that a methanolextract of M. citrifolia leaves and certain chlorophyll-derived compounds, such as pheophorbide a andpyropheophorbide a, possess anti-HCV activities. Thesecompounds would be good candidates for seed com-pounds for developing novel antivirals against HCV.

ACKNOWLEDGMENTS

The authors are grateful to Dr. C.M. Rice (RockefellerUniversity, New York, NY, USA) for providing Huh-7.5cells and pFL-J6/JFH1. This study was supported in partby Science and Technology Research Partnerships forSustainable Development from JST and JICA. It was alsoperformed as part of Japan Initiative for Global ResearchNetwork on Infectious Diseases (J-GRID), Ministry ofEducation, Culture, Sports, Science and Technology,Japan.

DISCLOSURE

The authors have no conflicts of interest to declare.

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Antiviral activity of extracts from Morinda citrifolia leaves and chlorophyll catabolites,...

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