The 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 to study. Morinda citrifolia is a common plant distributed widely in Indo-Pacific region; its fruits and leaves are food sources and are also used as a treatment in traditional medicine. In this study, using a HCV cell culture system, it was demonstrated that a methanol extract, its n-hexane, and ethyl acetate fractions from M. citrifolia leaves possess anti-HCV activities with 50%-inhibitory concentrations (IC50) of 20.6, 6.1, and 6.6 μg/mL, respectively. Bioactivity-guided purification and structural analysis led to isolation and identification of pheophorbide a, the major catabolite of chlorophyll a, as an anti-HCV compound present in the extracts (IC50 = 0.3 μg/mL). It was also found that pyropheophorbide a possesses anti-HCV activity (IC50 = 0.2 μg/mL). The 50%-cytotoxic concentrations (CC50) of pheophorbide a and pyropheophorbide a were 10.0 and 7.2 μg/mL, respectively, their selectivity indexes being 33 and 36, respectively. On the other hand, chlorophyll a, sodium copper chlorophyllin, and pheophytin a barely, or only marginally, exhibited anti-HCV activities. Time-of-addition analysis revealed that pheophorbide a and pyropheophorbide a act at both entry and the post-entry steps. The present results suggest that pheophorbide a and its related compounds would be good candidates for seed compounds for developing antivirals against HCV.
hepatitis C virus
nuclear magnetic resonance
thin layer chromatography
Hepatitis C virus (HCV) belongs to the Hepacivirus genus within the Flaviviridae family. The viral genome, a single-stranded, positive-sense RNA of 9.6 kb, encodes a polyprotein precursor consisting of about 3000 amino acid residues . The polyprotein is cleaved by the host and viral proteases to generate 10 mature proteins, namely core, envelope (E) 1, E2, a putative ion channel p7, and nonstructural proteins NS2, NS3, NS4A, NS4B, NS5A and NS5B. Core, E1, and E2 are the structural proteins and form the infectious virus particle together with the viral genome. The nonstructural proteins play essential roles in viral RNA replication. Based on a considerable degree of sequence heterogeneity of its genome, HCV is currently classified into seven genotypes [1-7] and more than 70 subtypes (1a, 1b, 2a, 2b, etc.) .
Hepatitis C virus is a major cause of chronic liver diseases, such as hepatitis, cirrhosis, and hepatocellular carcinoma, with substantial morbidity and mortality [3, 4]. The prevalence of HCV is about 2%, representing 120 million people worldwide. Current standard treatment using pegylated interferon and ribavirin is effective in only half the patients infected with HCV genotype 1, which is the most resistant of all HCV genotypes to interferon-based therapy. Therefore, development of complementary and/or alternative drugs for treatment of HCV infection is still needed from both clinical and economic points of view. In this regard, antiviral substances obtained from medicinal plants are potentially good targets to study [5-7], as has also been reported for other viruses .
Morinda citrifolia belongs to the Rubiaceae family and is thought to have originated in Indonesia. This common plant is distributed widely in the Indo-Pacific region. The fruits and leaves of M. citrifolia are food sources for local people and are also used as a treatment for infections and inflammatory diseases  in traditional medicine. Nowadays, the juice from the ripe fruits, traditionally known as “noni,” is sold as a health food even in industrialized countries. It has been reported that methanol or ethanol extracts of M. citrifolia fruits and/or leaves have antibacterial activities against some bacteria, such as Staphylococcus aureus  and Mycobacterium tuberculosis . Using an HCV subgenomic replicon, anti-HCV activity has also been reported for both methanol and ethanol extracts of M. citrifolia fruits . In this study, we used an HCV infection system in cultured cells to explore the anti-HCV activities of methanol extracts of the fruits (ripe and frozen), leaves, roots, and branches of M. citrifolia. We report here that a methanol extract of M. citrifolia leaves and its subfractions, as well as an isolated compound, pheophorbide a, and its catabolite, pyropheophorbide a, possess antiviral activities against HCV.
MATERIALS AND METHODS
Cells and viruses
Huh7.5 cells and the plasmid pFL-J6/JFH1 to produce the J6/JFH1 strain of HCV genotype 2a  were kindly provided by Dr. C.M. Rice (Rockefeller University, New York, NY, USA). The J6/JFH1-P47 strain of HCV was prepared as described previously . Huh7.5 cells were 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), and streptomycin (100 μg/mL) (Invitrogen) at 37 °C in a 5% CO2 incubator.
Extraction of various parts of M. citrifolia and further fractionation and purification of the samples
M. citrifolia fruits (ripe and frozen), leaves, roots, and branches were collected in Okinawa Prefecture, Japan. Methanol extracts of each of these components of M. citrifolia were prepared and subjected to purification procedures, as described previously [15-18]. In brief, the plant components were dried at room temperature, pulverized according to their characteristics, and then extracted with methanol at 50 °C for 6 hr. The extracts were then filtered and the filtrates concentrated by using an evaporator at temperatures not exceeding 40 °C. The residues thus obtained were resuspended in water and successively partitioned between n-hexane, ethyl acetate, and 1-butanol. Next, the n-hexane extracts were subjected to recycling preparative HPLC (solvent system, 100% methanol; column, GS-320; ID, 21.5 mm × 500 mm; flow rate, 5.0 mL/min; detection, UV 210 nm) to yield five fractions (Fr. 1–5). Fr. 5 was subjected to HPLC separation (solvent system, 100% methanol; column, TSK-gel GOLIGOPW [Tosoh Bioscience Diagnostics, Tessenderlo, Belgium]; ID, 4.6 mm × 250 mm; flow rate, 1.0 mL/min; detection, UV 210 nm) to yield three fractions (Fr. 5-1 to 5-3). Fr. 5-1 was rechromatographed by ODS column chromatography (Varian Mega Bond-Elut C18; Agilent Technologies Japan, Tokyo, Japan) with 100% methanol as an eluent to yield three fractions (Fr. 5-1-1 to 5-1-3). Fr. 5-1-3 was subjected to HPLC (solvent system, methanol–acetone [9:1]; column, Cosmosil Cholester [Nacalai Tesque, Kyoto, Japan]; ID, 4.6 mm × 450 mm; flow rate, 2 mL/min; detection, UV 400 nm) to obtain two fractions (Fr. 5-1-3-1 and 5-1-3-2).
The 1H-NMR spectra were measured with a JEOL ECA 500 spectrometer (500 MHz; Tokyo, Japan). HPLC was performed on a JASCO LC-2000 plus system (JASCO, Tokyo, Japan). A Merck TLC plate (Art. 5715; Merck, Darmstadt, Germany) was used for TLC comparisons.
Chlorophyll a (from spinach) and sodium copper chlorophyllin were purchased from Sigma–Aldrich (St Louis, MO, USA) and pheophytin a from Wako Pure Chemical Industries (Osaka, Japan). Pheophorbide a and pyropheophorbide a were purchased from Frontier Scientific (Logan, UT, USA).
Analysis of anti-HCV activities of plant extracts and purified compounds
For anti-HCV activity assay, test samples were weighed and dissolved in DMSO to obtain stock solutions, which were stored at –20 °C until used. Huh7.5 cells were seeded in 24-well plates (1.9 × 105 cells/well). A fixed amount of HCV was mixed with serial dilutions of the plant extracts (100, 30, 10, 3, and 1 μg/mL) and inoculated into the cells. After 2 hr, the cells were washed with medium to remove the residual virus and further incubated in medium containing the same concentrations of the plant extracts as those used during virus inoculation. In order to assess the mode of action of the samples examined, in some experiments treatment with the plant extracts was performed only during virus inoculation or only after virus inoculation until virus harvest. Culture supernatants were obtained at 1 and 2 days post-infection and titrated for virus infectivity, as described previously . Virus and cells treated with medium containing 0.1% DMSO served as controls. Percent inhibition of virus infectivity by the samples was calculated by comparing with the controls; IC50 were determined.
To determine whether anti-HCV activities of the test samples occurred at the entry or the post-entry step, time-of-addition experiments were performed as described previously [6, 7]. In brief:
1 HCV was mixed with each of the compounds and the mixture was inoculated into the cells. After virus adsorption for 2 hr, the residual virus and test sample were removed and the cells refed with fresh medium without the test sample for 46 hr. This experiment examines 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 residual virus was removed and the cells refed with fresh medium containing the test samples for 46 hr. This experiment examines 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 virus adsorption for 2 hr, the residual virus and test sample were removed and the cells refed with fresh medium containing the test samples for 46 hr.
WST-1 assay for cytotoxicity
WST-1 assay was performed as described previously with a slight modification . In brief, Huh7.5 cells in 96-well plates were treated with serial dilutions of the plant extracts or 0.1% DMSO as a control for 48 hr. After this treatment, 10 μL of WST-1 reagent (Roche, Mannheim, Germany) was added to each well and the cells cultured for 4 hr. The WST-1 reagent is absorbed by the cells and converted to formazan by mitochondrial dehydrogenases. The amount of formazan, which correlates with the number of living cells, was determined by measuring the absorbance of each well using a microplate reader at 450 and 630 nm. Percent cell viability compared to the control was calculated for each dilution of the plant extracts and CC50 were determined.
Immunoblotting analysis was performed as described previously [6, 7, 20]. In brief, cells lysed with an SDS sample buffer were subjected to SDS-PAGE and transferred onto polyvinylidene difluoride membranes (Millipore, Bedford, MA, USA). The membranes were incubated with the respective primary antibody, such as mouse monoclonal antibodies against HCV NS3 and GAPDH (Millipore). Horseradish peroxidase-conjugated goat anti-mouse immunoglobulin (Invitrogen) was used to visualize the respective proteins by means of an enhanced chemiluminescence detection system (GE Healthcare, Buckinghamshire, UK).
Real-time quantitative RT-PCR
Real-time quantitative RT-PCR was performed as described previously [6, 7, 20]. In brief, 1 µg of total RNA extracted from the cells using a ReliaPrep RNA cell miniprep system (Promega, Madison, WI, USA) was reverse transcribed using a GoScript Reverse Transcription system (Promega) with random primers. The resultant cDNA was subjected to real-time quantitative PCR analysis using SYBR Premix Ex Taq (TaKaRa, Kyoto, Japan) in a MicroAmp 96-well reaction plate and an ABI PRISM 7500 system (Applied Biosystems, Foster City, CA, USA). The HCV NS5A-specific primers used were 5′-AGACGTATTGAGGTCCATGC-3′ (sense) and 5′-CCGCAGCGACGGTGCTGATAG-3′ (antisense). Human GAPDH gene expression measured by using primers 5′-GCCATCAATGACCCCTTCATT-3′ (sense) and 5′-TCTCGCTCCTGGAAGATGG-3′ (antisense) served as internal controls.
Results are expressed as mean ± SEM. Statistical significance was evaluated by Student's t-test. P < 0.05 was considered statistically significant.
Anti-HCV activities of methanol extracts of M. citrifolia fruits, leaves, roots, and branches
Methanol extracts of the fruits (ripe and frozen), leaves, roots, and branches of M. citrifolia were examined for antiviral activities against the HCV J6/JFH1-P47 strain. It was found that a methanol extract of M. citrifolia leaves at a concentration of 30 μg/mL inhibits HCV infection by 98.3%, whereas extracts of ripe fruits, frozen fruits, roots and branches at the same concentration inhibit HCV infection by 23.4%, 34.0%, 59.6%, and 27.7%, respectively. The following analyses, therefore, focus on the extract from M. citrifolia leaves.
Anti-HCV activities of further purified samples of M. citrifolia leaves
The methanol extract of M. citrifolia leaves was further partitioned with different solvents, comprising n-hexane, ethyl acetate, 1-butanol and water, and their IC50, CC50, and SIs (SI: CC50/IC50) determined. The IC50 values of the partitions with n-hexane and ethyl acetate were 6.1 and 6.6 μg/mL, respectively; both of these showed stronger anti-HCV activities than did the methanol extract, which had an IC50 of 20.6 μg/mL (Table 1). The n-hexane-partitioned sample was fractionated into a further five fractions by a recycling preparative HPLC method; of these fractions, only Fr. 5 showed anti-HCV activity, the IC50 being 7.8 μg/mL (Table 2). Fr. 5 was therefore further purified by HPLC, ODS column chromatography and another HPLC and Fr. 5-1-3-2 identified as the most potent and purified fraction, having an IC50 of 4.6 μg/mL.
|Sample||IC50 (μg/mL)||CC50 (μg/mL)||SI|
|Ethyl acetate partition||6.6||>30b||>4.5|
|Sample||IC50 (μg/mL)||CC50 (μg/mL)||SI|
|ODS column chromatography|
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 of chlorophyll a and its degraded products. The presence of pheophorbide a, which is reportedly the major catabolite of chlorophyll a [21, 22], was confirmed by direct comparison with a standard sample by TLC analysis. Structural analyses using HPLC and NMR identified the purified compound as pheophorbide a .
Anti-HCV activities of pheophorbide a and pyropheophorbide a
Pheophorbide a is a breakdown product of chlorophyll. In its breakdown process, chlorophyll loses the Mg2+ ion through demetallation to generate pheophytin a in senescent leaves [21, 22]. Pheophytin a is catabolized to pheophorbide a through dephytylation. In fruits, dephytylation of chlorophyll takes place first to generate chlorophyllide, which is then catabolized to pheophorbide a through demetallation. Pheophorbide a is further catabolized to generate pyropheophorbide a. On the other hand, sodium copper chlorophyllin is a semi-synthetic compound in which the Cu2+ ion replaces the Mg2+ ion.
Anti-HCV activities of commercially available, reagent-grade chlorophyll a and its-related compounds were examined. It was found that chlorophyll a barely, and sodium copper chlorophyllin and pheophytin a only weakly, exhibit anti-HCV activities (IC50 = 220, 32.0, and 54.5 μg/mL, respectively). On the other hand, pheophorbide a and pyropheophorbide a showed potent anti-HCV activities with IC50 of 0.3 and 0.2 μg/mL, respectively (Table 3).
|Sample||IC50 (μg/mL)||CC50 (μg/mL)||SI|
|Sodium copper chlorophyllin||32.0||158||4.9|
Mode-of-action of pheophorbide a and pyropheophorbide a
To determine whether the anti-HCV effects of pheophorbide a and pyropheophorbide a are exerted during the entry or post-entry step, time-of-addition experiments were performed. It was found that, when added to the culture only during virus adsorption followed by virus entry, pheophorbide a (1.0 μg/mL) and pyropheophorbide a (0.5 μg/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 a and pyropheophorbide a act during both the entry and post-entry steps.
|Compound (μg/mL)||During virus inoculation||After virus inoculation||During & after virus inoculation|
|Pheophorbide a (1.0)||64.0||95.3||98.7|
|Pyropheophorbide a (0.5)||53.0||98.1||99.7|
Inhibition of HCV RNA replication and HCV protein synthesis by pheophorbide a and pyropheophorbide a
To further confirm that pheophorbide a and pyropheophorbide a exert their anti-HCV activities not only during the virus entry step but also during the post-entry step (after the virus has entered the cells), Huh7.5 cells were inoculated with HCV for 2 hr in the absence of the test samples, and then treated with either one of the compounds for 1–2 days. Real-time quantitative RT-PCR and immunoblotting analyses demonstrated that both pheophorbide a and pyropheophorbide a inhibit HCV RNA replication in a dose-dependent manner (Fig. 1a) and, consequently, inhibit HCV protein synthesis in the cells (Fig. 1b).
It has been reported that methanol and ethanol extracts of M. citrifolia fruits show anti-HCV activities in a HCV subgenomic replicon system . In the present study, we found that a methanol extract of M. citrifolia leaves inhibits HCV replication in an HCV cell culture system more efficiently than do M. citrifolia fruits extracts (98% vs. ca. 30% inhibition at 30 μg/mL), the IC50 of the M. citrifolia leaves extract being 20.6 μg/mL (Table 1). Subsequent bioactivity-guided purification and structural analysis demonstrated that pheophorbide a, known to be the major catabolite of chlorophyll a (21, 22), and its related catabolite, pyropheophorbide a, have potent anti-HCV activities (Table 3). A time-of-addition study suggested that pheophorbide a and pyropheophorbide a act during both the entry and post-entry steps (Table 4). It should be noted that, although Fr. 5-1-3-2 purified from the M. citrifolia leaves extract consists almost entirely of pheophorbide a, its anti-HCV activity is much weaker than that of reagent-grade pheophorbide a (Tables 2, 3). One possible explanation for this apparent discrepancy is that a copurified small molecule(s) in the fraction interfered with the anti-HCV activity of pheophorbide a. Further studies are needed to clarify this issue.
Pheophorbide a reportedly inhibits influenza A virus infection . Pheophorbide a and pyropheophorbide a also reportedly show antiviral activities against herpes simplex virus type 2 and influenza A virus, but not poliovirus . Given that herpes simplex virus type 2 and influenza A virus are envelope viruses whereas poliovirus is a non-envelope virus, Bouslama et al. speculate that pheophorbide a and pyropheophorbide a inhibit envelope viruses, targeting specific envelope proteins and thereby interfering with viral binding to the host cell receptors (). On the other hand, our present results suggest that pheophorbide a and pyropheophorbide a inhibit HCV infection not only during the viral binding/entry step but also during the post-entry step (Table 4, Fig. 1). The post-entry step can be further divided into the following stages of the HCV lifecycle: (i) uncoating of the viral particles and capsid; (ii) synthesis and processing of the viral proteins and replication of the viral genome; and (iii) assembly, intracellular transport, and release of the viral particles [1, 26, 27]. Now that we have shown that pheophorbide a and pyropheophorbide a inhibit HCV infection during the post-entry step, it is important to elucidate the specific molecular mechanism(s) in the viral lifecycle targeted by those compounds.
Pheophorbide a and pyropheophorbide a are known to induce photosensitivity: the resultant photo-activated characteristics play important anti-tumor roles in photodynamic therapy using pheophorbide a and its derivatives [28-30]. Pheophorbide a also reportedly induces apoptosis of cancer cells and potentiates immunostimulating functions of macrophages [31, 32]. However, photosensitivity can cause serious adverse effects when these agents are used to treat cancer and viral infections. Importantly, the photosensitizing effect can be separated from anti-tumor effect . It is therefore tempting to speculate that a new derivative(s) with more potent anti-HCV activities and less capacity to induce photosensitivity could be synthesized from the seed compounds of pheophorbide a and pyropheophorbide a.
It has previously been reported that pheophytin a shows anti-HCV activities with IC50 of 4.97 μM (equivalent to 4.3 μg/mL) . Also chlorophyllin, a semi-synthetic derivative of chlorophyll, reportedly has antiviral activities against poliovirus and bovine herpesvirus, with IC50 of 19.8 and 8.6 μg/mL, respectively . However, in our study, compared with the more potent anti-HCV activities of pheophorbide a and pyropheophorbide a, pheophytin a and sodium copper chlorophyllin exhibited only marginal anti-HCV activity, the IC50 being 54.5 and 32.0 μg/mL, respectively (Table 3).
In this study, we demonstrated anti-HCV activities of pheophorbide a and pyropheophorbide a using the J6/JFH1 strain of HCV genotype 2a ([13, 14]). Whether these compounds inhibit replication of other HCV strains of different genotypes is an important question to answer. Currently, some other HCV genotypes, such as genotypes 1a, 1b and 3a to 7a, are available for drug screening tests [2, 35]. Such in vitro cell culture systems would help in determining the possible anti-HCV activities of pheophorbide a and pyropheophorbide a against different genotypes of HCV.
In conclusion, we have demonstrated that a methanol extract of M. citrifolia leaves and certain chlorophyll-derived compounds, such as pheophorbide a and pyropheophorbide a, possess anti-HCV activities. These compounds would be good candidates for seed compounds for developing novel antivirals against HCV.
The authors are grateful to Dr. C.M. Rice (Rockefeller University, New York, NY, USA) for providing Huh-7.5 cells and pFL-J6/JFH1. This study was supported in part by Science and Technology Research Partnerships for Sustainable Development from JST and JICA. It was also performed as part of Japan Initiative for Global Research Network on Infectious Diseases (J-GRID), Ministry of Education, Culture, Sports, Science and Technology, Japan.
The authors have no conflicts of interest to declare.