Med J Indones 12 Basic Medical Research Vol. 27, No. 1, March 2018

Antiviral effect of pauciflorum leaves extract to hepatitis C virus: An in vitro study in JFH-1 strain

1 2 1 1 3

4 1 Sri Hartati, Chie Aoki, Muhammad Hanafi, Marissa Angelina, Pratiwi Soedarmono, Hak Hotta 2

3 Research Centre for Chemistry, Indonesian Institute of Science (LIPI), Kawasan PUSPIPTEK, Tangerang, Department of International Health, Kobe University, Graduate School of Health Sciences, Kobe, Japan

4 Department of Microbiology, Faculty of Medicine, Universitas Indonesia, Jakarta, Indonesia Department of Oral Vaccine and Drug Development, Kobe University, Graduate School of Health Sciences, Kobe, Japan

ABSTRACT

Background:

A. Hepatitis pauciflorum C virus (HCV) is a leading cause of chronic liver diseases. Drug resistance to the regimen is also increasing. Hence, there is a need for new anti-HCV agents that are less toxic and more efficacious. The aim of this study is to evaluate the Methods:possibility of extracts can be a antiviral drug. Archidenron pauciflorum n n

Huh-7it cells were infected50 with the HCV genotype 2a strain JFH-I in the presence of methanol extracts of . The methanol extract further partition used -hexane, ethyl acetate, -butanol, and water showed in which butanol Results:extracts exerted the strongest IC (6.3 g/ml). Further,A. pauciflorum the butanol fraction was fractionated and yielded into 13 fractions. 50 50 The methanol extract of the leaves of exhibited concentration dependent inhibition against the JFH1 strain of50 HCV genotype 2a with an IC is 72.5 μg/ml. The butanol fraction exhibited the highest anti-HCV activity with an IC is 6.3 μg/ml. The butanol fraction was fractionated which yielded 13 fractions. Fractions 5 and 13 exhibited high anti-HCV activities with IC is 5.0 μg/ml and 8.5 μg/ml and a time-of-addition study demonstrated that fraction 5 inhibited viral infection at the Conclusion:post-entry step, whereas A.fraction pauciflorum 13 primarily inhibited the viral entry step.

The extract can be used as a herbal-based antiviral drug.

Keywords: , HCV, JFH-1

12–8

pISSN: 0853-1773 • eISSN: 2252-8083 • https://doi.org/10.13181/mji.v27i1.2189 • Med J Indones. 2018;27: • Received 03 Aug 2017 • Accepted 12 Jan 2018

Corresponding author: Muhammad Hanafi [email protected]

Copyright @ 2018 Authors. This is an open access article distributed under the terms of the Creative Commons Attribution-NonCommercial 4.0 International License (http://creativecommons.org/licenses/by-nc/4.0/), which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original author and source are properly cited.

Medical Journal of Indonesia Hartati, et al. 13 Antiviral effect of A. pauciflorum

jiringa

Hepatitis C infection disease caused by has been found to have high polyphenolic the hepatitis C virus (HCV) primarily affects the contents (>150 g gallic acid equivalents/mg function of liver. HCV is a major cause of chronic dried ) and antioxidant activities measured liver diseases and linked to the development using ferric reducing antioxidant power (FRAP) of cirrhosis and hepatocellular1 carcinoma. method, also have high potent to 2,2-diphenyl-19,20 Approximately 130–200 million people worldwide 1-picrylhydrazyl(DPPH) scavenging activity. are infected with HCV, which is spread primarily The of this plant are used as food sources in by blood-to-blood contact which associated with Indonesia and contain djenkolic acid and sulfur. intravenous drug use, contaminated medical Those elements can inhibit the pests as21 well as equipment, transfusion, and organ transplants. vitamin C and poly phenolic compounds, which Boceprevir or telaprevir plus pegylated have potential anti-oxidant activities. interferon alpha (IFN-α) and ribavirin are the of the familyGlycyrrhiza have been uralensis reported to standard therapies for patients with chronic exhibit various anti-viral activities. For example, HCV infections. This triple combination therapy glycyrrhizic acid from Fisch increases the sustained viral, with the response was reported to exert anti-viral activities against rates up2 to 75% in patients with HCV genotype 1 coxsackievirus A1620 and enterovirusAcacia 71, which confusa are infections, which respond relatively poor to IFN-α the most common causative agents of hand, foot, therapy. However, it is still important to search and mouth disease. Extracts from 21–24 , for new and effective anti-HCV drugs to increase were Mimosashown to scabrella exhibit anti-HCV activities. inA sustained viral responses as well as reduce the vitrosulfate prepared from galactomannan derived25 treatment side effects and costs. from was reported to exert A. 3 pauciflorumanti-herpetic and anti-rotaviral activities. Medicinal plants are attractive and However, the possible anti-viral activities of important resources of bioactive compounds. extracts have not yet been reported. Natural molecules derived from medicinal plants and natural products have been4–12 identified as In the course of our screening program of anti-HCV agents in recent years. Among them, the anti-HCV activities of Indonesian medicinal silymarin, ladanein, and (-)-epigallocatechin have plants, we A.observed pauciflorum the potential of the methanol been shown13–15 to be bioactive via oral administration crude extract and butanol fraction from the in mice, healthy individuals, or HCV-infected leaves of as anti-viral activities patients, representing potential therapeutic against HCV. Bioactivity-guided fractionation use in clinical trials. was performed and the mode of action of these Archidendron pauciflorum fractions with anti-HCV activities was analyzed. METHODS Abrema Djengkol jiringa Kosterm, is the common jiringa name of Albizia lucida sensu. auct. It is Archidendron also called puuciflorum Feuileea jiringa(Jack) Study Design Kurz, jiringa 16 A. in vitro jiringa (Benth.) I. C. Nielsen, Kuntz, (Jack) Parin etc. This was an study in human is a leguminous plant belonging to the hepatocyte culture, Huh-7it cells using hepatitis C family of Fabaceae. This plant has been used17 as a virus genotype-2a JFH-1 strain. Huh-7it cells were traditional medicine for a range of ailments and used to propagate HCV. All of the experiments consumed as a raw vegetable in . Some Collectionwere conducted of A. in triplicate. pauciflorum , extraction of chemical constituents is djenkolic acid (sulfur- crude extracts, and preparation of fractions containing amino acid), from leaves, have been A. pauciflorum reported containing five flavan-3-ol derivatives and new flavan-3-ol galates, gallocatechin-3’ leaves used in this study and 4’-O-gallates, gallocatechin7,3’ and were obtained from the Research Center for 7,4’di-O-gallates.A. jiringa Procyanidinds-B3 and B-4, Chemistry, Indonesian Institute of Sciences prodelphinidineStaphylococcus B-1. Methanol aureus S. extract epidermidis, leaves (LIPI), Kawasan Puspiptek Serpong, Indonesia. of Microsporum active asgypsum antibacterial18 were mostlyA. The leaves samples were verified by botanical active for , researchers at Herbarium, Research Center for and . The shoot of Biology, LIPI, Cibinong, Indonesia. A herbarium Medical Journal of Indonesia Med J Indones Vol. 27, No. 1, March 2018 14

A. pauciflorum specimen was deposited in the Research Center were adsorbed to the cells for 4 h at 37°C. Then, the for Chemistry-LIPI. leaves were cells were incubated with a medium containing dried, pulverized, and subsequently macerated in 0.4% methylcellulose (Sigma-Aldrich) for 40 h. 100% methanol for 24 h. The extracts were filtered HCV titers were determined using27,28 a focus-forming and the residue was resuspended in fresh 100% assay as previously described. Antigen-positive methanol. This maceration process was repeated cells were stained with anti-HCV anti-serum and over three days. The combined filtrates were horseradish peroxidase-conjugated goat anti- evaporatedn at room temperature,n not exceeding human IgG (MBL, Tokyo, Japan). Infectious foci were 40°C. The extract (80 g) was initially partitioned visualized using the Metal Enhanced DAB Substrate with -hexane, ethyl acetate, -butanol, and Kit (Pierce-Thermo Scientific). Foci in each well water to obtain a hexane fraction (18.2 g), ethyl were counted and normalized to untreated controls acetate fraction (3.0 g), butanol fraction (30.0 g), Evaluationto calculate the of anti-viraltitre of virus. activities and water fraction (25.3 g). Based on bioactivity results, the butanol fraction (2.5 g) was subjected 4 nto silica gel 60 column chromatography (0.063– Huh7it-1 cells (5.0×10 cells/well) which 0.200 mm; Merck, Darmstadt, Germany) with cultured intoA. a pauciflorum48-well plate were infected with -hexane: ethyl acetate and ethyl acetate: HCV at a multiplicity of infection of 0.1 in the methanol by gradient elution, which yielded 13 presence of extracts or fractions Samplesubfractions preparation (fraction number 1–13). (100, 50, 20, 10, 5, and 1 μg/ml) for 2 h at 37°C. After removing the residual viruses by rinsing the wells twice with serum-free medium, The methanol crude extracts and the cells were incubated for 46 h at the same subfractions were dissolved in DMSO to obtain sample concentrations. At 48 h p.i., the culture stock solutions at concentrations of 100 and 50 supernatants were collected and used for viral mg/ml, respectively. The stock solutions were titration. In some experiments, the percent Cellstored culture at −30°C and until virus used. preparation infectivity of the fractions at a concentration of 20 µg/ml was determined. Cells were treated with fractions 5 or 13 only during or only after viral Huh7it-1 is a well differentiated inoculation for26 the remaining culture period until hepatocyte derived from cellular carcinoma cell viral harvest to assess the mode of action of the line that was originally taken from a liver tumor25 Cytotoxicityplant extracts. assay in a 57 year-old Japanese male in 1982. The cell line was established by Nakabayashi and Sato J. 26 Cytotoxicities of the crude extracts and the The human adapted variants of the fractions against Huh7it-1 cells were evaluated HCV strain JFH-1 (JFH1a) were inoculated using the MTT [3-(4,5-dimethylthiazol- into the Huh7it-1 cells. Culture supernatants 2-yl)-5(3-carboxymethonyphenol)-2-(4- were collected at 3–5 days post infection (p.i.), sulfophenyl)-2H-tetrazolium] assay (Sigma- concentrated using Amicon 100K centrifugal Aldrich) following the4 manufacturer’s filters, and stored at −80°C. Huh7it-1 cells were instructions. Cells (2×10 cells/well) were cultured in Dulbecco’s modified Eagle’s medium placed in a 96-well plate and treated with a (DMEM, Invitrogen, Carlsbad, CA) supplemented medium containing serial dilutions of the plant with 10% fetal bovine serum (Biowest, Nuaille, extracts or fractions (1 to 300 μg/ml) for 48 France), 100 µg/ml kanamycin (Sigma-Aldrich, h. Cyclosporine A were used as a comparator.

St. Louis, MO, USA), 2 and non-essential amino The medium was replaced with 100 μl of MTT acids (Invitrogen). Cells were grown at 37°C in an solution and incubated at 37°C for an additional Virusatmosphere titration of 5% CO . 4 h. The medium was removed and 100 μl of dimethyl sulfoxide (DMSO, Sigma-Aldrich) was 4 added to dissolve the generated precipitate. Cells (2.4×10 cells/well) were placed in Absorbance at 550 nm was measured using a a 96-well plate 1 day before the virus infection. GloMax-Multi Microplate Multimode Reader Culture supernatants were serially diluted 10-fold (Promega). The percentage of viable cells with medium in a 96-well plate, and 50 μL of viruses versus the concentration of the test compound

http://mji.ui.ac.id Hartati, et al. 15 Antiviral effect of A. pauciflorum

50 50 was plotted. The concentration by which to 6.3 μg/ml (Table 1). The ethyl acetate fraction mediate 50% inhibition (CC ) was determined exhibited moderate anti-HCV activity (IC , 18.6 Databy non-linear analysis regression analysis. μg/ml), whereas the hexane and water fractions

exhibited weak or no anti-HCV50 activities. Cytotoxicities of these fractions to non-infected The data were presented in mean standard cells were determined and a CC value of the 50 deviation. The data were analyzed using non- butanol fraction was 167.2 μg/ml, whereas those linear regression analysis. IC were calculated of other fractions were >300 µg/ml. We selected from the formula of non-linear regression. the butanol fraction for further fractionation due RESULTS Anti-HCVto the high activities SI value (26.5). of the isolated fractions

Anti-HCV activities of extracts from A. pauciflorum Furthermore, the butanol fraction was separated using silica-based open column chromatograph which yielded 13 fractions as We examined crude extracts of 250 described in figure 2. Cells were infected with medicinal plants for anti-HCV effects using a HCV in the presence of each fraction (20 μg/ml) cell-culture HCV24 system to investigate activities and the effects of each fraction on HCV infection against HCV. Huh7it-1 cells, which are highly were examined. All fraction exhibited varying permissive and obtained by curing replicon levels of inhibition of HCV infection. However, cells with IFN-α, were challenged with the HCV we observed that fraction 5 and 13 exhibited a 50 genotype 2a strain JFH1 in the presence of strong inhibition of 100% and 91%, respectively, CC50 extracts. Virus titer in the culture supernatants of the untreated control (Table 2). The IC and at 48 h p.i. wasA. determined pauciflorum using a focus-forming values of fraction 5 were 5.0 and 33.2 μg/ml, assay. Screening results revealed that a crude respectively, whereas those of fraction 13 were extract from dose-dependently50 8.5 and 62 μg/ml respectively. suppressed production of infectious virus particles Table 2. A. Pauciflorum [50% inhibitory concentration (IC ): 72.5 μg/ml] without cytotoxicity at the concentrations tested. Effects of the butanol fractions from on HCV JFH1 and cell viabilities IC50 CC50 Cytotoxicity 50 of a crude extract to non-infected cells was50 determined50 using the MTT assay, which Inhibition Cell viability Samples showed a CC of 190 μg/ml and a selectively index (%) (%) (μg/ml) (μg/ml) (SI, CC /IC ) of 2.6 (Table 1). Fraction 1 -37±5.5 102±2.0 NT NT Fraction 2 -26±6.7 99±0.7 NT NT The crude extract was partitioned into Fraction 3 7±3.1 97±0.1 NT NT n-hexane, ethyl acetate, butanol, and water 100 Fraction 4 26±6.1 101±1.2 NT NT fractions and subsequently investigated50 for anti-HCV activities. The butanol fraction had the Fraction 5 108±0.2 5.0 33.2 highest anti-HCV activity with an IC value of Fraction 6 11±5.5 108±1.0 NT NT Fraction 7 59±5.5 84±4.9 NT NT A. pauci- Table 1. Fraction 8 24±1.7 104±1.3 NT NT florum 50 Fraction 9 28±2.5 112±3.2 NT NT The effects50 of methanol crude extract from and its fractions on anti-HCV activities (IC ) and cell Fraction 10 36±4.9 113±1.5 NT NT viabilities (CC ) IC50 CC50 SI Fraction 11 44±6.1 114±0.4 NT NT 190 62 Samples (μg/ml) (μg/ml) Fraction 12 47±4.3 112±0.4 NT NT Crude extract 72.5 2.6 Fraction 13 91±0.6 92±1.4 8.5 Hexane fraction >60 415.2 >6.9 Cyclosporin A 88±2.0 102±2.1 NT NT Ethyl acetate fraction 18.6 361.2 16.7 Inhibition and cell viability at the concentration of 20 ug/ml Butanol fraction 6.3 167.2 26.5 were shown except for the concentration of cyclosporin A as

Water fraction 50 45.3 >600 50>13.2 an inhibitor control, which is 1 ug/ml. Values were shown as mean ±SD values of two independent experiments; NT= SI: Selectively index, CC value was divided by the IC value not tested Medical Journal of Indonesia Med J Indones 16 Vol. 27, No. 1, March 2018

Table 3. A. pauciflorum

Mode of action studies of butanol fractions 5 and 13 from

% Inhibition Mode of action During + During Post-inoculation post-inoculation inoculation Fraction 5 98.9±0.5 63.4±4.1 95.4±1.2 Post-entry inhibition Fraction 13 95.7±0.9 85.6±1.4 66.4±0.5 Entry inhibition -

Fractions 5 and 13 at the concentration of 10 µg/ml and 20 µg/ml, respectively. Values presented the mean ± SD from three in dependent experiments Commented [MJI1]: Mohon kirimkan figure dalam format jpg Butanol fraction 5 (μg/ml) Butanol fraction 13 (μg/ml) denganCommented resolusi minimal[MJI1]: Mohon 300 dpi.kirimkan figure dalam format jpg R Mohondengan sumbu resolusi x danminimal y diberi 300 dpi. keterangan R Mohon sumbu x dan y diberi keterangan CommentedCommented [MJI2]: [MJI2]: Mohon Mohon kirimkan kirimkan figure dalam figure format dalam jpg format jpg e e dengandengan resolusi resolusi minimal minimal 300 300 dpi. dpi. MohonMohon sumbu sumbu x xdan dan y y diberi diberi keterangan keterangan l l µg/ml) a

a ( Ap-B Fr5

ti infectivity Relative ti subfr µg/ml) µg/ml) Figure 1. Ap-B Fr5 ( actio Ap-B Fr13 ( Synergetic effects of the butanol fraction 5 and 13 (μg/ SButanol fractionsubfr 5 (μg/ml) Butanol Fraction 13 (μg/ml)

Mode-of-action actioof butanol fraction 5 and 13 concentrations of fraction 13, demonstrating a To determine whether the anti-HCV synergistic effect of the two fraction against HCV effects of the two(μ fractiong/Butanol were inhibited Fraction at the 13 infection. This synergistic effect was further SButanol fraction 5 (μg/ml) entry or the post-entry step, time-of-addition evaluated by classic isobologram analysis and experiments were performed. As(μg/ml) presented in the dose-effect plots were drawn. The results Table 3, fraction 5 exhibited anti-HCV activity demonstrated that the curve was below the line at the post-entry step, whereas fraction 13 showing an additive effect, indicating that the exhibited anti-HCV activity primarily at the effect of a combination of the two fractions on entry step. These findings suggested that each HCV infection was strongly synergistic (Figure fraction contained different compounds with 1b). Cytotoxicity was not observed at the tested different mechanism of action. concentrations via isobologram analysis (data not shown). These A.results pauciflorum collectively suggested The percent infectivity by treatment that the strong anti-viral activities from the with fraction 5 was plotted for each fixed butanol fraction of leaves extracts concentration of fraction 13 (Figure 1a). have been contributed to several compounds The curves shifted to the left with increasing with different anti-viral mechanisms.

http://mji.ui.ac.id Hartati, et al. 17 Antiviral effect of A. pauciflorum

DISCUSSION not done the analysis of contents from butanol fraction 13. A. pauciflorum Due to the high rate of pre-existing or In conclusion, crude extract from emerging viral resistance, effective treatment leaves, and the butanol fraction of HCV-infected patients will likely require a exhibited great potential as anti-HCV agents. combination of inhibitors targeting distinct The butanol fraction yielded a higher SI value viral or host functions. As described above, compared to the crude extracts and other we demonstrated that both fractions 5 and 13 fractions. Among 13 butanol fractions, there were exerted inhibition activities with different modes two fractions only fractions exhibited strong anti- of action. On the other hand, the anti-HCV effect viral activities. Synergetic effects were observed by mono-treatment of each butanol fractions by a combination treatment of these two fractions. were not stronger than that of treatment with The results of the present investigation provide the butanol fraction alone. It was possible that evidence of the potential ofin medicinal vivo plants for fractions have synergistic mode in butanol herbal drug as anti-HCV. However, further study fractions than each fraction alone. Therefore, we for the use of this fraction is still need to examined the anti-HCV effects of the two fractions Acknowledgmentsevaluate its efficacy and toxicity. in combination. Cells were infected with HCV and treated with combinations of fractions 5 and 13 at various A.concentrations. pauciflorum A. jiringa We thank to Dr. Takaji Wakita for providing Pithecellobium jiringa pJFH1. This work was supported in part by grants (known as , from the Science and Technology Research in Malaysia) is a typical Partnership for Sustainable Development plant in including Indonesia. (SATREPS) from the Japan Science and Technology The of this plant contains jenkolic acid, Agency (JST) and Japan International Cooperation sulfur, vitamin21–24 C and E, flavonoid, , and ConflictAgency (JICA). of Interest fatty acids. The is used traditionally for the treatment of hypertension25 and diabetes, whereas the old leaves burnt to ashes were The authors affirm no conflict of interest used against itching parts. This plant was in this study. REFERENCES also found27 a high contain of polyphenolic compounds which has a potent antioxidant activity. Although the compounds responsible for anti-HCV activities have not yet been 1. Rosen HR. Clinical practice. Chronic hepatitis C infection. isolated in this study, we observed that fraction N Engl J Med. 2011;364(25):2429–38. 5 contained chlorophyll-related compounds, 2. Ghany MG, Nelson DR, Strader DB, Thomas DL, Seeff known to show antioxidant activity as major LB. An update on treatment of genotype 1 chronic constituents (obtained from our preliminary hepatitis C virus infection: 2011 practice guideline by study). We isolated a chlorophyll-related the American Association for the study of liver diseases. Hepatology. 2011;54(4):1433–44. compound from another Indonesian plant by 3. Balunas MJ, Kinghorn AD. Drug discovery from activity-guided purification and its compound medicinal plants. Life Sci. 2005;78(5):431–41. exhibited anti-HCV activity (manuscript in 4. Polyak SJ, Morishima C, Shuhart MC, Wang CC, Liu Y, preparation). Also, different groups previously Lee DY. Inhibition of T-cell inflammatory cytokines, reported that pheophytin and chlorin e6, which hepatocyte NF-kB signaling, and HCV infection are chlorophyll degradation products, exhibited by standardized silymarin. Gastroenterology. potential anti-HCV activities. The mechanism 2007;132(5):1925–36. 5. Polyak SJ, Morishima C, Lohmann V, Pal S, Lee is through the inhibition of NS3 protease and DY, Liu Y, et al. Identification of hepatoprotective probably disruption28,29 of the envelope protein, flavonolignans from silymarin. Proc Natl Acad Sci U S A. respectively. The activity of fraction 5 may 2010;107(13):5995–9. have contributed to the presence of chlorophyll- 6. Ciesek S, von Hahn T, Colpitts CC, Schang LM, Friesland related compounds. Further activity-guided M, Steinmann J, et al. The green tea polyphenol, purification and analytical studies are required epigallocatechin-3-gallate, inhibits hepatitis C virus entry. Hepatology. 2011;54(6):1947–55. to explore these anti-HCV substances. We have Medical Journal of Indonesia Med J Indones 18 Vol. 27, No. 1, March 2018

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