The Alkaloid Fraction from Melicope latifolia Leaves Inhibits Hepatitis C Virus

Hepatitis C Virus is an RNA virus that has a diameter of 50 nm and a length of 9.6 kb. Hepatitis C virus is classified in the Hepacivirus genus and Flaviviridae family. This virus has varied genotypes (1-7) and more than 70 subtypes (1a, 1b, 2a, 2b etc).2,3 The viral genome encodes polyprotein precursor consisting of about 3000 amino acid residues, which is cleaved by the host and viral protease to generate 10 mature protein, structural proteins namely core E1, E2, a putative ion channel p7 and nonstructural proteins NS2, NS3, NS4, NS4B, NS5A, and NS5B.2


INTRODUCTION
Hepatitis C Virus is one of the major health problems in the world that causes millions of people to die annually. According to WHO, it was estimated that the number of deaths from hepatitis C is 333,000 in 1990, 499,000 in 2010 and 704,000 in 2013. The increasing number of deaths illustrates the high incidence rates of hepatitis C virus over the decades. 1 Hepatitis C Virus is an RNA virus that has a diameter of 50 nm and a length of 9.6 kb. Hepatitis C virus is classified in the Hepacivirus genus and Flaviviridae family. This virus has varied genotypes (1-7) and more than 70 subtypes (1a, 1b, 2a, 2b etc). 2,3 The viral genome encodes polyprotein precursor consisting of about 3000 amino acid residues, which is cleaved by the host and viral protease to generate 10 mature protein, structural proteins namely core E1, E2, a putative ion channel p7 and nonstructural proteins NS2, NS3, NS4, NS4B, NS5A, and NS5B. 2 The recommended HCV therapy is direct-acting antiviral drugs (DAAs). Several inhibitors of viral nonstructural protein successfully improved sustained virology response (SVR) but the resistant factor, expensive price, and limited access to the treatment need to be considered as important problems. 4,5 In order to circumvent those problems, the development of new antiviral drugs is very much required.
Some plants have been reported to have hepatitis C antiviral activity. Plant extracts from Toona sureni, 6 Artocarpus heterophyllus, 7 Ficus fistulosa, 8 Alectron serratus, 9 and Melicope latifolia 4 significantly inhibited HCV activity in vitro. A variety of active phytochemicals such as flavonoids, terpenoids, lignins, sulphides, polyphenolics, coumarins, saponins, furyl, alkaloids, polylines, thiophenes, protein, and peptides have been identified to inhibit various viruses. 10 Melicope latifolia belongs to the Rutaceae family, commonly known in Indonesia as Ki Sampang and is traditionally used to cure fever and abdominal cramps. The tree typically has a height of about 20 m and is commonly found in primary and secondary open forests. M. latifolia plants are distributed in Malaysia, Philippines, Java-Indonesia, Kalimantan-Indonesia and Papua New Guinea. 11 The chemical compounds which were contained in the M. latifolia plant, are flavonoids, alkaloid, coumarins and terpenoids. 10,12 In general, plant species in the Rutaceae family contain the chemical constituent of alkaloid group, including the Melicope genus.
For instance, M. ptelefolia leaves were reported to contain the alkaloid compounds of N-methylflindersine and two new alkaloids named melicobisquinolinone A and B. 13 Our previous study showed that 80% ethanol extract of M. latifolia leaves exhibited potent activity as anti-HCV with IC 50 of 3.5 ± 1.4 μg/mL against J6/JFHI virus by inhibiting hepatitis C virus mainly at the entry step. Moreover, we also reported that M. latifolia inhibited various genotype of HCV with a percentage inhibition of higher than 87% against all genotypes i.e 1a,1b, 2a, 2b, 3a, 4a, 5a, 6a, 7a. 10 However, further studies to identify the active fraction which are responsible for anti-HCV activities have not been conducted yet. Therefore, this study was conducted to identify active compound from M.latifolia and analyzed their anti-HCV activity and cytotoxicity.

MATERIALS AND METHODS
General NMR spectra were recorded on a JEOL ECS-400, using CdCl 3 as the solvent. The HPLC system also includes two LC-10AD pumps and a SCL-10A controller. An Agilent RP-18 XDB column 4.6 x 250 mm was eluted with CH 3   Extraction, fractionation, and identification of major compound M. latifolia leaves were dried at room temperature then extracted using 80% ethanol by ultrasonic assisted extraction for two minutes at three times of replications. The M.latifolia ethanol extract (B1F) was further fractionated by liquid-liquid fractionation using dichloromethanewater to obtain dichloromethane fraction (B1F D). Further separation of B1F D by vacuum liquid chromatography using a gradient solvent of chloroform-methanol (100%-97%) resulted in 9 subfractions (B1F D1-D9). The anti-HCV active subfraction (B1F D2) was then separated by semi preparative HPLC using acetonitrile : water (7-3 v/v), 2 mL/ min of flow rate and resulted 5 subfractions (B1F D2H.1-B1F D2H.5). The active subfraction profile was analyzed using silica gel 60 F 254 plate, scanned by TLC Visualizer on UV 254 nm and 366 nm, and sprayed with dragendorf, a specific reagent for alkaloid compound. The chemical structure of active subfraction was identified using NMR-JEOL ECS-400 and the spectra were then analyzed using MNova program.

Anti-HCV activity and cytotoxicity assay
Cell culture preparation Huh7it cells were cultivated in DMEM (Dulbeco's Modified Eagle Medium) (GIBCO Invitrogen) and supplemented with 10% Fetal Bovine Serum (FBS), 1x Non-Essential Amino Acids (NEAA, GIBCO Invitrogen) 5 mL, and 0.15 mg/mL Kanamycin solution (SIGMA) in 5% CO 2 at 37 °C. The culture condition of Huh7it cells was observed under a microscope every day. The passage was performed while cells confluent >80%. 7

HCV propagation
HCV genotype 2a (JFH1) propagation was performed on hepatocyte Huh7it cells (1.8x10 7 cells). Infected cells were incubated at 37 °C in 5% CO 2 for 4 hours with agitation every 30 minutes. HCV supernatants were harvested on day 3 and were concentrated through an Amicon Ultra-15 centrifugal filter (Millipore) by centrifugation at 3500 rpm, 15 min, 4 o C. DAB staining was used to visualize infected cells for virus titration. 7

Cytotoxicity assay
The cytotoxicity analysis of the sample was assessed by MTT assay. Huh7it cells in 96 well plates were treated with serial dilution of the sample or control. The condition of the cells was observed after 48 hours incubation and the toxicity was checked under microscope. The medium was removed from 96 well plates and then MTT 10% 150µl/ well was put by multichannel pipette and incubated for 4 hours at 37 °C. MTT solution was removed from 96 well plates and 1 DMSO 100% was added to dissolve formazan. The absorbance of sample was measured at 560 nm and 750 nm. The percentage of inhibition was calculated by comparing the absorbance sample with control. Probit analysis was conducted to calculate the CC 50 . 7

Data analysis
The 50% growth inhibition (IC 50 ) and cytotoxicity (CC 50 ) was determined using an SPSS probit analysis by creating a curve relationship between the percentage of inhibition or cytotoxicity and the logs of doses.

Anti-HCV activity
The fraction and subfractions were subjected in vitro anti-HCV and cytotoxicity assay. First fraction (B1F D), and subfractions (B1F D1-B1F D9) were tested at a concentration of 30 μg/mL. The result showed that subfractions B1F D2, B1F D7, and B1F D9 exhibited strong activity against hepatitis C virus with inhibition percentages of 97.05%, 98.31%,and 98.30%, respectively. On the other hand, the same concentration of B1F D1, B1F D3-D6, and B1F D8 showed lower anti-HCV activity with percentage inhibition less than 28%. To determine the cytotoxicity effect, MTT test was performed in Huh7it. The results showed no toxic effect among all subfractions with mediated the cell viability higher than 85%, while the active subfractions B1F D2, B1F D7 and B1F D9 had viability levels of 99.77%, 93.63%, and 95.99%, respectively (Table 1).
To determine the IC 50 value, the active fraction and subfractions were evaluated for their inhibition percentages at various concentrations. The result showed that B1F D, B1F D2, B1F D7 and B1F D9 exhibited strong anti-HCV activity with an IC 50 value of 11.38 μg/mL, 13.33 μg/mL, 4.5 μg/mL, and 2.9 μg/mL, respectively. The cytotoxicity assay revealed that   all active fractions mediate CC 50 value of >100 μg/mL. There was no cytotoxicity with exposure of sample up to 100 µg/mL for 48 h. The selectivity index (SI) that determined the effectiveness of a potential medicinal drug was analyzed by the ratio of CC 50 and IC 50. It was shown that the highest SI value of >96.56 belongs to subfraction B1F D9 followed with B1F D7 and B1F D2 (Table 2). Due to a high amount of chlorophyl on B1F D7 and B1F D9, we focused to further separate B1F D2 using semi preparative HPLC to obtain the subfraction.
We obtained 5 subfractions (B1F D2H.1-B1F D2H.5) and analyzed their activity and toxicity as anti-HCV. The results determined that B1F D2H.3 had a strong active anti-HCV activity with an IC 50 value of 6.29 μg/mL, a CC 50 value of 82.64 μg/mL, and an SI >13.31 (Table 3). This result showed that subfraction D2H.3 may a potential target for separating the active anti-HCV constituent.

DISCUSSION
Developing anti-HCV agents from medicinal plants has become a currently significant issue. In recent years, many compounds were isolated from medicinal plants reported to be active anti-HCV agents. Chalepin and pseudane IX from Ruta angustifolia (leaves) revealed anti-HCV activities with IC 50 value of 1.7±0.5 and 1.4±0.2 µg/mL by inhibiting HCV replication and decreasing the NS3 protein level, 15 Embelin and 5-O-Methyl embelin were isolated from Embelia schimper (Fruit) with IC 50 of 21 µM and 46 µM, by inhibiting HCV replication and decreasing the NS3 protein level, 16 and Saikosaponin b2 from Bupleurum kaoi root with IC 50 16.1 µg/mL inhibited HCV by the neutralization of virus particle, attachment, and fusion. 17 Anti-HCV activity and the mechanism of action from the ethanol extract of M. latifolia leaves had been reported in previous studies. 10 In this study, we have fractionated and identified the active compound as anti-HCV agent from M. latifolia. The active fraction was contained major compound which similar with a known alkaloid compound called N-methylflindersine. This fraction has anti-HCV activity with IC 50 value of 6.2 µg/mL and less cytotoxicity effect with CC 50 value of 82.64 µg/mL. N-methylflindersine is the characteristic constituent of Rutaceae and Meliaceae families. It is known to possess insect growth inhibitor, antifeedant, and fungistatic activities. 13,18 The antiviral activity of N-methylflindersine in M.latifolia has not been reported yet.
Melicope species have been investigated for their chemical compounds. Melicope ptelefolia leaves were reported to contain N-methylflindersine and two new alkaloids named melicobisquinolinone A and B. Another alkaloid were obtained from Melicope semicarfolia, there were 2-acetylevolitrine (1), 2-acetylpteleine (2), and semecarpifoline (3). Several of these alkaloid exhibited significant antiplatelet aggregation activities in vitro. 19 There was APS, an alkaloid from Maytrenus ilicifolia (root bark), which has known activity as anti-HCV with EC 50 of 2.3 µM by decreasing HCV replication and NS5A level. 20 Although the mechanism of ethanol extract from M.latifolia was known to inhibit mainly at the entry step with 90.8 ± 0.2% and post entry step 60.6 ± 4.9%, the mechanism of action of its alkaloid fraction, N-methylflindersine was still unknown. Further analysis of the mechanism of N-methylflindersine was necessary.