IN-VITRO ANTIVIRAL ACTIVITY OF PISTACIA CHINENSIS FLAVONOIDS AGAINST HEPATITIS C VIRUS (HCV)

The present study was designed to evaluate anti-HCV activity of Pistacia chinensis aerial parts and identify the responsible bioactive constituents of anti-hepatitis ethnomedicinal plant P. chinensis methanol 80% extract. This has led to the isolation and characterization of gallic acid and eight flavonoids, apigenin, diosmetin, myricetin, apigenin 7-O-β-glucoside, quercetin 3-O-βglucoside myricetin 3-O-α-rhamnoside, myricetin 3-O-β-glucuronide and quercetin 3-O-βglucoside-7-O-α-rhamnoside from the plant, using various chromatographic procedures and the interpretation of spectral data in comparison with already existing data reported in the literature. Methanol 80% extract of P. chinensis and some isolates were tested for their anti-HCV activity using HCV cell culture (HCVcc) system. The results have shown that diosmetin and apigenin, significantly reduce HCV infection while myricetin sugars (myricetin 3-O-α-rhamnoside and myricetin 3-O-β-glucuronide) had no significant effect on HCV infection. The half maximal inhibitory concentration (IC50) of diosmetin and apigenin were calculated to be 42.5 μM and 39.9 μM respectively. However, cell viability assays demonstrated that apigenin was toxic in cell culture in the same range of concentrations that show HCV inhibition. This is the first time report for anti-HCV activity of diosmetin isolated from P. chinensis and this could become a molecular template for the development of new anti-HCV drugs.


INTRODUCTION
Hepatitis C virus (HCV) is a major cause of chronic liver disease which can lead to permanent liver damage, hepatocellular carcinoma and death [1]. Egypt reports the highest prevalence of HCV worldwide [2,3]. The presently available treatment with pegylated interferon plus ribavirin, has limited benefits due to adverse side effects and high cost. The recent addition to the therapy of direct acting antivirals (DAA), specific inhibitors of the viral protease NS3/4A, concerns only HCV genotype 1 infected patients and are also accompanied by numerous adverse effects [4]. It is important to note that, in Egypt, HCV genotype 4 is the most prevalent. Hence, there is a need to develop anti-HCV agents that are less toxic and cost-effective [5,6]. In spite the highly vigorous and extensive research in this field, a protective vaccine and effective treatment for HCV genotype 4 infected patients are not yet available [7,8].
Herbal medicines have been used for centuries against different ailments including viral diseases and become a focal point to identify, isolate and purify new entities to treat diseases like hepatitis C. In the course of our studies on development of HCV inhibitors from naturally occurring products, we focused on medicinal plants. Pistacia chinensis is a deciduous and small to medium-sized tree from Anacardiaceae family. It is widely distributed in China and North America. It is well known as a landscape and shade tree [9]. In Chinese traditional medicine, the oil from its seeds is used for biodiesel production in China and it shows a high resistance to various pests in the United States [9]. There are very few reports about chemical constituents and biological activities from P. chinensis plant. Two 4-arylcoumarin moieties (neoflavone) dimers were isolated from of P. chinensis leaves with estrogen-like activity [10]. Also some phenolic compounds such as gallic acid, m-digallic acid, quercetin, 6-O-galloyl arbutin-quercitrin and quercetin-3-O(6''-galloyl)-β-D-glucosides were isolated from the leaves [11] and a new pyrrolidone derivative was isolated from P. chinensis tender burgeon and anthotaxy [12]. P. chinensis has the ability to inhibit NO production as anti-inflammatory potential of this plant [13]. This research was carried out to evaluate antiviral activity of naturally derived extract and bio-active compounds on HCV from P. chinensis plant.

Plant identification and collection
Aerial parts of P. chinensis were collected from Al-Zohiriya garden, Giza, Egypt in May 2012. The plant was identified by Dr. Mohammed El-Gebaly, Department of Botany, National Research Centre (NRC) and by Mrs. Tereeza Labib consultant of plant taxonomy at the Ministry of Agriculture and director of Orman botanical garden, Giza, Egypt. A voucher specimen is deposited in the herbarium of Al-Zohiriya garden, Giza, Egypt.

Preparation of the extract
Air-dried powder of P. chinensis aerial parts (800 g) was extracted with methanol 80% several times at room temperature until exhaustion by maceration method. The extract was concentrated under reduced pressure to give 42 g of the crude extract.
Isolation of bioactive compounds from methanol 80% extracts of P. chinensis aerial parts 40 g of methanol extract was subjected to silica gel column chromatography eluting with hexane, dichloromethane, ethyl acetate and methanol gradually.
One hundred and eighty fractions of 100 ml conical flask were collected. The fractions that showed similar Paper Chromatography (PC) in Butanol-Acetic acid-Water 4:1:5 (BAW) and 15% acetic acid were combined to give 4 fractions (I, II, III, and IV). Fraction I (1.2 g) was subjected to sub-column of silica gel eluted with dichloromethane: ethyl acetate (60:40) gave compound 1 and elution with dichloromethane: ethyl acetate (80:20) gave compound 2. Fraction II (928 mg) was subjected to sub-column of silica gel eluted with dichloromethane: ethyl acetate (95:5) yielded compound 3 and elution with ethyl acetate solvent gave compound 4. Compound 5 yielded from elution with ethyl acetate : methanol (95: 5) and compound 6 was obtained from elution with ethyl acetate: methanol (90:10) and also compound 7 was obtained by elution with ethyl acetate: methanol (85:15) from fraction III (1.45 g). Compound 8 yielded by elution with ethyl acetate: methanol (75:25) and compound 9 was obtained by elution with ethyl acetate: methanol (60:40) from fraction IV (1.35 g). All the isolated compounds were purified on sephadex LH-20 column using different systems of methanol and distilled water.
General method for acid hydrolysis of flavonoid glycosides 5 mg of each flavonoid glycoside 5, 6, 7, 8 and 9 in 5 ml 10% HCl was heated for 5 h. The aglycones were extracted with ethyl acetate and identified by co-TLC with authentic standards. The sugars in the aqueous layer were identified by co-paper chromatography (co-PC) with authentic markers on Whatman No. 1 sheets in solvent system (n-BuOH-AcOH-H 2 O 4:1:5 upper layer).

Anti-HCV inhibition study
The day before infection, Huh-7 cells were plated in 96-well plates at a density of 6.000 cells/well. Huh-7 cells were infected with HCVcc at a multiplicity of infection of 0.7 in the presence of given concentrations of plant extracts or molecules. The inoculum was removed and cells were overlaid with fresh medium. After 28 h, infected cells were processed for immunofluorescent detection of E1 envelope glycoprotein as previously described by Rouillé et al., 2006 [18]. Quantity of cell/well and multiplicity of infection (MOI) were adjusted to obtain 20-40% of infected cells at 30 h post infection, allowing the automated quantification. The plates were analyzed with a High Content Screening (HCS) Operetta machine (Perkin Elmer) and the signals quantified with the Harmony software.

RESULTS AND DISCUSSION
The present investigation was focused on the evaluation of anti-HCV activity of P. chinensis methanol 80% extract and of flavonoids isolated from the extract. We investigated the presence of phytochemicals and bioactive constituents in P. chinensis aerial parts methanol extract. The major bioactive components of P. chinensis are gallic acid and eight flavonoids, apigenin, diosmetin, myricetin, The chemical structures of the bio-active components were elucidated by different spectroscopic analyses and shown in Figure 1.

Identification of the isolated compounds of P. chinensis methanol extract
Compound 1 (gallic acid) showed a single violet spot under short UV light and it gave black green colour with ferric chloride reagent confirming the presence of the phenolic gallic moiety [19] and confirmation of the chemical structure of compound 1 was proved by comparison of spectral data with spectra of Naira and Karvekar 2010 [20]. Compound 2 (apigenin) was obtained as a deep purple spot and the compound gave yellow colour when exposed to ammonia vapour and gave a bright yellow colour when spraying with AlCl 3 [21], spectral data of this compound is very close to spectra of Fatemeh et al. 2006 [22]. Compound 3 (Diosmetin, 4'methoxy luteolin) has appeared as a single deep purple spot when exposed to ammonia vapour, this compound gave yellow colour and also it gave a bright yellow colour when spraying with AlCl 3 (Mabry et al., 1970). The compound spectral data was very similar to spectra of Lunesa et al. 2011 [23]. Compound 4 (myricetin) was obtained as yellow green spot and gave a bright yellow colour when spraying with AlCl 3 [21] and its spectral data is identical to that of Chhagan et al. 2011 [24]. Compound 5 (apigenin 7-O-β-glucoside) is obtained as a deep purple spot and Journal of Applied Pharmacy (ISSN 19204159); www.japharmacy.ca the compound gave yellow colour when exposed to ammonia vapour and gave a bright yellow colour when spraying with AlCl 3 [21], acid hydrolysis of the compound gave apigenin as an aglycone and glucose as sugar moiety. Spectral data of this compound is very close to spectra of Ahmad et al. (2011 [ 25]. Compound 6 (quercetin 3-O-β-glucoside) is obtained as deep purple spot and the compound gave yellow colour when exposed to ammonia vapour and gave a bright yellow colour when spraying with AlCl 3 [21], acid hydrolysis of the compound gave quercetin as an aglycone and glucose as sugar moiety. Spectral data of this compound is very close to spectra of Ning et al. 2007 [26]. Compound 7 (myricetin 3-O-α-rhamnoside) and compound 8 (myricetin 3-O-β-glucuronide) both gave deep purple spot and both compounds gave yellow colour when exposed to ammonia vapour and also gave a bright yellow colour when spraying with AlCl 3 [21], acid hydrolysis of the two compounds gave myricetin as an aglycone and rhamnose and glucuronic acid as sugar moieties, respectively, and spectral data of the two compounds are very close to spectra of Rashed et al. 2012 [27]. Compound 9 (quercetin 3-O-β-glucoside-7-O-αrhmanoside) was isolated as yellow powder with a deep brown spot under UV light and this compound gave yellow orange colour when exposed to ammonia vapour and gave a bright yellow colour when spraying with AlCl 3 [21]. Complete acid hydrolysis of compound 9 yielded quercetin (Co-PC) as an aglycone and glucose and rhamnose as the sugar moieties. UV spectral data suggested that compound 9 is quercetin with substitution in positions 3 and 7 with free hydroxyl groups at 5, 3′, 4′ positions [21]. 1 H-NMR spectral data of compound 9 showed very similar proton signals to quercetin 3-O-β-glucoside-7-O-α-rhamnoside [28].

Anti-HCV activity of P. chinensis methanol extract and some isolated compounds
To determine the anti-HCV capacity of P. chinensis methanol extract, it was added at 25 µg/ml during inoculation of Huh-7 hepatoma cells with HCVcc and during the 28h post-inoculation. The number of infected cells was quantified at 30 h post infection. EGCG was used as a positive control, since it was recently identified as a potent inhibitor of HCV entry [29,30]. Our results show that P. chinensis extract at 25 µg/ml reduces HCV infection from 100% in the control to 70% (Figure 2A). Although this decrease was not statistically significant, it nonetheless suggested the presence of active compounds in the extract. Then, the purified compounds apigenin, diosmetin, myricetin 3-O-α-rhamnoside and myricetin 3-O-β-glucuronide were challenged for their anti-HCV capacities. compounds were added at 50 µM during all the infection process as described above. Apigenin and diosmetin reduced significantly HCV infection, whereas myricetin 3-O-α-rhamnoside and myricetin 3-O-β-glucuronide had no effect on HCV infection (Figure 2A). To further characterize the anti-HCV activity of apigenin and diosmetin, a dose-response study was performed ( Figure 2B). The half maximal inhibitory concentrations (IC 50 ) of the two compounds were calculated as IC 50 =39.9 µM for apigenin, and IC 50 = 42.5 µM for diosmetin. Because a number of dead cells were observed in the first experiment (data not shown), the cytotoxicity of these two compounds was also determined. The cellular toxicity experiments showed that aginenin is toxic for the cells at low concentration.
Only 66 % and 41% of the cells were viable after 72h treatment with 50 µM and 200 µM of apigenin, respectively ( Figure 2C). On the other hand, diosmetin was not toxic for Huh-7 cells at concentrations up to 100 µM, and had a limited effect at 200 µM ( Figure 2D). Taken together,