VIRAL IMMUNOLOGY Volume 28, Number 5, 2015 ª Mary Ann Liebert, Inc. Pp. 282–289 DOI: 10.1089/vim.2014.0079
Portulaca oleracea L. as a Prospective Candidate Inhibitor of Hepatitis C Virus NS3 Serine Protease Sobia Noreen,1 Ishtiaq Hussain,1 Muhammad Ilyas Tariq,1 Bushra Ijaz,2 Shahid Iqbal,1 Qamar-ul-Zaman,3 Usman Ali Ashfaq,4 and Tayyab Husnain 2
Abstract
Hepatitis C virus (HCV) infection is a worldwide health problem affecting about 300 million individuals. HCV causes chronic liver disease, liver cirrhosis, hepatocellular carcinoma, and death. Many side effects are associated with the current treatment options. Natural products that can be used as anti-HCV drugs are thus of considerable potential significance. NS3 serine protease (NS3-SP) is a target for the screening of antiviral activity against HCV. The present work explores plants with anti-HCV potential, isolating possible lead compounds. Ten plants, used for medicinal purposes against different infections in rural areas of Pakistan, were collected. The cellular toxicity effects of methanolic extracts of the plants on the viability of Huh-7 cells were studied through the Trypan blue dye exclusion method. Following this, the anti-HCV potential of phytoextracts was assessed by infecting liver cells with HCV-3a-infected serum inoculum. Only the methanolic extract of Portulaca oleracea L. (PO) exhibited more than 70% inhibition. Four fractions were obtained through bioassayguided extraction of PO. Subsequent inhibition of all organic extract fractions against NS3 serine protease was checked to track the specific target in the virus. The results showed that the PO methanolic crude and ethyl acetate extract specifically abridged the HCV NS3 protease expression in a dose-dependent fashion. Hence, PO extract and its constituents either alone or with interferon could offer a future option to treat chronic HCV.
Introduction
H
epatitis C virus (HCV) infection is an emerging global health problem over the last decade, found to be even more serious than human immunodeficiency virus infections. Presently, HCV affects approximately 3% of the global population and around 6% of the Pakistani population (2,18). HCV, a silent killer, is prone both to acute diseases with milder symptoms and to severe chronic liver diseases such as liver cirrhosis and hepatic cancer. After 10–15 years of infection, more than 50% of infected persons succumb to chronic disease (14). No anti-HCV vaccine has been developed to date because of the diversity of viral isolates. The present standard treatment is pegylated interferon (IFN)-a along with ribavirin and direct-acting antiviral (DAAs) products such as telaprevir and boceprevirare that are effective for a finite period based on the viral genotype (20,28). Pegylated IFN-a plus ribavirin can lead to a sustained virologic response in up to 80% of patients of genotypes 2 and 3, while in some cases, options are limited, especially for genotype 1 (3). A marked difference in response is also
observed in different races, with high rates prevailing among blacks. Elevated doses and widespread treatment options result in a transitory virologic reaction coupled with anemia, depression, fatigue, insomnia, fever, flu-like symptoms, hair loss, and cytopenia, among others. (3). Hence, there is a dire need for the development of novel, nontoxic agents in this era of HCV treatment. The genome of HCV is positive sense RNA strand with 9.6 kb nucleotides (25,26). In human hepatocytes, it encodes a precursor polyprotein (*3,000 amino acids) that is proteolytically processed into at least 10 distinct proteins by cellular and viral proteases. These comprise four structural core proteins, as well as envelope (C, E1, E2, and P7) and nonstructural (NS2, NS3, NS4A, NS4B, NS5A, and NS5B) proteins (24,31). All the NS proteins are indispensable for HCV infection or replication in chimpanzee models (15). NS3-4A serine protease and NS5B are the best targets for development of anti-HCV drugs; as both play key role in replication of HCV RNA. NS3 protein is a bifunctional protein with N-terminal serine protease and C-terminal ATPase. The NS3 protease belongs to
Departments of 1Chemistry and 3Pharmacy, University of Sargodha, Sargodha, Pakistan. 2 Center of Excellence in Molecular Biology (CEMB), University of the Punjab, Lahore, Pakistan. 4 Department of Bioinformatics and Biotechnology, Government College University Faisalabad, Faisalabad, Pakistan.
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the chymotrypsin family, and has the catalytically active triad of His-57, Ser-139, and Asp81 at the interface of two subdomains of b-barrel (5). It is smaller, and its structural stability depends on binding of a zinc atom. Its full expression requires the involvement of NS4A protein or at least a peptide of 14 amino acids in the form of N-terminal b-barrel (27). NS3-4A also weakens the host innate immune system by inhibiting the release of a host antiviral signaling factor. Intrahepatic INF-c production is also greatly reduced, and may weaken the hepatic inflammatory response and thus ensuing viral persistence (7). NS3-4A inhibition arrests the viral replication process and possibly restores repressed INF pathways. The use of herbs as medicine is a primitive tradition that is well known to all human civilizations since ancient times (4). Medicinal plants comprise of massive reservoir of constituents such as antioxidants, flavonoids, steroid, terpenoids, phenols, proteins, and so on, along with diverse chemical and biological potential against life-threatening diseases such as cancer, hepatitis, AIDS, malaria, and so on (35). Scientists have continually strived to examine precisely the active fragments of traditional medicines such as aspirin (analgesic), quinine (antimalarial), and taxol (anticancer), among others. These drugs have been obtained from the bark of white willow, Cinchona officinalis, and the yew plant, respectively (23,34). Antiviral properties of thousands of phyto-compounds have been recognized globally. The current decade is reflected by the dozens of plant bioactive compounds possessing anti-HCV activities especially targeting NS3 proteins. The extracts of potential herbs such as Acacia nilotica, Solanum nigrum, Boswellia carterii, Piper cubeba, Embelia schimperi, Syzygium aromaticum, Quercusin fectoria, and Trachyspermum ammi have been explored for their in vitro anti-HCV potential (19,22,32). Curcumin appears as a potential chemopreventive mediator against human hepatocellular carcinoma that inhibits expression of HCV replicon via PI3K/ Akt-SREBP-1 pathway (22). Studies exploring phytochemical compounds as HCV NS3 serine protease inhibitors are quite preliminary and limited in number (17). In order to explore the naturally occurring novel anti-HCV agents by deciphering their potential against NS3 protease of the HCV genome, the current study screened 10 plants from indigenous medicinal plants against HCV NS3 protease, and herein the bioassay-guided extraction of Portulaca olaracea L is described. Materials and Methods Sample collection
Based on folklore, 10 medicinal plants were collected from Soon Valley Punjab, Pakistan. Samples were identified and authenticated by a taxonomist, and voucher samples were submitted at the herbarium of University of Sargodha. Freshly collected plant samples were rinsed with tap water before washing with deionized water to remove any dust particles. Samples were dried under shade at ambient temperature for a couple of weeks until a constant weight was achieved. The dried samples were pulverized to a coarse powder with an electric grinder, and were then stored in polyethylene airtight bags at room temperature until further assessment.
283 Preparation of plant extracts
All the dried samples were weighed accurately and immersed in methanol (5:25 w/v) followed by incubation at 37C, the ideal temperature for enzymatic action. Resulting mixtures were filtered through Whatmann filter paper no. 42 (125 mm) and cotton wool. Filtrates were collected, and the residue was redissolved in fresh methanol. This process was repeated three times, and all the filtrates were combined and subjected to rotary evaporation to remove the solvent. Finally, the obtained semi-solid extracts were weighed to calculate the yield, and were stored in a desiccator. The fractions were stored in preweighed airtight glass vials at 4C in a refrigerator until further assessment. Preparation of stock solution
About 50 mg of each dried plant extract was individually dissolved in 1.00 mL of dimethyl sulfoxide (DMSO) resulting in 50 lg/lL of stock concentration. Solutions were filtered through a 0.22 lm filter and were stored at - 20C. Cell culturing
Huh-7 cells were cultured in Dulbecco’s modified Eagle’s medium along with 10% fetal bovine serum, 100 IU/mL penicillin, and 100 lg/mL streptomycin before being incubated at 37C along with a continuous supply of 5.00% CO2. Huh-7 cell line was a gift generously offered by Dr. Zafar Nawaz (Department of Biochemistry and Molecular Biology, University of Miami, FL). Cellular toxicity analysis using the Trypan blue dye exclusion method
Trypan blue dye was utilized to assess Huh-7 cell viability. Liver cells were seeded in six-well plates with density of 3 · 105/well for toxicological assessment of the plant extracts. Different concentrations of test samples were added to the six-well culture plates. After 24 h of incubation, the cells were trypsinized, and a suspension of these cells, along with Trypan blue dye, was prepared with a 1:1 ratio. The cell suspension (10 lL) was dispensed onto a glass slide, and the number of viable cells was calculated using a hemocytometer. Antiviral analysis of compounds in liver cells
Huh-7 cells (3 · 105 cells/well) were cultured in a 60 mm culture dish. Cells were washed twice with phosphate-buffered saline (PBS) after 24 h. The infection experiment was conducted as described previously by El Awady et al. (13) and Zekri et al. (37). Briefly, Huh-7 cells were grown to 60–70% confluence and washed twice with PBS. Then, 500 lL (1 · 105 IU/well) of HCV-3ainfected sera and 500 lL of serum-free media were added to the plated cells. Cells were incubated with HCVinfected serum for 24 h. The next day, adherent cells were washed twice with PBS, and cells were allowed to grow for 48 h. To analyze the effect of medicinal herbs, the infected Huh-7 cells (described above) were allowed to grow to semi-confluence (60–70%) in a six-well plate with 2.00 mL media. This was followed by adding the amount of the herb
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(1.5 lg/well) that was nontoxic for the cells, as described above. RNA was extracted from the cell lysate 24 h posttreatment of the medicinal herb using a Gentra RNA isolation kit according to the manufacturer’s protocol. Briefly, 100 lL of cell lysis solution containing 5.00 lL of internal control (IC) from Sacace Biotechnologies (Caserta, Italy) was added to the cells. The RNA pellet was dissolved thoroughly in 1.00% diethylpyrocarbonate-treated water. The controls used in the experiments were naı¨ve Huh-7 cells, cells with HCV serum infection alone, and DMSO without any compound addition. For viral quantification, a Sacace HCV quantitative analysis kit (Sacace Biotechnologies) was used. For viral RNA quantification, 10 lL of extracted total RNA from cell lysate was mixed with IC (described above) and quantified on a Cepheid PCR SmartCycler II system (Cepheid, Sunnyvale, CA). Antiviral analysis of Portulaca olaracea extract against HCV NS3 protease
Huh-7 cells were cultured at a density of 3 · 105 in sixwell plates for 24 h. After the removal of media, cells were washed thoroughly with PBS and transfected immediately with the expression vector pCR3.1/Flag/NS3-NS4A containing NS3 full-length gene (accession # HCV-PKHQ202536) and NS4A gene(accession # HCV-PKHM135518) kindly provided by the Applied and Functional Genomics Lab, CEMB Pakistan. The cells were co-transfected with pCR3.1/Flag/NS3-NS4A in the presence and absence of 1.5 lg Portulaca olaracea extract and INF along with Lipofectamine 2000 (Invitrogen Life Technologies, Carlsbad, CA) as per the company’s standard procedure. Total RNA was extracted 24 h post-transfection using Trizol reagent (Invitrogen Life Technologies) in accordance with the manufacturer’s protocol. cDNA synthesis was carried out with 1.00 lg RNA, using a Revert Aid H-minus First Strand cDNA Synthesis Kit (Fermentas, St. Leon-Rot, Germany) in order to determine the potential result of Portulaca olaracea against HCV NS3 gene. Relative expression analysis in control and PO-treated cells was performed using conventional PCR (Applied Biosystems, Inc., Foster City, CA). Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as internal control. First, HCV NS3 RNA expression was normalized with GAPDH expression within each condition, and then compared HCV NS3 gene RNA levels between conditions. Primers used for the amplification of HCV NS3 and GAPDH genes included NS3 forward primer: GGA CGA CGA TGA CAA GGA CT; NS3 reverse: CCT CGT GAC CAG GTA AAG GT; GAPDH forward: ACC ACA GTC CAT GCC ATC AC; and GAPDH reverse: TCC ACC ACC CTG TTG CTG TA. PCR analysis was performed at an initial denaturation temperature of 95C for 5 min followed by 40 cycles, each denaturation at 95C for 35 sec; annealing at 58C for 40 sec; and extension at 72C for 45 sec, with final extension at 72C for 10 min. The amplified DNA samples were examined on 2.00% agarose gel. The DNA bands were visualized under UV light, and images of gels were captured by a Gel Doc System. Relative quantification was also carried out using an ABI 7500 real-time PCR machine with SYBR Green Chemistry (Fermentas).
NOREEN ET AL. Bioassay-directed extraction and fractionation of Portulaca olaracea methanolic extracts
For bioassay-directed extraction, 150 g of each crude methanolic extract was immersed in deionized water and fractionated with hexane, ethyl acetate, chloroform, and nbutanol (150 mL · 3 each), respectively. Polar and nonpolar organic layers were concentrated in vacuum to secure four fractions (H, E, C, and B), which were first screened for their phyto-constituents using standard qualitative procedures (11,16) and then assayed for their HCV NS3-SP inhibition activities. Western blotting
The inhibitory effect of extracted Portulaca olaracea methanolic extraction protein expression level of NS3 transfected in Huh-7 cells was checked using Western blot. Total protein was extracted from the cell lysate using ProteoJET protein extraction reagent (Fermentas). Equal amounts of proteins extracted from Huh-7 cells transfected with NS3 vector, NS3 vector and DMSO, NS3 and IFN, NS3 and POE, NS3 and POM, in comparison to mock were subjected to 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The separated protein on the gel was transferred to nitrocellulose membrane (Amersham Pharmacia Biotech, San Francisco, CA). The nonspecific sites were blocked using 5.00% skimmed milk before treatment with specific antibodies. Blots were treated with primary monoclonal antibodies specific to HCV NS3 protein and GAPDH (Santa Cruz Biotechnology, Inc., Dallas, TX), which was used as internal control. Then blots were treated with secondary horseradish peroxidase-conjugated antigoat antimouse antibody (Sigma Aldrich, St. Louis, MO). The protein expression levels were checked using a chemiluminescent detection kit (Sigma Aldrich). The protein was quantified from X-ray images using freely available software Gel-quant-express (http://gel-quant-express.software.informer.com/4.1/). Statistical analysis and data presentation
Statistical analyses of experimental data (all in triplicate) was conducted and results were expressed as mean – standard deviation. One-way analysis of variance was also used, and results were considered significant at p < 0.05, and highly significant at < 0.01 or < 0.001. GraphPad Prism v5.00 for Windows (San Diego, CA) was used. Results
Based on cultural information, 10 medicinal plants were collected from the Soon Valley of Punjab, Pakistan, to evaluate their antiviral potential against NS3-SP of HCV. The botanical and taxonomic data of the plants are compiled in Table 1. Toxicological study of medicinal plants in liver cell line
Prior to antiviral screening against HCV, the cytotoxic effects of all the samples were determined on liver Huh-7 cells using Trypan blue exclusion method. After 24 h incubation of Huh-7 cells at different concentrations (1.5, 3.125, 6.25, 12.5, 25, 50, 100, and 200 lg/well) of extracts, cell viability assay was performed using Trypan blue dye, and
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Table 1. Botanicals Preferred for Anti-HCV Action and Their Methanolic Extract Percentage Yield Sr. no. 1 2 3 4 5 6 7 8 9 10
Botanical name Achyranthus aspera Bacop amonnieri
Common name
Family
Prickly chaff flower Brahmiboti
Local use
Amaranthaceae Diuretic, purgative, hepatoprotective, laxative, anti-allergic Plantagenaceae Ulcers, tumors, ascites, leprosy, enlarged spleen, inflammation, anemia, biliousness Chichorium intybus Kasni Compositae Gallstones, jaundice, hypertension Chlorophytum Safedmoosli Liliaceae Diabetes, sex tonic, preborivilianum and postnatal problems, arthritis Corchorus Bauphali, Tiliaceae Tracheal injury, antipyretic, depressus (Linn.) Munderi diabetes, laxative, gonorrhea, chronic cystitis Myristica Nutmeg, Jaiphal, Myristiceae Carminative, liver diseases, fragrans Houtt. Javitri respiratory ailments, tuberculosis Portulaca Purslane, Hurfa Portulacaceae Anticancer, boils, oleracea L. hemorrhoids, dysentery Sesamum indicum Sesame, Til Pedaliaceae Anticancer, laxative, emollient Sonchus arvensis Field milk thistle, Asteraceae Gallstone, gout hemorrhoids, Dodhak hypertension, wound healing Zizyphus jujube Chinese Rhamnaceae Antiulcer, sedative, immunostimulant, date, Unab anti-inflammatory
Part(s) Extract used % yield Leaves
12.65
Leaves
29.65
Leaves Roots
24.25 15.14
Leaves
8.77
Leaves
16.3
Leaves
19.3
Seed Leaves
31.2 24.9
Seeds
9.45
HCV, hepatitus C virus.
cells were counted using a hemocytometer. The concentration of Portulaca oleracea did not affect the viability of Huh-7 cells (Fig. 1). Similar results were observed for other nine extracts. Anti-HCV assay
It has been reported that HCV can replicate in Huh-7 cells by the detection of viral genes as well as viral copy number by real-time PCR in both cells and supernatant, whereas
FIG. 1. Cellular toxicity analysis using the Trypan blue exclusion method. Huh-7 cells were grown at density of 3 · 105 in six-well plates. After 24 h, cells were treated with different plant extracts (Table 1) over a range of concentrations. A representative graph with Portulaca oleracea L. (PO) extract is shown. After overnight incubation, survived cells were counted with a hemocytometer. For the treatment, mean (n = 3) – SD (n = 3) with a p-value of < 0.001 was found to be significant compared with the control (Ctrl.) Huh-7 cells without any treatment.
HCV replication in cell culture is strictly limited to human hepatocytes and their derivatives. In the present study, methanolic extracts from 10 different plants were tested to determine the antiviral activity against HCV. Before analyzing the antiviral activity of medicinal plants in infected Huh-7 cells, cell viability and proliferation was checked. To check the viability of cells post-HCV serum infection, the Trypan blue exclusion method was used, and cells were counted regularly for 3 days for the effect of HCV. Figure 2 shows that cell viability and proliferation increased post-HCV serum inoculum. These infected cells were sub-cultured, and
FIG. 2. Viability and proliferation of cells post-HCV infection. Huh-7 cells were inoculated with HCV serum (1 · 105/mL) for 24 h. The following day, HCV serum was changed with regular cell culture medium. The cells were allowed to grow for 3 days. The survived cells were counted with a hemocytometer, and the Trypan blue dye exclusion method was used. For the treatments, mean (n = 3) – SD (n = 3) with p-value of < 0.05 was found to be significant compared with the control. HCV, hepatitis C virus.
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larity. Significant inhibition (74%) in HCV genotype 3a viral titer was observed in the cells treated with ethyl acetate extract (Fig. 4). Inhibition of HCV NS3 gene expression by Portulaca oleracea
FIG. 3. Anti-HCV activity of 10 different plants. Huh-7 cells infected with HCV serum were cultured and incubated with 1.5 lg/well concentration of different plant extracts for 24 h. After incubation, total RNA was extracted, and the levels of HCV RNA were determined by quantitative realtime polymerase chain reaction (PCR) assay compared with the control (without treatment of plant extract). Achyranthus aspera (AAL), Bacop monnieri (BMB), Chichorium intybus (CIL), Chlorophytum borivilianum (CBR), Corchorus depressus (Linn.) (CDL), Myristica fragrans Houtt (MFHL), Portulaca oleracea L (PO), Sesamum indicum (SIS), Sonchus arvensis (SAL), Zizyphus jujuba (ZJB). Portulaca oleracea L (PO) shows a maximum inhibition of more than 70% compared with the control. All the treatments were done in triplicate (n = 3). Error bars indicate mean – SD. ***p < 0.001.
As only Portulaca oleracea significantly reduced viral RNA, only this plant was analyzed further. In order to determine the effect of Portulaca oleracea extract against HCV NS3 protease, Huh-7 cells were transfected with NS3 protease plasmid in the presence and absence of herbal extracts. After 24 h incubation, cells were harvested, RNA was extracted, and cDNA was generated by oligodT primers. cDNA was amplified by PCR using primers specific to the HCV NS3 protease. Figure 5A and B represents the qualitative and quantitative inhibition of HCV NS3 gene by Portulaca oleraceae extracts. The methanolic extract of Portulaca oleracea reduced the NS3 gene expression by 80%, whereas the ethyl acetate extract reduced the level by up to 85%. The effect of Portulaca oleracea on NS3 gene was also compared to IFN (101 U), and it is shown that
the effect of medicinal plants on viral RNA reduction was analyzed. Real-time PCR results showed that only methanolic extracts of Portulaca oleracea out of 10 medicinal plants exhibited more than 70% inhibition of HCV-RNA at nontoxic concentration (Fig. 3). Methanolic extract was further fractionated in different solvents on the basis of po-
FIG. 4. Anti-HCV activity of organic extracts of Portulaca oleracea. HCV infected Huh-7 cells were incubated with 1.5 lg/well concentration of PO methanolic (M) and PO ethyl acetate (E) extracts for 24 h. After incubation, total RNA was extracted, and the levels of HCV RNA were determined by quantitative real-time PCR assay, and are shown as percentages to the levels of HCV RNA in cells incubated without PO compound (control, Ctrl), and DMSO-treated cells. All the treatments were done in triplicate (n = 3), and findings were found to be statistically significant (***p £ 0.001).
FIG. 5. Antiviral activity of methanolic and ethyl acetate extracts of Portulaca oleracea against HCV NS3 protease at mRNA level. (A) Huh-7 cells were transfected with 0.5 lg of constructed HCV NS3 protease vector in the presence and absence of PO and interferon (INF; Roche) for 24 h. Cells were harvested, and relative RNA determinations were made using semi-quantitative real-time PCR. The results reflected that PO and INF inhibited HCV NS3 expression, while expression of GAPDH remained constant. Lane 1, NS3 gene alone; Lane 2, mock cells; Lane 3, IFN; Lane 4, POM; Lane 5, POE; Lane 6, DMSO. (B) Quantitative inhibition of HCV NS3 protease by methanolic and ethyl acetate extracts of Portulaca oleracea studied through real-time PCR assay by using SYBR green master mix. Statistically significant inhibitions (***p £ 0.001) in mRNA expression levels of HCV NS3-SP expression in POM-, POE-, and IFN-treated cells relative to control were determined experimentally (n = 3).
HCV NS3 PROTEASE INHIBITOR
FIG. 6. (A) Antiviral activity of methanolic and ethyl acetate extracts of Portulaca oleracea against HCV NS3 protease at protein level. Huh-7 cells were transiently transfected with NS3 gene of HCV and subsequently treated with PO methanolic (POM), ethyl acetate (POE) extracts, DMSO, and INF. Total protein was isolated 24 h post-transfection and subjected to SDS-PAGE, and Western blot. Results show that POM and POE significantly reduced the expression of NS3 gene compared with the control, DMSO-, and INF-treated cells. Lane 1, NS3 transfected cells as control; Lane 2, DMSO; Lane 3, IFN; Lane 4, POM; Lane 5, POE; Lane 6, mock. Blots were also normalized by measuring the expression of GAPDH used as internal control. (B) The quantitative expression of protein on blots was measured using freely available software Gel-quant-express. All experiments were performed in triplicate. Error bars indicate mean – SD; ***p £ 0.001. DMSO, dimethyl sulfoxide; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; SDS-PAGE, sodium dodecyl sulfate polyacrylamide gel electrophoresis. Portulaca oleracea is more active in the reduction of NS3 gene expression (Fig. 5A and B). GAPDH mRNA expression was used as internal control, which remained unaffected after the addition of the extract. Portulaca oleracea extract inhibitory effect against HCV was also checked at protein level using NS3 transfected Huh-7 cells. Dramatic reduction in the protein expression of NS3 gene was found after treatment of Portulaca oleracea extracts. There was 60% and 70% inhibition in Huh-7 cells treated with POM and POE respectively (Fig. 6). Discussion
The spread of a life-threatening disease such as HCV coupled with insufficient virus-targeted treatment, a dearth of vaccination, great side effects, and high cost of therapy prompted us to explore better-targeted approaches for the control of HCV. The present study was designed to screen medicinal plants for their therapeutic potential against nonstructural proteins of HCV. Medicinal plants have been used for the treatment of different diseases for centuries. Based on folklore and Eastern medicine, the exploration of plants from valleys and unexplored areas is the focus of research nowadays (1). The plant communities are losing
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their species richness at high rate due to intensive deforestation and unlimited expansion of urban areas. Through bioassay-directed screening, structure–activity relationship, and biochemical investigation, identification of bioactive compounds as potent molecular targets of HCVNS3 is hot topic, which may expand future anti-HCV regimens. Methanolic extracts of 10 plants (Table 1) and their fractions were scrutinized for their anti-HCV activities. After the toxicological analysis of plant extract (Fig. 1), the basic methanolic extract of Portulaca oleracea at 1.5 lg revealed antiviral action against expression of HCV-RNA through real-time PCR (Figs. 3 and 4). The viral load was reduced to almost 70% showing that Portulaca oleracea possessed appreciable antiviral activity. Moreover, the present finding is in agreement with an earlier report demonstrating 37% and 50% reduction in HCV titer by methanolic and chloroform extracts of Solanum nigrum seeds, respectively (19). Portulaca oleracea (a member of the Poaceae family) is a succulent, herbaceous plant growing in warm climates, having a cosmopolitan distribution. In Chinese folklore, Portulaca oleracea is recognized as a vegetable for long life, so it is extensively used in Chinese conventional herbal remedies. It has been frequently used as a vegetable in Asia, Oman, and the United Arab Emirates, and in traditional medicines around the globe. It has a profound role as a hepatoprotective, antigastric ulcer, diuretic, anticancer, antidiabetic, antimicrobial, and inflammatory agent (8,12,21). It is rich in vitamins (A, B, C, and E), minerals (mainly potassium reservoir), fatty acids (particularly omega-3 acids glutathione, glutamic acid, and aspartic acid), citric, malic acids, calcium oxalate, coumarins, dopamine, alkaloids, flavonoids, urea, and saponins (29). To gain more insight into its bioactive compounds, methanolic extract of Portulaca oleracea was partitioned into fractions through successive extraction with four solvents on polarity basis. Among these, the ethyl acetate fraction and crude methanolic extract exhibited antiviral activity specifically against NS3-SP (Fig. 5A and B). The extract of Portulaca oleracea not only inhibited the NS3 mRNA level, but there was also significant inhibition at the protein level (Fig. 6). This preliminary phytochemical screening demonstrates that hexane fractions are rich in nonpolar compounds such as steroids, for example, while ethyl acetate fractions sustain high concentration of flavonoids. The presence of these compounds provides a foundation for the identification of unknown therapeutic agents present in the extract through spectral data. This response is likely to be comparable to in vitro inhibition of HCV NS3-SP with ethanolic extract of Rhodiola kirilowii (Chinese herb) and Camellia sinensis Epigallocatechin-3-gallate compound (9,38). NS3-mediated processing of protein junctions among the nonstructural proteins is indispensible for HCV replication and hence recognized as a target for the development of antiviral agents (30). Moreover, NS3 may also interfere with host cell functions like protein kinase A arbitrated signal transduction and inhibition of cell transformation (6,33). Besides Portulaca oleracea, a number of studies have explored bioactive products from Galla Chinese, Rhodiola kirilowii, Lonicera hypoglauca Miq., Swietenia macrophylla, Solanum nigrum, and Camellia sinensis with antiNS3 protease activity (10,36,38). Previous reports have verified that Epicatechin and Epigallocatechin compounds
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from rhizomes of the Chinese therapeutic herb have revealed remarkable activity against NS3 serine protease of HCV (9). Novel antiviral activity of compounds from Portulaca oleracea presents an attractive lead for the development of potential anti-HCV agents. Additionally, if natural molecules could be used in combination or supplementation to INF, thus reducing the overall cost of treatment. The above data also suggest that the therapeutic importance of extracts may lead to an alternative, more potent oral treatment for chronic HCV.
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14. 15. 16. 17.
Acknowledgments
Financial support from the Higher Education Commission, Islamabad, Pakistan, under the Indigenous Fellowship Scheme is gratefully acknowledged. Author Disclosure Statement
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19.
No competing financial interests exist. 20. References
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Address correspondence to: Dr. Sobia Noreen Department of Chemistry University of Sargodha Sargodha 40100 Pakistan E-mail:
[email protected];
[email protected]
