Candidate plants in the present study were selected on the basis of following one or more criteria: i) Claimed efficacies in treating various liver diseases in folk or traditional medicine; ii) reported in vitro or in vivo hepatoprotective potentials; iii) published antiviral activities against genetically-close human viruses, such as human immunodeficiency virus (HIV) and herpes simplex virus (HSV); and iv) taxonomically related to plants known for their antiviral activities.

A total of 60 medicinal plants were collected from different regions of the Kingdom of Saudi Arabia (n=57) as well as Sudan (n=3) (Table I). Plants were identified by an experienced plant taxonomist at the College of Pharmacy, King Saud University, (Riyadh, Saudi Arabia) and voucher specimens (Table I) were deposited at the college herbarium.

Dried plant materials were ground to a coarse powder using a mortar and pestle, extracted with 80% ethanol for 3 days followed by filtering with Whatman Grade 1 paper and were concentrated using a rotary evaporator (Buchi Labortechnik AG, Flawil, Switzerland) under reduced pressure at 4°C. Plants extracts showing anti-HBV activity were further extracted sequentially with different organic solvents of increasing polarity: Hexane, ethyl acetate, dichloromethane, methanol (all from Merck, Darmstadt, Germany), including the aqueous phase. Briefly, 100 g of each plant powder was soaked in a suitable volume of hexane with intermittent shaking for 24 h, and filtered using Whatman Grade 1 paper. Each of the residues were further extracted twice with fresh solvent, and the filtrates were pooled together. The residue was air-dried followed by sequential extractions with dichloromethane, ethyl acetate, methanol and double-distilled water similar to the procedure performed for hexane. Finally, solvents were removed under reduced pressure at 4°C using a rotary evaporator (Buchi Labortechnik AG). Following complete drying, the yield percentage of each extract was calculated (Table II). For biological screening, each extract was dissolved in dimethyl sulfoxide (DMSO; Sigma-Aldrich, Merck KGaA), and the stocks (100 mg/ml) were stored at −20°C until subsequent use.

The HBV reporter cell line (HepG2.2.15) was a generous gift from Dr Shahid Jameel (International Center for Genetic Engineering & Biotechnology, New Delhi, India). HepG2.2.15 cells were grown in RPMI-1640 medium (Gibco; Thermo Fisher Scientific Inc., Waltham, MA, USA) supplemented with 10% heat-inactivated bovine serum (Gibco; Thermo Fisher Scientific, Inc.), 1X penicillin-streptomycin, and 1X sodium pyruvate streptomycin (HyClone; GE Healthcare Life Sciences, Logan, UT, USA) at 37°C in a humidified chamber (5% CO₂). Lamivudine (Sigma-Aldrich; Merck KGaA), the approved anti-HBV nucleoside analog-based drug was used as a standard.

Cytotoxicity of extracts was tested on HepG2.2.15 cells using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) cell proliferation assay kit (Tervigen, Gaithersburg, MD, USA) to determine the extract concentrations (doses) that did not affect cell viability, and were used in subsequent assays. MTT assay is based on the metabolic reduction of soluble MTT by mitochondrial enzyme activity of viable cells, into an insoluble colored formazan product which can be measured optically (14). Cells were seeded (0.5×10⁵ cells/100 µl/well) in flat-bottom 96-well tissue culture plates (Corning Inc., Corning, NY, USA). Following 24 h of incubation, the cells were treated (in triplicate) with various concentrations of test samples (0, 6.25, 12.5, 25, 50 and 100 µg/ml) prepared in culture media, and incubated at 37°C for 48 h. The final concentration of DMSO never exceeded 0.1% in any of the assays and therefore, had no signs of toxicity. Blank (only media) and untreated/negative (0.1% DMSO in media) controls were also included. Cells were treated with MTT reagent (10 µl/well) and further incubated at 37°C for 3–4 h. Upon appearance of a purple color, the detergent solution (100 µl) from the kit was added to each well and further incubated at 37°C for 1 h. Optical density (OD) was recorded at 570 nm using a microplate reader (ELx800; BioTek Instruments, Inc., Winooski, VT, USA). Non-linear regression analysis was performed using Excel software (Microsoft Corp., Redmond, WA, USA) to determine the concentration that resulted in a 50% cytotoxicity concentration (CC₅₀) using the following equation:

Where OD(s), OD(b) and OD(c) are the absorbances of the sample, blank and negative control, respectively.

At 24 and 48 h post-treatment, cells were visually monitored for morphological changes, such as lesions of the cell membrane and the compactness of cytoplasmic components under an inverted microscope (Bio Optical, Milan, Italy) at a magnification of ×200.

HepG2.2.15 cells were seeded in 96-well plates (0.5×10⁵/well) and incubated at 37°C. The following day, the culture media was replaced with fresh media (150 µl, each in triplicate) containing various doses (0, 6.25, 12.5, 25 and 50 µg/ml) of the test samples and controls, and incubated at 37°C for 48 h. The culture supernatants of each sample were collected and stored at −20°C. The secreted HBsAg in the culture supernatants was analyzed using an ELISA kit (cat. no. 72348; Monolisa HBsAg ULTRA; Bio-Rad Laboratories Inc., Hercules, CA, USA) according to the manufacturer's instructions. OD was recorded using an ELx800 microplate reader and analyzed according to the manufacturer's instructions. Non-linear regression analysis was performed using Excel software to determine the half maximal (50%) inhibitory concentration (IC₅₀) of HBsAg secretion.

Based on the dose-dependent inhibition results, time-course (day 1, 3 and 5) analyses were performed to further investigate the antiviral potential of the most active extracts. The HBsAg expression study was performed by treating cells with the safest single-dose (50 µg/ml), as determined by the IC₅₀ values.

Extracts exhibiting the most promising inhibitory effects on HBsAg secretions were further subjected to time-course (day 1, 3 and 5) analyses of HBeAg expression at a dose of 50 µg/ml. ELISA was performed on the culture media using an HBeAg/anti-HBe ELISA kit (cat. no. KAPG4BNE3; DIAsource ImmunoAssays; SA, Louvain-la-Neuve, Belgium) according to the manufacturer's instructions. OD was recorded using an ELx800 microplate reader, and analyzed following the DIASource manual.

Plants exhibiting the most promising anti-HBV activities were subjected to qualitative phytochemical screening for major secondary metabolites, including alkaloids, flavonoids, anthraquinones, tannins and saponins, following standard procedures (15–17) with minor modifications. Briefly, for alkaloids, the Mayer's test was performed. A total of 0.5 gm of the extract was dissolved in 2% hydrochloric acid (Sigma-Aldrich; Merck KGaA), and filtered. Mayer's reagent was freshly prepared by dissolving 0.68 g of mercuric chloride (Sigma-Aldrich; Merck KGaA) and 2.5 g of potassium iodide (Sigma-Aldrich; Merck KGaA) and made to 50 ml with distilled water. A few drops of the reagent were added to the 3 ml of extract solution in a test tube where formation of a yellowish precipitate indicated the presence of alkaloids. For flavonoids, the sodium hydroxide test was performed. A total of 5 ml of the extract solution was treated with few drops of 20% sodium hydroxide (Sigma-Aldrich; Merck KGaA) in a test tube. Formation of an intense yellow color, which becomes colorless on addition of diluted hydrochloric acid, indicated the presence of flavonoids. For tannins, the ferric chloride test was performed. A total of 0.25 gm of the extract was dissolved in 10 ml of distilled water in a test tube and few drops of 5% ferric chloride (Sigma-Aldrich; Merck KGaA) was added. Appearance of brownish green or blueish-black color indicated the presence of tannins. For saponins, the frothing test was performed. A total of 0.5 gm of the extract was dissolved in 10 ml of distilled water in a test tube and shaken vigorously. Formation of a thick persistent froth that persisted for at least 15 min indicated the presence of saponins. And lastly, for anthraquinone the Borntrager's test was performed. A total 0.5 gm of the extract residue was boiled with 5 ml of dilute hydrochloric acid and filtered while hot. The filtrate was combined with 5 ml of chloroform and shaken. The chloroform layer was transferred into a test tube and 2 ml of dilute ammonia solution (Sigma-Aldrich; Merck KGaA) was added. The appearance of a rose-pink to cherry-red color indicated the presence of anthraquinones.

CC₅₀ values of different plants extracts were calculated (Table II), which allowed for the determination of the single optimal dose (50 µg/ml) with no sign of cytotoxicity. This observation was confirmed by microscopic observation of the cell morphology/growth at 24 and 48 h post-treatment with different concentrations of each extract (data not shown). Therefore, extracts at 50 µg/ml were used in the subsequent antiviral assays.

Firstly, the total ethanolic-extracts of 60 medicinal plants were screened for anti-HBV activity by measuring the expression levels of viral HBsAg at 48 h. Of these, 9 plants showed inhibition of HBsAg production in a dose-dependent manner. These were Abutilon figarianum, Acacia oerfota, Capparis decidua, Coccinea grandis, Corallocarpus epigeus, Fumaria parviflora, Guiera senegalensis, Indigofera caerulea and Pulicaria crispa with IC₅₀ values of 106.46, 101.46, 76.85, 31.57, 71.90, 79.84, 73.21, 84.62 and 23.10 µg/ml, respectively (Table II). Based on these results, 5 sequential extracts (hexane, dichloromethane, ethyl acetate, methanol and aqueous) of each of the 9 selected plants were prepared, and tested for cytotoxicity. The sequential extracts were further evaluated for dose-dependent HBsAg inhibition. The extraction yield percentage, IC₅₀, CC₅₀ and their corresponding TI values were calculated (Table II). Of these, 24 different extracts (from the 9 selected plants) that showed marked HBsAg inhibition were evaluated in a time-course study.

The selected extracts (of 9 plants) that showed marked HBsAg inhibition were evaluated in a time-course study, using 50 µg/ml doses for 5 days (Fig. 1). While prolonged treatment beyond day 5 did not show any notable inhibitory effect, further continuation of the culture resulted in cell overgrowth and death (data not shown). The optimal antiviral activities, in order, on day 5 post-treatment were: G. senegalensis (dichloromethane extract; IC₅₀=10.65), P. crispa (ethyl acetate extract; IC₅₀=14.45), C. gardis (total ethanol extract; IC₅₀=31.57), F. parviflora (hexane extract; IC₅₀=35.44), C. decidua (aqueous extract; IC₅₀=66.82), C. epigeus (total ethanol extract; IC₅₀=71.9), I. caerulea (methanol extract; IC₅₀=73.21), A. figarianum (dichloromethane extract; IC₅₀=99.76) and A. oerfota (total ethanol extract; IC₅₀=101.46) (Table II).

The HBeAg is a processed product of the ‘pre-Core’ gene that is co-translated with the ‘Core’ gene by bicistronic subgenomic-RNA. Therefore, in a natural infection, seropositivity of HBeAg is a hallmark of active viral DNA replication. Notably, this is analogous to the HIV ‘p24’ antigen where ELISA is a valid tool to monitor retroviral RNA replication. Therefore, the most promising active extracts that greatly suppressed HBsAg synthesis (Fig. 1), were analyzed for time-course effect on HBeAg production in the culture supernatants. While HBeAg secretion was inhibited maximally at day 3, there were no further improvements in antiviral activities at day 5 (Fig. 2). Limited by cell overgrowth and death, and unaffected virus replication (inhibition of HBeAg), the study was terminated at day 5. Furthermore, the antiviral activities on downregulating virus replication, as measured by HBeAg production, were: G. senegalensis (dichloromethane extract; 66%); I. caerulea (methanol extract; 58.60%); P. crispa (ethyl acetate extract; 55.30%); F. parviflora (hexane-extract; 54.68%); C. decidua (aqueous extract; 51.52%); C. epigeus (total ethanol extract; 43.31%); C. grandis (total ethanol extract; 35.51%); A. oerfota (total ethanol extract; 35.28%); and A. figarianum (dichloro methane extract; 27.56%) compared to the untreated control (Fig. 2). It is noteworthy that, with the exception of C. grandis and A. figarianum, all plant extracts (50 µg/ml) showed greater antiviral activity than lamivudine (2 µM).

Plants that exhibited anti-HBV activity showed the presence of alkaloids, flavonoids, tannins, saponins and anthraquinones (Table III), which have been previously reported for their antiviral activities (7,8).