Antiviral activity and possible mode of action of ellagic acid identified in Lagerstroemia speciosa leaves toward human rhinoviruses

Background Human rhinoviruses (HRVs) are responsible for more than half of all cases of the common cold and cause billions of USD annually in medical visits and school and work absenteeism. An assessment was made of the cytotoxic and antiviral activities and possible mode of action of the tannin ellagic acid from the leaves of Lagerstroemia speciosa toward HeLa cells and three rhinoviruses, HRV-2, -3, and -4. Methods The antiviral property and mechanism of action of ellagic acid were evaluated using a sulforhodamine B assay and real-time reverse transcription-PCR (RT-PCR) with SYBR Green dye. Results were compared with those of the currently used broad-spectrum antiviral agent, ribavirin. Results As judged by 50% inhibitory concentration values, natural ellagic acid was 1.8, 2.3, and 2.2 times more toxic toward HRV-2 (38 μg/mL), HRV-3 (31 μg/mL), and HRV-4 (29 μg/mL) than ribavirin, respectively. The inhibition rate of preincubation with 50 μg/mL ellagic acid was 17%, whereas continuous presence of ellagic acid during infection led to a significant increase in the inhibition (70%). Treatment with 50 μg/mL ellagic acid considerably suppressed HRV-4 infection only when added just after the virus inoculation (0 h) (87% inhibition), but not before -1 h or after 1 h or later (<20% inhibition). These findings suggest that ellagic acid does not interact with the HRV-4 particles and may directly interact with the human cells in the early stage of HRV infections to protect the cells from the virus destruction. Furthermore, RT-PCR analysis revealed that 50 μg/mL ellagic acid strongly inhibited the RNA replication of HRV-4 in HeLa cells, suggesting that ellagic acid inhibits virus replication by targeting on cellular molecules, rather than virus molecules. Conclusions Global efforts to reduce the level of antibiotics justify further studies on L. speciosa leaf-derived materials containing ellagic acid as potential anti-HRV products or a lead molecule for the prevention or treatment of HRV infection.


Background
Human rhinoviruses (HRVs) (Picornaviridae) are the most frequent cause of mild upper respiratory tract infection, or common cold. They are responsible for more than half of all cases of the common cold [1,2]. HRVs are also associated with more severe diseases such as acute otitis media in children [3] and sinusitis in adults [4]. HRVs can also cause severe lower respiratory tract infections such as pneumonia [5], wheezing [6], bronchiolitis, and exacerbations of asthma and chronic obstructive pulmonary disease [2] in infants and children as well as fatal pneumonia in elderly and immunocompromised adults [7]. Although HRV-induced upper respiratory illness is often mild and self-limiting, the socioeconomic impact caused by medical visits and school and work absenteeism by HRV infection is considerable and the degree of inappropriate antibiotic use is significant [2,8,9]. More than 100 serotypes of HRV become an obstacle of development of a unifying vaccine [10]. There is, therefore, a high need for the development of selective antiviral agents with novel target sites to establish an effective HRV management strategy and tactics because there are currently no approved antiviral therapies for the prevention or treatment of HRV infection [2].
Plants have been suggested as alternative sources for antiviral products largely because they constitute a potential source of bioactive secondary substances that have been perceived by the general public as relatively safe, with minimal impacts to human health, and often act at multiple and novel target sites [11][12][13][14]. Much effort has been focused on plants and their constituents as potential sources of commercial antiviral products for prevention or treatment of HRV infection. In the screening of plants for anti-HRV activity, a methanol extract from the leaves of Giant Crapemyrtle (called banaba in the Phillippines), Lagerstroemia speciosa (L.) Pers. (Lythraceae), was shown to have good antiviral activity toward HRV-4. Very little information has been done to consider potential use of L. speciosa to manage HRV, although the plant leaves possess antidiabetic and hypoglycemic [15], antiobesity [16], antioxidant [17], antigout [18], antiinflammatory [19], and antibacterial activities [20].
The aim of the study was to assess the cytotoxic and antiviral effects on HeLa cells and three HRV serotypes (HRV-2, HRV-3, and HRV-4) of the tannin ellagic acid from L. speciosa leaves, compared to commercial pure ellagic acid and ribavirin, a currently used broadspectrum antiviral agent [21]. The antiviral property and mechanisms of action of the constituent were elucidated using sulforhodamine B (SRB) assay and real-time reverse transcription-PCR with SYBR Green dye.

Instrumental analysis
1 H and 13 C NMR spectra were recorded in DMSO-d 6 on a JNM-ECX 400 spectrometer (Jeol, Tokyo, Japan) at 400 and 100 MHz, respectively, using tetramethylsilane as an internal standard, and chemical shifts are given in δ (ppm). UV spectra were obtained in methanol on a BioMate 5 spectrophotometer (Thermo Spectronic, Rochester, NY), Fourier transform infrared (FT-IR) spectra on a Nicolet Magna 550 series II spectrometer (Midac, Atlanta, GA), and mass spectra on a Jeol GSX 400 spectrometer. Silica gel 60 (0.063-0.2 mm) (Merck, Darmstadt, Germany) was used for column chromatography. Merck precoated silica gel plates (Kieselgel 60 F 254 , 0.20 mm) were used for analytical thin-layer chromatography (TLC). A SCL-10 AVP high-performance liquid chromatograph (Shimadzu, Kyoto, Japan) was used for isolation of active principles.

Materials
Commercially available pure ellagic acid (≥95% purity) and SRB were purchased from Sigma-Aldrich (St. Louis, MO). The antiviral agent ribavirin was supplied by Tokyo Chemical Industry (Tokyo). Anitbiotic-antimycotic and minimum essential medium (MEM) were purchased from Invitrogen (Grand Island, NY). Fetal bovine serum was supplied by PAA Laboratories (Etobicoke, Ontario, Canada). All of the other chemicals and reagents used in this study were of analytical grade quality and available commercially.

Plants
Air-dried leaves of L. speciosa were purchased from a local Giant Crape-myrtle farm in the Philippines. A certified botanical taxonomist was used to identify the plant. A voucher specimen (LS-1 L) was deposited in the Research Institute of Agriculture and Life Sciences, College of Agriculture and Life Sciences, Seoul National University.

Bioassay-guided fractionation and isolation
Air-dried leaves (2 kg) of L. speciosa were pulverized, extracted with methanol (2 × 10 L) at room temperature for 2 days, and filtered. The combined filtrate was concentrated to dryness by rotary evaporation at 40°C to yield 112 g of a dark greenish powder. The extract (100 g) was sequentially partitioned into hexane-(9.35 g), ethyl acetate-(14.4 g), butanol-(39.35 g), and water-soluble (36.9 g) portions for subsequent bioassay. The organic solvent-soluble portions were concentrated under vacuum at 40°C and water-soluble portion was concentrated at 50°C. For isolation of active principles, viral CPE inhibition assay described previously [22,23] toward HRV-4 in HeLa cell was used.
The ethyl acetate-soluble fraction (10 g) was most biologically active (Table 1)  to give an active fraction (45 mg, R f = 0.12). For further separation of the constituents from the active fraction, a high-performance liquid chromatography was performed. The column was a 4 mm i.d. × 200 mm EC 200/4 Nucleodex alpha-PM (Macherey-Nagel, Easton, PA) using a mobile phase of methanol and water (9:1 by volume) at a flow rate of 1 mL/min. Chromatographic separations were monitored using a UV detector at 340 nm. Finally, an active principle (35 mg) was isolated at a retention time of 2.61 min. The isolate was obtained as green amorphous powder and identified by instrumental analyses, including MS and NMR. The mass spectrum exhibited a molecular ion at m/z 302 [M] + and IR absorption at 3380, 1720, and 1690-1610 cm −1 indicates the presence of phenolic hydroxyl, α-pyrone C = O, and benzonoid C = C groups, respectively. This compound (1) was thus identified as ellagic acid (2,3,7,8-tetrahydroxychromeno[5,4,3-cde] chromene-5,10-dione) ( Figure 2). The interpretations of proton and carbon signals were largely consistent with those of Nawwar and Souleman [24]. Ellagic acid was identified on the basis of the following evidence: green amorphous power. UV (MeOH) λ max nm: 255, 360. FT-IR: ν max cm −1 : 3380, 1720, 1690, 1610. EI-MS (70 eV)

Antiviral assay
The antiviral activity of L. speciosa leaf-derived materials toward three HRVs tested was evaluated by the SRB method using CPE reduction [22,23]. In brief, HeLa cells were seeded onto a 96-well microtiter plate at a concentration of 3 × 10 4 cells per well for 1 day. (OD tV -OD cV )/(OD cd -OD cV ) × 100, where OD tV is the optical density measured with a given concentration of the test material in HRV infected cells; OD cV is the optical density measured for the control untreated HRV infected cells; OD cd is the optical density measured for the control untreated HRV uninfected cells.

Cytotoxicity assay
HeLa cells were seeded onto a 96-well microtiter plate as stated previously. The culture medium was removed and the plates were replaced with media containing different concentrations of the test materials in DMSO, based on the preliminary test results. After incubation at 37°C with 5% CO 2 for 2 days, the cytotoxicity of the test materials to HeLa cells was evaluated using a SRB assay [22,23] as stated previously.

Infectivity of HRV particles
The effects of ellagic acid and ribavirin on the infectivity of HRV-4 particles were evaluated as described previously by Choi et al. [23]. Approximately twofold quantities of the IC 50 values of each test compound were applied. HRV-4 was preincubated with 50 μg/mL ellagic acid or 100 μg/mL ribavirin for 1 h at 4°C. Monolayers of HeLa cells were infected with the pretreated or untreated HRV-4 for 1 h at 37°C. Unbound virus was removed by washing the wells with 2 × PBS, and then cells were incubated in fresh medium supplemented with or without test compound at 37°C. After 2 days, SRB test and antiviral activity were carried out as stated previously.

Time course
The time-of-addition effects of ellagic acid were tested according to the method of Choi et al. [23]. In brief, monolayers of HeLa cells were seeded onto a 96-well microtiter plate as stated previously. After washing with 1 × PBS, 50 μg/mL ellagic acid were added onto the culture cells at before (−1 h), during (0 h), or after (1, 2, 4, and 6 h) HRV-4 infection at 37°C. Ribavirin served as a positive control and was similarly prepared. After 2 days, SRB test and antiviral activity were carried out as stated previously.

Reverse transcription-PCR analysis
To evaluate the level of gene expression, quantitative real-time reverse transcription-PCR (RT-PCR) with SYBR Green dye was performed. HRV-4 infected and noninfected cultures of HeLa cells grown in Corning 25 cm 2 cell culture flasks (Corning, NY) were treated

Data analysis
Cytotoxicity was expressed as 50% cytotoxic concentration (CC 50 ) of the compound that reduced the viability of cells to 50% of the control. Fifty percent inhibitory concentration (IC 50 ) was defined as the compound concentration required to reducing the viral CPE to 50% of the control. The CC 50 and IC 50 values were calculated using GraphPad Prism 5 software (GraphPad Software, La Jolla, CA). Therapeutic index was determined as the ratio of CC 50 to IC 50 . Results were expressed as mean ± SD of triplicate samples of three independent experiments. Statistical analyses were carried out using SAS 9.13 program (SAS Institute, Cary, NC). Data from two groups were analyzed by a Student's t-test, and multiple groups were analyzed by a one-way analysis of variance and Bonferroni multiple comparison test.

Effect on the infectivity of HRV particles
The effects of ellagic acid and ribavirin on the infectivity of HRV-4 particles were likewise examined ( Figure 3). The inhibition rates of preincubation with 50 μg/mL ellagic acid and 100 μg/mL ribavirin were 17 and 5.7%, respectively. Continuous presence of ellagic acid and ribavirin during infection led to a significant increase in the inhibition rate (70 and 65.7%).

Time course of compound addition
To investigate the mode of action of ellagic acid and ribavirin, time course of the inhibition of these compounds at different periods (before, during, and after) of HRV-4 infection was likewise investigated (Figure 4). Treatment with 50 μg/mL ellagic acid considerably suppressed HRV infection only when added just after the virus inoculation (0 h) (87% inhibition). The inhibition rate of ellagic acid declined to 20% or less when added at either prior (−1 h) or post (1, 2, 4, and 6 h) infection. However, the inhibitory effect of 100 μg/mL ribavirin on HRV-4 infection occurred between 0 and 6 h.

Effect on the level of HRV replication
The RNA replication level of HRV-4 in HeLa cells was remarkably inhibited in the cell cultures treated with 50 μg/mL ellagic acid ( Figure 5). The RNA replication level of HRV-4 was reduced by 5 fold, compared to the level in the cell cultures without ellagic acid.

Discussion
The current in vitro study indicates that materials derived from L. speciosa leaves exhibited antiviral activity and selectivity toward three rhinoviruses HRV-2, HRV-3, and HRV-4. The plant grows widely in tropical countries, including the Philippines, India, Malaysia, China, and Australia and is a popular folk medicine in Southeast Asia. L. speciosa leaves contain acetal, alkaloids, sterols, tannins, and triterpenoids [26,27]. Excellent antidiabetic properties of a standardized extract from L. speciosa leaves have been well noted [28,29]. Various compounds, including phenolics, terpenoids, and alkaloids, exist in plants, and jointly or independently they contribute to antiviral efficacy [14]. Many plants and their constituents manifest antiviral activity toward different viruses [14,30] and have been proposed as alternatives to conventional antiviral drugs. Anti-HRV  [39]), organic acid (e.g., raoulic acid, IC 50 0.51 μM [40]; gallic acid, IC 50 ≈ 294.55 μM [41]), and thiosulfinates (allicin and allyl methylthiosulfinate [42]). It has been reported that HRV capsid-binding compounds toward all HRV serotypes showed the existence of group A and B, based on a wide range of susceptibilities to antiviral compounds [43]. In the current study, the antiviral principle was determined to be the tannin ellagic acid. The constituent exhibited antiviral activity toward both group A (HRV-2) and group B (HRV-3 and HRV-4). IC 50 of ellagic acid was between 95.9 and 125.8 μM toward three HRVs, although IC 50 of the natural compounds stated previously is between 0.22 and 294.55 μM. Ellagic acid exhibited greater antiviral activity than ribavirin toward three HRVs and high selectivity. This original finding indicates that materials derived from L. speciosa leaves can hold promise for the development of novel and effective naturally occurring antiviral agents for two different HRV groups (A and B). In addition, ellagic acid was reported to possess anti-HIV activity, through inhibition of HIV protease [44]. Orobol 7-O-D-glucoside from L. speciosa leaves is also known to have broad-spectrum antiviral  activity toward various HRVs including group A and B, as well as pleconaril-resistant HRV-5 [39].
Investigations on the modes of antiviral action of naturally occurring compounds may contribute to the development of selective HRV therapeutic alternatives with novel target sites. The modes of anti-HRV action of plant secondary substances have been well reviewed by Rollinger and Schmidtke [45]. Targeting virus molecules is likely more specific and less toxic, but there is a narrow spectrum of viruses and a higher risk of creating resistant viruses [46]. On the contrary, chemicals which target cellular molecules may possess a broader antiviral activity spectrum and less risk of developing virus resistance, but may be more toxic to the host cell [46]. In the current study, ellagic acid does not interact with the HRV-4 particles, as preexposure of the virus to the constituent did not alter the infectivity of HRV-4 particles. Based on time-of-addition experiments, ellagic acid significantly suppressed HRV-4 infection only when added just after the virus inoculation (0 h), but not before −1 h or after 1 h or later. This finding suggests that ellagic acid may directly interact with the human cells in the early stage of HRV infections to protect the cells from the virus destruction. In addition, RT-PCR analysis revealed that ellagic acid strongly inhibited the RNA replication of HRV-4 in HeLa system, suggesting that ellagic acid inhibit virus replication by targeting on cellular molecules, rather than virus molecules. Detailed tests are needed to fully understand the anti-HRV mode of action of ellagic acid.

Conclusions
L. speciosa leaf-derived preparations containing ellagic acid could be useful as an antiviral agent in the prevention or treatment of HRV infection. The antiviral action of ellagic acid may be an indication of at least one of the pharmacological actions of L. speciosa. For the practical use of L. speciosa leaf-derived preparations as novel anti-HRV products to proceed, further research is needed to establish their human safety and whether this activity is exerted in vivo after consumption of L. speciosa leaf-derived products by humans. Historically, a tea from the plant leaves has been used for the treatment of diabetes mellitus in the Philippines [28]. Rats fed ellagic acid at doses as high as 50 mg/day up to 45 days did not cause any signs of systemic toxicity [47]. Lastly, detailed tests are needed to understand how to improve anti-HRV potency and stability for eventual commercial development.  Software. Each bar represents the mean ± SD of duplicate samples of three independent experiments. *** Significant at P < 0.001, according to a Student's t-test.