anti adenovirus properties allicin by Kirk Andrew

Journal of the Taiwan Institute of Chemical Engineers 42 (2011) 228–232

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In vitro anti-adenoviral activity of ﬁve Allium plants Chien-Hsien Chen, Tzu-Wei Chou, Li-Hui Cheng, Chin-Wen Ho * Department of Bioengineering, Tatung University, Taipei 104, Taiwan

A R T I C L E I N F O

A B S T R A C T

Article history: Received 27 April 2010 Received in revised form 21 June 2010 Accepted 5 July 2010

This study aimed to evaluate the antiviral activity of extract of five Allium plants (shallots, garlic, onions, leeks, and green onions) and pure compounds of quercetin, zalcitabine (ddC), allicin, and ribavirin against adenovirus. With regard to the antiviral activity of ddC, both the MTT and PRD (Plaque reduction) methods showed a high correlation (R2 = 0.8952) with no significant difference (paired t-test, p > 0.05). Most of the Allium plants tested were non-toxic to human lung carcinoma (A549) cells, and shallots exhibited the highest level of antiviral activity for both ADV41 and ADV3, followed by garlic and onions. Shallots exhibited the highest level of antiviral activity against ADV3 and ADV41 infection from 0 to 2 h, during the early period of virus replication. MTT assay with A549 cells proved to be a rapid and sensitive assay system for screening anti-adenoviral drugs. The potential of shallots for use in treating adenoviral infection merits further studies. To our knowledge, this is the first report to describe the anti-adenoviral activities of the Allium plant. ß 2010 Taiwan Institute of Chemical Engineers. Published by Elsevier B.V. All rights reserved.

Keywords: In vitro Anti-adenoviral activity Allium plant Quercetin

1. Introduction Adenoviruses cause a variety of human diseases, including respiratory illness, gastroenteritis and related syndromes, and neurologic disease (Cherry, 2004; Gray et al., 1999; Lin et al., 2007). In particular, adenovirus type 3 (ADV3) and adenovirus type 41 (ADV41) have been indicated as etiological agents of respiratory and gastrointestinal infections in infants and young children (Haramoto et al., 2007; Slatter et al., 2005). Chang et al. (2008) also reported that between 1981 and 2005, except 2001, ADV3 was the predominant serotype responsible for the outbreak of respiratory adenovirus infections in children in Taiwan. Furthermore, multiple and often genetically divergent adenovirus serotypes can cause similar diseases (Kinchington et al., 2005; Lenaerts et al., 2008). Treatment for adenoviral infection is currently limited to symptomatic therapy, and there is no effective treatment for adenoviral disease (Kinchington et al., 2005; Lenaerts et al., 2008). Moreover, there is currently no licensed systemic or topical treatment available (Kinchington et al., 2005; Lenaerts et al., 2008). Thus, it is necessary to develop an optimal antiviral agent having broad serotype speciﬁcity to treat infections caused by adenoviruses. Medicinal plants have been widely used in Asian countries like China, India, Japan, and Taiwan for many years (Clardy and Walsh, 2004; Mukhtar et al., 2008). Hence, in these regions, natural

* Corresponding author. Tel.: +886 2 25925252; fax: +886 2 25854735. E-mail address: cwho@ttu.edu.tw (C.-W. Ho).

products are still a major source of antiviral therapeutic agents for treating infectious diseases (Clardy and Walsh, 2004; Mukhtar et al., 2008). Therefore, it is useful to search for new antiviral agents from natural sources (Clardy and Walsh, 2004; Mukhtar et al., 2008). Members of the Allium species have many traditional dietary and medicinal applications as anti-infective agents (Lanzotti, 2006). For example, garlic (Allium sativum L.) and onions (Allium cepa L.) have been used for a long time as foods and for the treatment of many diseases (Lanzotti, 2006). Other Allium species such as leeks (Allium porrum L.) and shallots (Allium ascalonicum L.) are also rich sources of biological volatile and nonvolatile compounds, especially quercetin (Block et al., 1992; Fattorusso et al., 2002). These compounds (allicin, diallyl trisulfide, and ajoene) derived from garlic extract have been shown to have antiviral activity (Harris et al., 2001). Previous studies have also shown that onions are characterized by the highest total flavonoid content (including quercetin and its conjugates) among 62 vegetables investigated (Capasso et al., 2003; Miean and Mohamed, 2001). Furthermore, many studies have demonstrated the antiviral activity of quercetin (Cai et al., 2006; Lyu et al., 2005; Tait et al., 2006). As mentioned above, the antiviral activity of fresh and freeze-dried Allium extracts supports the study of these extracts for anti-adenoviral activity. This study aimed to (1) establish a sensitive and accurate method for screening anti-adenovirus agents and (2) to evaluate the antiviral activity of the aqueous extracts of five Allium species (shallots, garlic, onions, leeks and green onions) and pure compounds of quercetin, zalcitabine (ddC), allicin, and ribavirin.

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2. Materials and methods 2.1. Cells and viruses Human lung carcinoma cell line A549 was used to evaluate the cytotoxicity of shallot extracts and as a target for virus infection in the MTT assay. The A549 cell lines were grown as adherent cells in DMEM supplemented with 10% (v/v) foetal bovine serum (FBS), 100 units/ml penicillin G, 100 mg/ml streptomycin, and 0.25 mg/ ml amphotericin B. In the antiviral and cytotoxicity assays, the medium was supplemented with 1% FBS and the abovementioned antibiotics. Clinical stool isolate of adenovirus ADV41 (strain HME460) used in this study was obtained from Taipei City Hospital (Zhongxiao Branch). The ADV3 strain (strain HME-351) was isolated from seawater in Taiwan. Both ADV strains were classified as ADV41 and ADV3 serotype by molecular typing. All ADV strains were propagated in A549 cells. Virus titres were determined by cytopathic effects in A549 cells and were expressed as 50% tissue culture infective dose (TCID50) per ml. All viruses were stored at 70 8C. 2.2. Test sample preparation and compounds All Allium vegetables (shallots (A. ascalonicum L.), leeks (A. porrum L.), garlic (A. sativum L.), green onions (Allium fistulosum L.), onions (A. cepa L.), were purchased fresh from Han-Kuang fruits and vegetables Cooperative, Yun-Lin, Taiwan. Allium plant samples were cleaned with distilled water, air-dried at 22 8C in darkness, frozen at 80 8C for 24 h, and were then vacuum-dried (Cheng, 2006). All lyophilized powder samples were stored at 20 8C. The lyophilized powder was dissolved in a 0.5% dimethyl sulfoxide (DMSO; Kanto Chemical Co., Tokyo, Japan) solution at 5 104– 5 106 mg/ml. Pure quercetin, allicin (Wako Chemicals Ltd., Osaka, Japan), ribavirin, and 2,3-dideoxycytidine (ddc) (Sigma Chemical Co., St. louis, USA) were also suspended in the DMSO solution. The reagents for cell culture were obtained from Hyclone Co. (Utah, USA). The MTT formazan reagent was purchased from MP Biomedicals, Inc. (Illkirch, France), and a 0.5 mg/ml stock solution was prepared in sterilized phosphate-buffered saline (PBS). 2.3. Cytotoxicity Cell viability was determined by Chiang’s method (Chiang et al., 2002). Briefly, the A549 cells were cultured overnight in 24-well tissue culture plates (Nunc, Roskilde, Denmark) at a concentration of 5 104 cells/ml and a volume of 1 ml per well. Various concentrations (80–3500 mg/ml) of lyophilized powder of each vegetable or isolated compound were applied to culture wells in triplicate and incubated at 37 8C in a humidified 5% CO2 atmosphere. Three days later, the culture medium was removed carefully without disturbing the attached cells, and each sample was assayed by the MTT formazan method (Plumb et al., 1989). The concentration of test substances with 50% cellular cytotoxicity (CC50) was calculated according to Chiang et al. (2002). The negative control was sterilized PBS. The morphology of the cells was examined daily and observed microscopically for any detectable change.

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5% CO2 incubator for 1 h under 37 8C and shake plates every 15 min. Then, to add overlay medium (DMEM medium containing 1% FBS (fetal bovine serum) and 0.3% agarose gel) to each well, and then to incubate in 37 8C, 5% CO2 incubator and to observe everyday for 4 days. After incubation, to fix the cells with 10% formalin for 2 h. Carefully remove the overlay medium and formalin solution, stain the cell sheets by 0.1% crystal violet solution. Pour off the stain by washing steadily with tap water and standing until air dry. Finally, to count the plaque number for each different dilution under light microscopy. The antiviral activity of a crude aqueous extract and pure compound against ADV41 and ADV3 viruses was evaluated by Chiang’s MTT method (Chiang et al., 2002). Briefly, 5 104 cells/ml per well treated with trypsin, infected or uninfected adenovirus cells were seeded in 24-well flat-bottomed microtitre plates. After incubation overnight at 37 8C with 5% CO2, cell monolayers were infected with viruses (adenovirus type 3 and type 41) at 24 TCID50 per well at room temperature and incubated for another 2 h. Different concentrations (80–3500 mg/ml) of test substances were then added to culture wells in triplicate. The maximum concentration of DMSO (0.1%) was used as a negative control for the ADV assays. The MTT assay was carried out after 3 days of incubation at 37 8C with 5% CO2. The antiviral concentration of 50% effectiveness (EC50) was defined as the concentration that achieved a 50% inhibition of virus replication, which was calculated as (Atv Acv)/(Acd Acv) 100%. Atv is the absorbance of the test compounds with virus-infected cells; Acv and Acd are the absorbance of the virus and cell control, respectively. 2.5. Dose response At first, A549 cells were cultured overnight in 24-well plates, at a concentration of 5 104 cells/ml and a volume of 1 ml per well. ADV41 and ADV3 (24 TCID50 per well) were absorbed onto confluent monolayers of A549 cells for 2 h. Finally, various concentrations (120–1500 mg/ml) of lyophilized shallot powder were added in triplicate. The MTT test and antiviral activity measurements were carried out after 3 days. 2.6. Time course A549 cells were cultured overnight, and various concentrations of lyophilized shallot powder (120–960 mg/ml) were added at various time intervals—pre-infection ( 1 h), co-infection (0 h), or post-infection (1 h, 2 h, 4 h, 6 h, 8 h, 12 h, or 24 h)—with adenovirus (ADV3 and ADV41) to A549 cells at 37 8C. The MTT test and antiviral activity measurements were carried out after 3 days. 2.7. Statistical analysis All values were expressed as the standard error of the mean (SEM). Antiviral activities were estimated with selectivity indices (SIs) calculated from CC50 and EC50 values. The correlation of different dose effects of lyophilized shallot powder on the inhibition of ADV3 or ADV41 was evaluated by linear regression. Differences with p 0.05 were considered to be statistically significant.

2.4. Antiviral assay 3. Results The antiviral activity of ddC was determined using a standard plaque reduction assay (Cooper, 1967). Brifely, add 24 TCID50 per well adenovirus concentration and two-fold serial dilutions of ddC into conﬂuent cell monolayers in a 6-well plate in triplicate, simultaneously. Both the virus suspension and medium were served as the virus control and cell control. Incubate the plates in

3.1. Compare the activity of 2,3-dideoxycytidine (ddc) determined by MTT method with plaque reduction assay To determine whether the MTT assay using A549 cells is practically useful and reliable for the screening of anti-ADV

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[()TD$FIG]

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Table 1 Assessment of antiviral activity of Allium plant extract and other chemical compound. Test compounds

ADV41 SIa (CC50b/EC50c)

ADV3 SI (CC50/EC50)

Shallot Garlic Onion Green onion Leek Quercetin Allicin Ribavirin 2,3-Dideoxycytidine

3.7 (2696.8/733.9) (2649.6/>3500) (>960/>960) (>960/>960) (>960/>960) 12.6 (417.0/33.0) (0.7/–d) (119.5/–d) >423.7(>1000/2.36)

2.4 (2696.8/1137.6) (2649.6/>3500) (>960/>960) (>960/>960) (>960/>960) 12.4 (417.0/33.6) ND ND ND

Selectivity index (SI) = CC50/EC50. The concentration (mg/ml) of 50% cellular cytotoxicity (CC50) of test substances was calculated according to Chiang et al. (2002, 2003). c The concentration (mg/ml) of 50% antiviral effectiveness (EC50) was calculated according to Chiang et al. (2002, 2003). d Maximum concentration of compound to test did not ﬁnd EC50. b

compounds, ddc was evaluated for its inhibitory effect on the cytopathogenicity of the ADV41 strain. In this study, ddC showed the highest and most selective anti-ADV activity (Table 1) and inhibited replication in the conventional plaque reduction assay (PRD). The anti-ADV activity was conﬁrmed by the MTT method using A549 cells (Table 1). The inhibitory effect (EC50) of ddC in both methods was found to be in a similar range of 2.384– 2.360 mg/ml. Both the MTT and PRD methods showed a high correlation (R2 = 0.8952), with no signiﬁcant difference in the paired t-test (p > 0.05). Therefore, all of the other antiviral tests samples in this study were all determined by the MTT method.

Fig. 1. Inhibitory effect of adding shallot at various times pre-infection or postinfection of adenovirus (ADV-41) to A549 cells. Different concentrations of shallot [120 mg/L (open triangles), 240 mg/L (ﬁlled triangle), 480 mg/L (open circles), 960 mg/L (ﬁlled circles)], were added at various times pre-infection ( 1 h), coinfection (0 h) or post-infection (1–24 h) of adenovirus (ADV-41) to A549 cells at 37 8C. The x-axis indicates the time course of adding shallot. Each point represents the mean S.E.M. of triplicate samples of three independent experiments.

[()TD$FIG]

3.2. Assessment of anti-adenoviral activity In this study, except shallots, most of the Allium plants tested were non-toxic to human lung carcinoma (A549) cells but showed antiviral activity near cytotoxic concentrations (Table 1). Shallots showed the highest level of antiviral activity for both ADV41 (3.8– 57.83%) and ADV3 (5.47–46.67%) with a range of 80–960 mg/ml, followed by garlic (3.17–20% and 8.2–16.44%, respectively), and onions (1.2–15.7% and 0–13.64%, respectively). However, green onions and leeks showed a maximum antiviral activity of 2% for both adenoviruses. With regard to the other compounds, ddC showed the strongest activity against ADV41 (SI > 423), and quercetin showed similar activity against both ADV41 and ADV3 (SI = 12.4–12.6). No antiviral activity against ADV41 and ADV3 was noted for allicin and ribavirin (Table 1). 3.3. Dose effect of shallot extract on adenoviruses A strong correlation between drug concentration and antiviral activity was observed for shallot powder at concentrations between 80 and 1500 mg/ml (the correlation coefﬁcient (r) was 0.96 for ADV3 and 0.95 for ADV41).

Fig. 2. Inhibitory effect of adding shallot at various times pre-infection or postinfection of adenovirus (ADV-3) to A549 cells. Different concentrations of shallot [120 mg/L (filled squares), 240 mg/L (open triangles), 480 mg/L (filled triangle), 960 mg/L (open circles), 1500 mg/L (filled circles)], were added at various times pre-infection ( 1 h), co-infection (0 h) or post-infection (1–24 h) of adenovirus(ADV-3) to A549 cells at 37 8C. The x-axis indicates the time course of adding shallot. Each point represents the mean S.E.M. of triplicate samples of three independent experiments.

4. Discussion 3.4. Time-course effect for shallot extract To investigate the mechanism underlying the inhibition of adenoviral infection by shallot extracts, we investigated the timecourse effect at 1 h before and at 24 h after infection with the adenoviruses and in contact with various concentrations of shallot extract (120–960 mg/ml). The results showed that shallots exhibited the highest level of antiviral activity on both ADV3 and ADV41 infections from 0 to 2 h, which was during the early period of virus replication (Figs. 1 and 2).

In this study, we selected ﬁve medicinal plant extracts derived from plant species of the family Alliaceae, including shallots (A. ascalonicum L.), leeks (A. porrum L.), garlic (A. sativum L.), green onions (A. ﬁstulosum L.), and onions (A. cepa L.) to determine their potential for use as anti-adenoviral agents for the treatment of adenovirus infections. The shallot powder showed the most potent and selective anti-ADV activity among the extracts tested. Extracts of garlic and onions showed little activity against ADV. Extracts of green onions and leeks were completely ineffective against ADV

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replication. Previous studies have also reported that dried and autoclaved shallot extracts showed more antibacterial and antifungal activity than extracts of onion and garlic (Amin and Kapadnis, 2005). Among the Allium vegetables, shallots contain the highest concentrations of quercetin and allicin compounds (Cheng, 2006; Fattorusso et al., 2002). It was reported that quercetin and its conjugates are found in rich supply in Allium plants (Hollman and Arts, 2000). Some naturally occurring quercetin compounds were shown to possess antibacterial or antiviral activity (Cheng, 2006; Chiang et al., 2003; Mahmood et al., 1996). Furthermore, the correlation coefficient (r) between quercetin content and antiviral activity for both adenovirus types was >0.95 (p < 0.05). Studies have also proven that quercetin has a similar mode of action against ADV-3 and ADV41 (Chiang et al., 2002). We could infer that the lyophilized shallot powder containing quercetin possesses anti-adenoviral activity. Cheng (2006) reported that the highest content of quercetin in lyophilized shallot powder was equal to only 35.7 mg of quercetin/g fresh weight, which implies that some constituent(s) of shallots (other than quercetin) might contribute to the anti-ADV activity. A phytochemical analysis of shallot extracts has also confirmed the presence of flavone, ascalin, and furostanol saponins (Fattorusso et al., 2002; Wang and Ng, 2002). Furthermore, Fattorusso et al. (2001) and Cheng (2006) showed that the quercetin content of onions was greater than that in garlic, green onions, or leeks, but that the fresh garlic extract had greater anti-adenoviral activity than onion extract in this study. Therefore, some constituents of the Allium plants that confer anti-adenoviral activity may not be derived from quercetin alone, and the antiviral activity may be due to a combination of phytochemicals rather than the action of a single compound (Liu, 2003). Thus, shallots may be regarded as effective agents for antiviral activity, and further isolation and purification of the active compounds is warranted. Previous studies showed that ddC successfully suppresses virus production. Similarly, ddC was also appeared as the strongest antiadenoviral activity either in ADV3 or ADV41. However, ddC may infrequently cause severe side effects, including pancreatitis and peripheral neuropathy, which could prove fatal (Lenaerts et al., 2008; Opii et al., 2007). Quercetin was also found to possess antiadenoviral activity against ADV3 and ADV41, which was higher than that for shallots. Previous studies have also proved that quercetin possesses antiviral activity against HIV, HSV, ADV3, ADV8, and ADV11 (Chiang et al., 2003; Mahmood et al., 1996). Studies have suggested that for all anti-infective bioassays, EC50 values should be below 25 mM for pure compounds (Cos et al., 2006). The EC50 values of quercetin against ADV3 and ADV41 in this study were 100 mM. Thus, the present study findings reveal that the antiviral compounds (ddC and quercetin) are inappropriate for use as antiviral drugs to treat respiratory or gastrointestinal infections in infants or young children. Primary bioassays are generally designed for the rapid screening of large numbers of products or extracts, and they should meet the following criteria: simple, easy to implement, and yield results quickly and preferably at low cost (Cos et al., 2006). The MTT (3-(4.5-dimethylthiazol-2-y1)-2,5-diphenyltetrazolium bromide) assay is based on the fact that live cells reduce tetrazolium salts into coloured formazan compounds (Kodama et al., 1996; Pauwels et al., 1988; Plumb et al., 1989); however, this assay has not been applied to the evaluation of anti-ADV41 activity. In 1996, Kodama et al. (1996) established a sensitive and accurate method for the screening of anti-adenovirus agents using the MTT method. Liu et al. (2005) compared the antiviral effects in vitro using an MTT assay and a plaque reductive assay to evaluate Chinese herbal medicines. They observed no difference between the plaque reductive assay and the MTT assay for antiviral activity

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against coxsackievirus. In the anti-adenoviral assay in the present study, we also obtained the same results that the MTT and PRD methods showed a high correlation (R2 = 0.8952), with no signiﬁcant difference in a paired t-test (p > 0.05). Thus, our study proved that an anti-ADV screening method with A549 cells using a MTT assay could be widely used for rapid screening of natural products for anti-adenoviral activity.

5. Conclusion The present study showed that the powdered form of aqueous extracts of shallot exhibited potent anti-ADV3 and ADV41 activity. Furthermore, pure compounds of antivirals are not appropriate for use as antiviral drugs even if some of them appeared to exhibit the strongest antiviral activity. Therefore, the potential of shallots for treating adenoviral infection merits further studies. An MTT assay on the A549 cell proved to be a rapid and sensitive assay system for screening anti-adenoviral drugs. Finally, our results suggest that the anti-adenoviral activity of some Allium plants may be due to a combination of photochemicals rather than due to quercetin alone.

References Amin, M. and B. P. Kapadnis, ‘‘Heat Stable Antimicrobial Activity of Allium Ascalonicum against Bacteria and Fungi,’’ Indian J. Exp. Biol., 43, 751 (2005). Block, E., S. Naganathan, D. Putman, and S. H. Zhao, ‘‘Allium Chemistry: HPLC Analysis of Thiosulﬁnates from Onion, Garlic, Wild Garlic, Leek, Scallion, Shallot, Elephant (Great-headed) Garlic, Chive, and Chinese Chive; Uniquely High Allyl-to-methyl Ratios in Some Garlic Samples,,’’ J. Agric. Food Chem., 40, 2418 (1992). Cai, S. Q., R. Wang, X. Yang, M. Shang, C. Ma, and Y. Shoyama, ‘‘Antiviral Flavonoid-type C-glycosides from the Flowers of Trollius chinensis,’’ Chem. Biodivers., 3, 343 (2006). Capasso, F., T. S. Gaginella, G. Grandolini, and A. A. Izzo, Phytotherapy. A Quick Reference to Herbal Medicine. , Springer-Verlag, Berlin, Heidelberg (2003). Chang, S. Y., C. N. Lee, P. H. Lin, H. H. Huang, L. Y. Chang, W. Ko, S. F. Chang, P. I. Lee, L. M. Huang, and C. L. Kao, ‘‘A Community-derived Outbreak of Adenovirus Type 3 in Children in Taiwan between 2004 and 2005,’’ J. Med. Virol., 80, 102 (2008). Cheng, L. H., ‘‘Analysis of Allicin and Quercetin and Functional Food Development from Several Allium Vegetables’’, M.S. Thesis, Department of Bioengineering, Tatung University, Taipei, Taiwan (2006). Cherry, J. D., Adenoviruses, 5th ed., p. 1843, Feigin, R.D., J.D. Cherry, G.J., Demmler, S.L. Kaplan (Eds.), WB Saunders, Philadelphia, (2004). Chiang, L. C., W. Chiang, M. Y. Chang, L. T. Ng, and C. C. Lin, ‘‘Antiviral Activity of Plantago Major Extracts and Related Compounds in vitro,’’ Antiviral Res., 55, 53 (2002). Chiang, L. C., W. Chiang, M. C. Liu, and C. C. Lin, ‘‘In vitro Antiviral Activities of Caesalpinia pulcherrima and Its Related Flavonoids,’’ J. Antimicrob. Chemother., 52, 194 (2003). Clardy, J. and C. Walsh, ‘‘Lessons from Natural Molecules,’’ Nature, 432, 829 (2004). Cooper, P. D., The Plaque Assay of Animal Viruses, p. 243, Maramorosch, K., H. Koprowski (Eds.), Methods in Virology, vol. 3. Academic Press Inc., New York, USA, (1967). Cos, P., A. J. Vlietinck, D. V. Berghe, and L. Maes, ‘‘Anti-infective Potential of Natural Products: How to Develop a Stronger in vitro ‘Proof-of-concept’,’’ J. Ethnopharmacol., 106, 290 (2006). Fattorusso, E., E. M. Iorizzi, V. Lanzotti, and O. Taglialatela-Scafati, ‘‘Chemical Composition of Shallot (Allium ascalonicum Hort.),’’ J. Agric. Food Chem., 50, 5686 (2002). Fattorusso, E., V. Lanzotti, O. Taglialatela-Scafati, and C. Cicala, ‘‘The Flavonoids of Leek, Allium porrum L,’’ Phytochemistry, 57, 565 (2001). Gray, G., C. J. D. Callahan, A. W. Hawksworth, C. A. Fisher, and J. C. Gaydos, ‘‘Respiratory Diseases among U.S. Military Personnel: Countering Emerging Threats,’’ Emerg. Infect. Dis., 5, 379 (1999). Haramoto, E., H. Katayama, K. Oguma, and S. Ohgaki, ‘‘Quantitative Analysis of Human Enteric Adenoviruses in Aquatic Environments,’’ J. Appl. Microbiol., 103, 2153 (2007). Harris, J. C., S. L. Cottrell, S. Plummer, and D. Lloyd, ‘‘Antimicrobial Properties of Allium sativum (Garlic),’’ Appl. Microbiol. Biotechnol., 57, 282 (2001). Hollman, P. and I. Arts, ‘‘Flavonols, Flavones, and Flavanolssnature, Occurrence and Dietary Burden,’’ J. Sci. Food Agric., 80, 1081 (2000). Kinchington, P. R., E. G. Romanowski, and J. Y. Gordon, ‘‘Prospects for Adenovirus Antivirals,’’ J. Antimicrob. Chemother., 55, 424 (2005). Kodama, E., S. Shigeta, T. Suzuki, and E. D. Clercq, ‘‘Application of a Gastric Cancer Cell Line (MKN-28) for Anti-adenovirus Screening Using the MTT Method,’’ Antiviral Res., 31, 159 (1996). Lanzotti, V., ‘‘The Analysis of Onion and Garlic,’’ J. Chromatogr. A, 1112, 3 (2006). Lenaerts, L., E. De Clercq, and L. Naesens, ‘‘Clinical Features and Treatment of Adenovirus Infections,’’ Rev. Med. Virol., 18, 357 (2008).

232

C.-H. Chen et al. / Journal of the Taiwan Institute of Chemical Engineers 42 (2011) 228–232

Lin, C. H., Y. C. Huang, C. H. Chiu, C. G. Huang, K. C. Tsao, and T. Y. Lin, ‘‘A Cluster of Adenovirus Serotype 3 Infections in Children in Northern Taiwan: Clinical Features and Laboratory Findings,’’ J. Microbiol. Immunol. Infect., 40, 302 (2007). Liu, R. H., ‘‘Health Beneﬁts of Fruits and Vegetables are from Additive and Synergistic Combinations of Phytochemicals,’’ Am. J. Clin. Nutr., 78 (Suppl), 517 (2003). Liu, Y., Z. Yang, and J. He, ‘‘Comparison and Analysis of CPE Assay, MTT Assay and Plaque Reductive Assay for Evaluating Medicines in Antiviral Effects in vitro,’’ Med. J. Wuhan Univ., 26, 199 (2005). Lyu, S. Y., J. Y. Rhim, and W. B. Park, ‘‘Antiherpetic Activities of Flavonoids against Herpes Simplex Virus Type 1 (HSV-1) and Type 2 (HSV-2) in vitro,’’ Arch. Pharm. Res., 28, 1293 (2005). Mahmood, N., S. Piacente, C. Pizza, A. Burke, A. I. Khan, and A. J. Hay, ‘‘The Anti-HIV Activity and Mechanisms of Action of Pure Compounds Isolated from Rosa damascene,’’ Biochem. Biophys. Res. Commun., 229, 73 (1996). Miean, K. H. and S. Mohamed, ‘‘Flavonoid (Myricetin, Quercetin, Kaempferol, Luteolin, and Apigenin) Content of Edible Tropical Plants,’’ J. Agric. Food Chem., 49, 3106 (2001). Mukhtar, M., M. Arshad, M. Ahmad, R. J. Pomerantz, B. Wigdahl, and Z. Parveen, ‘‘Antiviral Potentials of Medicinal Plants,’’ Virus Res., 131, 111 (2008).

Opii, W. O., R. Sultana, H. M. Abdul, M. A. Ansari, A. Nath, and D. A. Butterfield, ‘‘Oxidative Stress and Toxicity Induced by the Nucleoside Reverse Transcriptase Inhibitor (NRTI)-20 ,30 -dideoxycytidine (ddC): Relevance to HIV-dementia,’’ Exp. Neurol., 204, 29 (2007). Pauwels, R., J. Balzarini, M. Baba, R. Snoeck, D. Schols, P. Hcrdewijn, J. Desmyter, and E. D. Clercq, ‘‘Rapid and Automated Tetrazolium-based Colorimetric Assay or the Detection of Anti-HIV Compounds,’’ J. Virol. Methods, 20, 309 (1988). Plumb, J. A., R. Milroy, and S. B. Kaye, ‘‘Effects of the pH Dependence of 3-(4,5dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium Bromide-formazan Absorption on Chemosensitivity Determined by a Novel Tetrazolium-based Assay,’’ Cancer Res., 49, 4435 (1989). Slatter, M. A., S. Read, C. E. Taylor, B. N. Crooks, M. Abinun, T. J. Flood, A. J. Cant, C. Wright, and A. R. Gennery, ‘‘Adenovirus Type F Subtype 41 Causing Disseminated Disease Following Bone Marrow Transplantation for Immunodeficiency,’’ J. Clin. Microbiol., 43, 1462 (2005). Tait, S., A. L. Salvati, N. Desideri, and L. Fiore, ‘‘Antiviral Activity of Substituted Homoisoflavonoids on Enteroviruses,’’ Antiviral Res., 72, 252 (2006). Wang, H. X. and T. B. Ng, ‘‘Ascalin, a New Anti-fungal Peptide with Human Immunodeficiency Virus Type 1 Reverse Transcriptase-inhibiting Activity from Shallot Bulbs,’’ Peptides, 23, 1025 (2002).