Fitoterapia 93 (2014) 47–53
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Comparison of in vitro antiviral activity of tea polyphenols against influenza A and B viruses and structure–activity relationship analysis Zi-Feng Yang a,c,1, Li-Ping Bai b,1, Wen-bo Huang a, Xu-Zhao Li a, Sui-Shan Zhao a, Nan-Shan Zhong a,c,⁎, Zhi-Hong Jiang b,⁎⁎ a
State Key Laboratory of Respiratory Disease (Guangzhou Medical University), The First Affiliated Hospital of Guangzhou Medical University, 151 Yanjiang Road, Guangzhou, China State Key Laboratory of Quality Research in Chinese Medicine, Macau Institute for Applied Research in Medicine and Health, Macau University of Science and Technology, Taipa, Macau SAR, China c National Clinical Research Center, Guangzhou, China b
a r t i c l e
i n f o
Article history: Received 17 September 2013 Accepted in revised form 7 December 2013 Available online 24 December 2013 Keywords: Tea polyphenols Antiviral Influenza virus Structure–activity relationship Chemical compounds studied in this article: (−)-Epicatechin (PubChem CID: 72276) (−)-Epigallocatechin (PubChem CID: 72277) (−)-Epicatechingallate (PubChem CID: 107905) (−)-Epigallocatechingallate (PubChem CID: 65064) Procyanidin B2 (PubChem CID: 122738) Theaflavin(PubChem CID: 114777) Theaflavindigallate (PubChem CID: 44448535) Kaempferol (PubChem CID: 5280863) Quercetin (PubChem CID: 5280343) Myricetin (PubChem CID: 5281672)
a b s t r a c t Influenza poses a particular risk of severe outcomes in the elderly, the very young and those with underlying diseases. Tea polyphenols are the natural phenolic compounds in teas, and principally consist of catechins, proanthocyanidins, flavonols, and theaflavins, which antiviral activities have been reported recently. This study is to gain a further insight into potential of various tea polyphenols for inhibiting influenza virus infection. Five tea polyphenols exhibited inhibitory activity against influenza A virus in the trend of theaflavin N procyanidin B-2 N procyanidin B-2 digallate N (−)-epigallocatechin(EGC) N (−)-epigallocatechingallate(EGCG) with IC50 values in the range of 16.2–56.5 μg/ml. Six of the tested compounds showed anti-influenza B virus activity in the order of kaempferol N EGCG N procyanidin B-2 N (−)-EGC ~ methylated EGC N theaflavin with IC50 values in the range of 9.0–49.7 μg/ml. Based on these results, the structure–activity relationship (SAR) was explained as follows. First, the dimeric molecules, such as theaflavin and procyanidin B-2, generally displayed more potent antiviral activity against both influenza A and B viruses than the catechin monomers. Second, the kaempferol for inhibition of influenza B virus indicated that the more planar flavonol structure with only one C-4′ phenolic hydroxyl group in the B ring is necessary for the anti-influenza B virus activity. A similar SAR can be drawn from the assays of another enveloped RNA virus, such as respiratory syncytial virus. These results are expected to provide guides for rational design of antiviral drugs based on polyphenols. © 2014 Elsevier B.V. All rights reserved.
1. Introduction
⁎ Correspondence to: N.-S. Zhong, State Key Laboratory of Respiratory Disease (Guangzhou Medical University), The First Affiliated Hospital of Guangzhou Medical University, 151 Yanjiang Road, Guangzhou, China. Tel.: + 86 20 83062888; fax: + 86 20 34282274. ⁎⁎ Corresponding author. Tel.: +853 88972777; fax: +853 28825886. E-mail addresses: [email protected] (N.-S. Zhong), [email protected] (Z.-H. Jiang). 1 These authors contributed equally to this paper. 0367-326X/$ – see front matter © 2014 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.fitote.2013.12.011
Influenza viruses bring about worldwide outbreaks and pandemics in humans and animals every year with high morbidity and mortality [1]. The severe “Spanish flu” pandemic occurred in 1918–1919 and resulted in at least 20 million human deaths [2]. The H1N1 virus, characterized by a unique triple-reassortant gene segment never previously identified in either animals or humans, caused a global pandemic in 2009 with a rapid spread of the virus in less than two months [3]. This forced the World Health Organization to raise the
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alert level of the influenza pandemic from phase 3 to 6 [4]. The recent outbreaks of novel avian-origin influenza A (H7N9) virus had caused 135 confirmed cases in China as of August 12, 2013 [5]. According to structural analysis of the H7N9 virus, a single glutamate change to serine at residue 228 in HA, would be close to human adaptation [6]. It is anticipated that future pandemics will be due to highly pathogenic variations of the influenza virus. Currently, the United States Food and Drug Administration lists two classes of antiviral drugs that are approved for prevention and treatment of influenza virus; these are M2 ion-channel inhibitors (amantadine and rimantadine) and neuraminidase inhibitors (oseltamivir, zanamivir and paramivir). The former is only effective against influenza A viruses and drug resistance has become widespread [7], while the latter, which is effective against both influenza A and B viruses, is also confronting drug resistance in new strains, such as pandemic H1N1 and novel human avian influenza H7N9 virus [7,8].These combined reasons have motivated scientists to explore novel antiviral drugs for activity against influenza virus, including natural products [9]. Plant polyphenols are a class of natural compounds characterized by the presence of multiple phenol structural units. They are usually astringent and bitter in taste with molecular weights between 500 and 3000. Plant polyphenols are also well known for the ability to react with proteins, given that they precipitate albumin, gelatin, and other proteins in aqueous solutions, and are used to transform animal hide to leather by cross-linkage of collagen molecules [9,10]. Of popular and great interest are tea polyphenols. These compounds are well known through their potential to contribute to better health. Tea polyphenols are a group of relatively small polyphenols, mainly consisting of catechins, flavonols, proanthocyanidins, and theaflavins. Tea
catechins, part of theflavanol group by chemical classification, account for around 70% of green tea polyphenols. These teacatechins include (−)-epigallocatechingallate (EGCG), (−)-epigallocatechin (EGC), (−)-epicatechingallate (ECG), (−)-epicatechin (EC), (−)-catechin, and (+)-catechin (Fig. 1) [11]. Proanthocyanidins, such as procyanidin B-2 and its digallate derivative (Fig. 1), are a group of natural polyphenols derived from the dimerization of flavan-3-ols (i.e., green tea catechins) through carbon–carbon linkage. In addition, green tea also contains small amounts of flavonols, such as kaempferol, quercetin, and myricetin (Fig. 1), which are similar to catechins but with a more planar chemical structure. Catechins, proanthocyanidins, and flavonols together are regarded as the principal biologically active components of green tea. Theaflavins are major active constituents of black tea, which are produced by the enzymatic oxidation and dimerization from flavan-3-ols during the manufacture of black tea and oolong tea from green tea. The most abundant theaflavins in black tea are theaflavin, theaflavin-3-gallate, theaflavin-3′-gallate, and theaflavin-3,3′-digallate (Fig. 1) [9]. All the above-mentioned natural polyphenols that are present in various teas were generally described as tea polyphenols, and show a variety of potent pharmacological activities including antioxidative activity [12], cancer prevention [13,14], antibacterial activity [15], reduction of blood cholesterol [16], antiangiogenesis activity [17], inhibition of fibrillogenesis of amyloid β-peptide [18,19], and anti-HIV replication activity [20].Tea catechins show promise as an intervention for clinically preventing influenza infection. A prospective clinical study exhibited that gargling with tea catechin extracts was effective in preventing influenza infection in elderly nursing home residents [21].The incidence of influenza infection was significantly lower in the tea catechin group (1.3%) than in the control group (10%).
Fig. 1. Chemical structures of tea polyphenols and their derivatives.
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A clinical trial of randomized, double-blind, placebo-controlled study reported that the consumption of green tea extracts can enhance systemic immunity and prevent the occurrence of upper respiratory tract infection and influenza symptoms in health adults [22]. Another randomized control trial of 200 healthcare workers reported that taking green tea catechins is effective prophylaxis for influenza infection [23]. Moreover, in the year of 2006, the U.S. FDA has approved the first new botanical drug, called VeregenTM which is principally made from green tea catechins, to be used as a topical treatment of external genital and anal warts caused by human papilloma viruses in adults [24]. A recent in vitro experimental study demonstrated that green tea extract exhibited inhibitory effects on a broad range of viruses including six enveloped viruses (including influenza virus H3N2 and H5N3) and three non-enveloped viruses [25]. In addition, EGCG has been found to exhibit inhibitory effects against influenza virus [26,27]. Recently, three gallate derivatives of theaflavin and an extract containing 80% theaflavin have been reported to show potent inhibitory effects against influenza virus in vitro [9]. Theaflavin-3,3′-digallate displayed the highest inhibitory activity against neuraminidase (NA) among three gallate derivatives of theaflavin, with an IC50 of 10.67 μM. These results suggest that tea polyphenols have significant potential for treatment of influenza virus infection. However, antivirus activities of other tea polyphenols, such as theaflavin, procyanidin B-2, and kaempferol, remain unclear. To the best of our knowledge, no comparative study has been carried out on the inhibition of influenza virus by the various tea polyphenols. Consequently, the structure–activity relationship of tea polyphenols in terms of antivirus activity is yet to be established. In the present study, we used the cytopathic effect (CPE) inhibition assay on Madin–Darby canine kidney (MDCK) cells to evaluate the antivirus effect of a series of tea polyphenols, including six catechins, two proanthocyanidins, two theaflavins, three flavonols, and one structurally modified derivative of epigallocatechin, toward a variety of influenza strains in vitro. In light of our results, the structure–activity relationship of their antiviral activity is summarized. 2. Materials and methods 2.1. Tea polyphenols, cells, reagents, and viruses Procyanidin B-2 and procyanidin B-2 3,3′–O-digallate were purified from grape seed extract (JF-Natural, Tian-Jin, China) by silica gel column chromatography using chloroform/methanol/ water from 7:3:0.5 to 5:5:1 as the mobile phase. Theaflavin and theaflavin-3,3′–O-digallate were purified from a commercial 80% theaflavins extract (Hangzhou Easily Biotechnology, China) by gel filtration chromatography (sephadex LH-20) and an elution gradient from 50 to 90% aqueous methanol. (−)-EC, (−)-EGC, (−)-ECG, and (−)-EGCG were purified from green tea extracts (JF-Natural) by silica gel column chromatography (chloroform/ methanol/water from 7:3:0.5 to 6:4:1 as mobile phase) and octadecylsilane (ODS) column chromatography (20 to 60% aqueous methanol as mobile phase). Kaempferol and quercetin were isolated and purified from flowers of Sophora japonica. These polyphenols were characterized by high-resolution mass spectrometry, 1H and 13C nuclear magnetic resonance (NMR) spectroscopy. (±)-Catechin was obtained from Sigma-Aldrich
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(USA). Myricetin was purchased from Shanghai Winherb Medical Science (Shanghai, China). (−)-Methylated epigallocatechin was prepared according to the literature [28]. The purity of all tested compounds exceeded 98% according to analysis by ultra-performance liquid chromatography–time-of-flight mass spectrometry. MDCK cells, human epidermis larynx carcinoma cells (HEp-2), influenza virus A/PR/8/34 (H1N1), and respiratory syncytial virus (RSV long strain) were purchased from the American Tissue Culture Collection (ATCC). Influenza B virus and adenovirus type 3 (ADV3) were isolated from clinical specimens in our laboratory. The influenza viruses were propagated and passaged in MDCK cells. HEp-2 cells were used as the host for RSV and ADV3. All cells were grown in Dulbecco's modified Eagle's medium (DMEM) with 10% heat-inactivated fetal calf serum (FCS). 2.2. Cytotoxicity assay Cells in 96-well plates were left untreated or treated with the indicated amounts of agents and cultured at 37 °C for 48 h. The viability was measured with the MTT assay as previously described [29]. The 50% toxic concentration (TC50) was calculated by Reed–Muench analysis [30]. 2.3. Cytopathic effect inhibition assay MDCK cells (1.0 × 104) were prepared in 96-well plastic plates and cultured at 37 °C for 24 h. To assess the anti-influenza activity, cells were washed with phosphate-buffered saline (PBS) and infected with 100 TCID50 (median tissue culture infective dose) of influenza virus at 37 °C for 2 h, followed by removal of the medium and addition of the indicated tea polyphenols at different concentrations with dilution (two fold) in serumfree Minimum Essential Medium supplemented with 2 μg/ml TPCK-trypsin. After incubation for 48 h at 34 °C, the CPE caused by the influenza virus was measured microscopically. The concentration required for 50% inhibition of the virus CPE (IC50) was calculated by the Reed–Muench method. The selection index (SI) was calculated by the ratio of TC50/IC50 [31]. 2.4. Activities against RSV and ADV3 The antiviral effects of tea polyphenols against RSV and ADV3 were performed by using a CPE inhibition assay [32,33]. Briefly, HEp-2 cells were washed with PBS and challenged with the virus. Following washing with serum-free MEM, HEp-2 cells were treated with tea polyphenols in the indicated concentrations. The CPEs were observed microscopically for 3–5 days, and the IC50 were determined as previously described [30]. 3. Results 3.1. Cytotoxicity of tea polyphenols to MDCK cells 12 tea polyphenols (1–5, 7–13) and methylated EGC (6) were examined for their cytotoxicity in confluent cultures of MDCK cells before determining the anti-influenza virus activity of these compounds. As shown in Table 1, no significant cellular
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toxic effect was observed within the concentration in the range of 38–204 μg/ml of these tea polyphenols.
3.2. Anti-influenza activity of tea polyphenols Tea polyphenols were assayed for inhibition of influenza virus in vitro by the CPE inhibition assay, as described above. MDCK cells were infected with influenza virus (100TCID50), and then compounds were added at various concentrations. After treatment for 48 h, the antiviral effect of each compound was evaluated. As shown in Table 1, the IC50value for the five polyphenols theaflavin (9), procyanidin B-2 (7), procyanidin B-2 digallate (8), EGC (3), and EGCG (5) against A/PR/8/34 virus were 16.21, 50.85, 35.32, 30.94, and 56.49 μg/ml, respectively. Theaflavin (9) demonstrated the most effective anti-influenza A virus activity with an SI value of 8.44. In addition, kaempferol (11), EGCG (5), procyanidin B-2 (7), EGC (3), methylated EGC (6), and theaflavin (9) exhibited anti-influenza B virus activity, with IC50 values ranging from 8.98 to 49.72 μg/ml. Kaempferol (11) displayed the most potent effect against influenza B virus with an SI value of 19.01. It was reported that tea polyphenols most likely bind to the glycoproteins of the viral envelope to inhibit influenza viral infection. The block of hemagglutinin (HA) domains by tea polyphenols binding inhibits the influenza virions to bind to and consequently enter host cells [24]. In order to explore the specificity of these tea polyphenols for the enveloped influenza viruses and get some insights into the preference of tea polyphenols between enveloped and non-enveloped viruses, RSV and ADV3 were respectively used as a representative of both enveloped and non-enveloped viruses to further examine inhibitory effects of these tea polyphenols on non-influenza viruses. Of all the tea polyphenols, theaflavin (9) showed the strongest anti-ADV3 virus activity with an SI value of 5.02, while kaempferol (11) exhibited the most potent anti-RSV virus effect with an SI value of 5.92. Moreover, theaflavin digallate (10) showed inhibitory effects against ADV and RSV viruses with SI values of 2.75 and 4.93, respectively (see Table 2).
4. Discussion Influenza is a disease with a high mortality rate throughout the world and the effects of vaccines against influenza virus infection are limited because of the frequent variation of viral antigens [27]. Therefore, scientists are expending considerable effort in searching for new antiviral molecules from natural products and synthetic compounds. Tea polyphenols are regarded as a class of biologically active compounds that are believed to be beneficial to human health and are the basis of many claims made about the health benefits of teas, red wine, fruits, vegetables, and some Chinese herbs. We conducted a comprehensive study on the in vitro antiviral activity of thirteen tea polyphenols against both influenza A and B viruses. The results showed that five compounds exhibited anti-influenza A virus activity, and six compounds exhibited anti-influenza B virus activity. Of note, kaempferol (11) showed potent inhibitory activity against influenza B virus with an IC50 value of 8.98 μg/ml (SI value of 19.01). In addition, kaempferol (11) and theaflavin digallate (10) also displayed strong antiviral activity toward RSV with IC50 values of 4.84 (SI value of 5.92) and 33.1 μg/ml (SI value of 4.93), respectively. Moreover, theaflavin (9) and its digallate derivative (10) showed anti-ADV virus activity with IC50 values of 13.62 (SI value of 5.02) and 59.34 μg/ml (SI value of 2.75), respectively. Based on the antiviral results of these tea polyphenols, we found that structures of dimeric flavan-3-ols without a galloyl group (i.e., theaflavin and procyanidin B-2) had potent antiviral activity against both influenza A and B viruses. Thus, the presence of the galloyl group in these dimeric flavanols is unnecessary for anti-influenza virus activity, and it was obvious that the dimers had better antiviral activity than the monomers. It was reported that quercetin showed weak antiviral activity against Japanese encephalitis virus (JEV) [34], and it also inhibits rhinovirus endocytosis and replication in airway epithelial cells [35]. In some previous reports, quercetin and its derivatives have been proved to be effective to lessen the impact of influenza infection in vitro or in vivo, probably partly due to its antioxidant and/or antihypoxant properties [36–39]. However, our results
Table 1 Antivirus effects of polyphenols against influenza A and B viruses. No.
Polyphenols
TC50 (μg/ml) MDCK
IC50 (μg/ml) A/PR/8/34
SI
IC50 (μg/ml) B/Lee/1940
SI
1 2 3 4 5 6 7 8 9 10 11 12 13
(±)-Catechin (−)-EC (−)-EGC (−)-ECG (−)-EGCG Methylated EGC Procyanidin B-2 Procyanidin B-2 3,3′-di-O-gallate Theaflavin Theaflavin-3,3′-di-O-gallate Kaempferol Quercetin Myricetin
144.56 145.09 38.11 55.16 57.55 94.58 203.87 55.156 136.94 54.31 143.79 42.28 79.82
N144.56 N145.09 30.94 N55.16 56.49 N94.58 50.85 35.32 16.21 N54.31 N143.79 N42.28 N79.82
b1 b1 1.23 b1 1.01 b1 4 1.56 8.44 b1 b1 b1 b1
N144.56 N145.09 13.47 N55.16 11.09 33.43 56.02 N55.15 49.72 N54.31 8.98 N42.28 N79.82
b1 b1 2.82 b1 5.18 2.82 3.63 b1 2.75 b1 19.01 b1 b1
a Mean of results from three independent experiments; TC50, the concentration required for 50% cytotoxicity of the polyphenols; IC50, the concentration required for 50% inhibition of the virus CPE; The selection index (SI) was calculated by the ratio of TC50/IC50.
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Table 2 Antivirus effects of polyphenols against non-influenza viruses. No.
Polyphenols
TC50 (μg/ml) HEp-2
IC50 (μg/ml) RSV
SI
IC50 (μg/ml) ADV3
SI
1 2 3 4 5 6 7 8 9 10 11 12 13
(±)-catechin (−)-EC (−)-EGC (−)-ECG (−)-EGCG Methylated EGC Procyanidin B-2 Procyanidin B-2 3,3′-di-O-gallate Theaflavin Theaflavin-3,3′-di-O-gallate Kaempferol Quercetin Myricetin
144.56 145.09 62.45 27.58 57.55 104.49 240.32 47.95 68.47 163.38 28.67 7.97 39.91
N144.56 N145.09 N62.45 8.68 21.8 N104.49 N240.32 N47.95 N68.47 33.1 4.84 N7.97 N39.91
b1 b1 b1 3.17 2.63 b1 b1 b1 b1 4.93 5.92 b1 b1
N144.56 N145.09 N62.45 10.96 N57.55 93.07 N240.32 N47.95 13.62 59.34 N28.67 N7.97 N39.91
b1 b1 b1 2.51 b1 1.12 b1 b1 5.02 2.75 b1 b1 b1
a Mean of results from three independent experiments; TC50, the concentration required for 50% cytotoxicity of the polyphenols; IC50, the concentration required for 50% inhibition of the virus CPE; The selection index (SI) was calculated by the ratio of TC50/IC50.
did not show that quercetin has a direct anti-influenza virus effect in our MDCK cells infection model, which may be probably due to different study design and starting point. Many in vitro studies have shown that tea polyphenols can bind to proteins [40], wherefrom the compounds might combine with the glycoproteins of the viral envelope. It is particularly important that kaempferol (11) can inhibit the enveloped viruses such as hepatitis B, herpes simplex virus (HSV), JEV, and so on [41–43]. Among the tea polyphenols, kaempferol (11) exhibited a significant antiviral activity (EC50 = 15 μM) against HSV-1 [42]. Moreover, the inhibition effect of kaempferol on JEV was due to its binding with the frame-shift site RNA (fsRNA) in the JEV serogroup [43]. In addition, kaempferol (11) has been shown to be the most effective compound against influenza viruses (H1N1 and H9N2) through inhibition of neuraminidase activity [44], but no similar activity was observed in our repeated assays. These contradictory results may be due to different assays and warrant further investigation. 4.1. Structure–activity relationship of tea polyphenols in antiviral activity On the basis of the results of the bioassays of the tea polyphenols, the structure–activity relationships (SAR) are summarized as follows. First, the dimers of tea polyphenols, such as theaflavin (9) and procyanidin B-2 (7), generally display more potent antivirus activity against both influenza A and B viruses than the catechin monomers [i.e., (−)-EC (2) and (±)-catechin (1)]. Second, the galloyl group of both theaflavindigallate (10) and procyanidin B-2 digallate (8) was found to play an uncooperative role in their antivirus effect, probably due to its steric hindrance, as compared to theaflavin (9) and procyanidin B-2 (7). This is in contrast with the result that anti-HIV activity of betulinic acid increased when it forms a gallate conjugate [45]. Third, the highest SI value of 19.01 of kaempferol (11) toward influenza B virus indicated that the more planar flavonol with only one C-4′ phenolic hydroxyl group in the B ring is necessary for the anti-influenza B virus activity of flavonols. Such SAR can also be drawn from the assay of anti-RSV activity of all the tested flavonols. Fourth,
theaflavin (9) showed selectivity for inhibiting influenza A virus, while kaempferol (11) and EGCG (5) exhibited specificity for inhibiting influenza B virus. In addition, the phenolic hydroxyl groups of EGC (3) in the B ring play an important role in anti-influenza A virus activity but not in anti-influenza B virus activity, as compared with methylated EGC (6). 4.2. Hypothesis on the significant SAR of tea polyphenols in anti-influenza virus activity The above strict SAR of tested tea polyphenols is attributed to the chemical differences between two subgroups of flavonols and flavanols. The former is chemically more planar and rigid than the latter due to the presence of a C-2–C-3 double bond and a ketone in C-4. In other words, flavanols are more flexible than flavonols. In vitro neuraminidase (NA) inhibition assay [46] revealed that rigid flavonols (kaempferol, quercetin and myricetin) show much stronger inhibitory effect than flexible flavanols (catechin and epicatechin). The functionality of one hydroxyl at C-4′ position in the B ring, a double bond at C-2–C-3, and a ketone at the C-4 position in kaempferol (11) is essential for inhibiting influenza virus as a NA inhibitor. Addition of one more hydroxyl in the B ring, such as quercetin (12) and myricetin (13), causes steric hindrance and disfavors the anti-influenza effect [46]. Based on the literature report, we summarized that the flavonols are strong NA inhibitors, compared with the flavanols. Kaempferol (11) exerts a significant antiviral effect against influenza B virus most likely by targeting the NA of influenza B virus. We speculated that the striking specificity of kaempferol (11) for influenza B virus but not for A virus is probably due to the discrepancy in the NAs between A and B virus. Kaempferol (11) may possess a high affinity with the NA of influenza B. The reported SAR of kaempferol (11) in NA inhibition assay [46] is in full coincidence with our finding in the cellular level. However, the following two mechanisms might be responsible for the antiviral effects of tea catechins and its dimers on both influenza A and B viruses: one is the blockage of viral binding to the cell receptors in the early stage of a virus infection, and the other is attenuation of viral replication after entry [9,26,27,47,48]. Both theaflavin (9) and procyanidin B-2 (7), two dimers of
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flavan-3-ol, inhibit both influenza A and B viruses probably due to the combinative results via above two mechanisms. Acknowledgments This study was financially supported by the Macao Science and Technology Development Fund, MSAR (039/2011/A2 to ZHJ), the Science and Technology Planning Project of Guangdong Province, China (Guangdong–Macau Joint Research Centre for New Drug Discovery Against Respiratory Pathogens, Grant no. 2010B091000018), and the National Key Technology R&D Program of the 12th National Five-year Development Plan of China (Grant no. 2012BAI05B01). Conflict of interest The authors have no conflict of interests. References [1] Miller M, Viboud C, Simonsen L, Olson DR, Russell C. Mortality and morbidity burden associated with A/H1N1pdm influenza virus: who is likely to be infected, experience clinical symptoms, or die from the H1N1pdm 2009 pandemic virus ? PLoS Curr 2009;1 (RRN1013). [2] Taubenberger JK, Reid AH, Janczewski TA, Fanning TG. Integrating historical, clinical and molecular genetic data in order to explain the origin and virulence of the 1918 Spanish influenza virus. Philos Trans R Soc Lond B Biol Sci 2001;356:1829–39. [3] Trifonov V, Khiabanian H, Greenbaum B, Rabadan R. The origin of the recent swine influenza A(H1N1) virus infecting humans. Euro Surveill 2009;14:191–3. [4] Authorizations, E.U. Update: infections with a swine-origin influenza A (H1N1) virus—United States and other countries, April 28, 2009. Morbidity and Mortality Weekly Report. 58; 2009. [5] World Health Organization. Number of confirmed human cases of avian influenza A(H7N9) reported to WHO. http://www.who.int/entity/ influenza/human_animal_interface/influenza_h7n9/09_ReportWebH7N9 Number.pdf; 2013. [6] Tharakaraman K, Jayaraman A, Raman R, Viswanathan K, Stebbins NW, Johnson D, et al. Glycan receptor binding of the influenza A virus H7N9 hemagglutinin. Cell 2013;153:1486–93. [7] Hurt AC, Chotpitayasunondh T, Cox NJ, Daniels R, Fry AM, Gubareva LV, et al. Antiviral resistance during the 2009 influenza A H1N1 pandemic: public health, laboratory, and clinical perspectives. Lancet Infect Dis 2012;12:240–8. [8] Hu Y, Lu S, Song Z, Wang W, Hao P, Li J, et al. Association between adverse clinical outcome in human disease caused by novel influenza A H7N9 virus and sustained viral shedding and emergence of antiviral resistance. Lancet 2013;38:2273–9. [9] Zu M, Yang F, Zhou W, Liu A, Du G, Zheng L. In vitro anti-influenza virus and anti-inflammatory activities of theaflavin derivatives. Antiviral Res 2012;94:217–24. [10] Zhu M, David Phillipson J, Greengrass PM, Bowery NE, Cai Y. Plant polyphenols: biologically active compounds or non-selective binders to protein? Phytochemistry 1997;44:441–7. [11] Vuong QV, Stathopoulos CE, Nguyen MH, Golding JB, Roach PD. Isolation of green tea catechins and their utilization in the food industry. Food Rev Int 2011;27:227–47. [12] Lambert JD, Elias RJ. The antioxidant and pro-oxidant activities of green tea polyphenols: a role in cancer prevention. Arch Biochem Biophys 2010;501:65–72. [13] Yang CS, Wang X. Green tea and cancer prevention. Nutr Cancer 2010;62:931–7. [14] Khan N, Mukhtar H. Multitargeted therapy of cancer by green tea polyphenols. Cancer Lett 2008;269:269–80. [15] Ferrazzano GF, Amato I, Ingenito A, Zarrelli A, Pinto G, Pollio A. Plant polyphenols and their anti-cariogenic properties: a review. Molecules 2011;16:1486–507. [16] Huang H-C, Lin J-K. Pu-erh tea, green tea, and black tea suppresses hyperlipidemia, hyperleptinemia and fatty acid synthase through activating AMPK in rats fed a high-fructose diet. Food Funct 2012;3:170–7. [17] Cao Y, Cao R. Angiogenesis inhibited by drinking tea. Nature 1999;398:381-381.
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Comparison of in vitro antiviral activity of tea polyphenols against influenza A and B viruses and structure–activity relationship analysis Zi-Feng Yang a,c,1, Li-Ping Bai b,1, Wen-bo Huang a, Xu-Zhao Li a, Sui-Shan Zhao a, Nan-Shan Zhong a,c,⁎, Zhi-Hong Jiang b,⁎⁎ a
State Key Laboratory of Respiratory Disease (Guangzhou Medical University), The First Affiliated Hospital of Guangzhou Medical University, 151 Yanjiang Road, Guangzhou, China State Key Laboratory of Quality Research in Chinese Medicine, Macau Institute for Applied Research in Medicine and Health, Macau University of Science and Technology, Taipa, Macau SAR, China c National Clinical Research Center, Guangzhou, China b
a r t i c l e
i n f o
Article history: Received 17 September 2013 Accepted in revised form 7 December 2013 Available online 24 December 2013 Keywords: Tea polyphenols Antiviral Influenza virus Structure–activity relationship Chemical compounds studied in this article: (−)-Epicatechin (PubChem CID: 72276) (−)-Epigallocatechin (PubChem CID: 72277) (−)-Epicatechingallate (PubChem CID: 107905) (−)-Epigallocatechingallate (PubChem CID: 65064) Procyanidin B2 (PubChem CID: 122738) Theaflavin(PubChem CID: 114777) Theaflavindigallate (PubChem CID: 44448535) Kaempferol (PubChem CID: 5280863) Quercetin (PubChem CID: 5280343) Myricetin (PubChem CID: 5281672)
a b s t r a c t Influenza poses a particular risk of severe outcomes in the elderly, the very young and those with underlying diseases. Tea polyphenols are the natural phenolic compounds in teas, and principally consist of catechins, proanthocyanidins, flavonols, and theaflavins, which antiviral activities have been reported recently. This study is to gain a further insight into potential of various tea polyphenols for inhibiting influenza virus infection. Five tea polyphenols exhibited inhibitory activity against influenza A virus in the trend of theaflavin N procyanidin B-2 N procyanidin B-2 digallate N (−)-epigallocatechin(EGC) N (−)-epigallocatechingallate(EGCG) with IC50 values in the range of 16.2–56.5 μg/ml. Six of the tested compounds showed anti-influenza B virus activity in the order of kaempferol N EGCG N procyanidin B-2 N (−)-EGC ~ methylated EGC N theaflavin with IC50 values in the range of 9.0–49.7 μg/ml. Based on these results, the structure–activity relationship (SAR) was explained as follows. First, the dimeric molecules, such as theaflavin and procyanidin B-2, generally displayed more potent antiviral activity against both influenza A and B viruses than the catechin monomers. Second, the kaempferol for inhibition of influenza B virus indicated that the more planar flavonol structure with only one C-4′ phenolic hydroxyl group in the B ring is necessary for the anti-influenza B virus activity. A similar SAR can be drawn from the assays of another enveloped RNA virus, such as respiratory syncytial virus. These results are expected to provide guides for rational design of antiviral drugs based on polyphenols. © 2014 Elsevier B.V. All rights reserved.
1. Introduction
⁎ Correspondence to: N.-S. Zhong, State Key Laboratory of Respiratory Disease (Guangzhou Medical University), The First Affiliated Hospital of Guangzhou Medical University, 151 Yanjiang Road, Guangzhou, China. Tel.: + 86 20 83062888; fax: + 86 20 34282274. ⁎⁎ Corresponding author. Tel.: +853 88972777; fax: +853 28825886. E-mail addresses: [email protected] (N.-S. Zhong), [email protected] (Z.-H. Jiang). 1 These authors contributed equally to this paper. 0367-326X/$ – see front matter © 2014 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.fitote.2013.12.011
Influenza viruses bring about worldwide outbreaks and pandemics in humans and animals every year with high morbidity and mortality [1]. The severe “Spanish flu” pandemic occurred in 1918–1919 and resulted in at least 20 million human deaths [2]. The H1N1 virus, characterized by a unique triple-reassortant gene segment never previously identified in either animals or humans, caused a global pandemic in 2009 with a rapid spread of the virus in less than two months [3]. This forced the World Health Organization to raise the
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alert level of the influenza pandemic from phase 3 to 6 [4]. The recent outbreaks of novel avian-origin influenza A (H7N9) virus had caused 135 confirmed cases in China as of August 12, 2013 [5]. According to structural analysis of the H7N9 virus, a single glutamate change to serine at residue 228 in HA, would be close to human adaptation [6]. It is anticipated that future pandemics will be due to highly pathogenic variations of the influenza virus. Currently, the United States Food and Drug Administration lists two classes of antiviral drugs that are approved for prevention and treatment of influenza virus; these are M2 ion-channel inhibitors (amantadine and rimantadine) and neuraminidase inhibitors (oseltamivir, zanamivir and paramivir). The former is only effective against influenza A viruses and drug resistance has become widespread [7], while the latter, which is effective against both influenza A and B viruses, is also confronting drug resistance in new strains, such as pandemic H1N1 and novel human avian influenza H7N9 virus [7,8].These combined reasons have motivated scientists to explore novel antiviral drugs for activity against influenza virus, including natural products [9]. Plant polyphenols are a class of natural compounds characterized by the presence of multiple phenol structural units. They are usually astringent and bitter in taste with molecular weights between 500 and 3000. Plant polyphenols are also well known for the ability to react with proteins, given that they precipitate albumin, gelatin, and other proteins in aqueous solutions, and are used to transform animal hide to leather by cross-linkage of collagen molecules [9,10]. Of popular and great interest are tea polyphenols. These compounds are well known through their potential to contribute to better health. Tea polyphenols are a group of relatively small polyphenols, mainly consisting of catechins, flavonols, proanthocyanidins, and theaflavins. Tea
catechins, part of theflavanol group by chemical classification, account for around 70% of green tea polyphenols. These teacatechins include (−)-epigallocatechingallate (EGCG), (−)-epigallocatechin (EGC), (−)-epicatechingallate (ECG), (−)-epicatechin (EC), (−)-catechin, and (+)-catechin (Fig. 1) [11]. Proanthocyanidins, such as procyanidin B-2 and its digallate derivative (Fig. 1), are a group of natural polyphenols derived from the dimerization of flavan-3-ols (i.e., green tea catechins) through carbon–carbon linkage. In addition, green tea also contains small amounts of flavonols, such as kaempferol, quercetin, and myricetin (Fig. 1), which are similar to catechins but with a more planar chemical structure. Catechins, proanthocyanidins, and flavonols together are regarded as the principal biologically active components of green tea. Theaflavins are major active constituents of black tea, which are produced by the enzymatic oxidation and dimerization from flavan-3-ols during the manufacture of black tea and oolong tea from green tea. The most abundant theaflavins in black tea are theaflavin, theaflavin-3-gallate, theaflavin-3′-gallate, and theaflavin-3,3′-digallate (Fig. 1) [9]. All the above-mentioned natural polyphenols that are present in various teas were generally described as tea polyphenols, and show a variety of potent pharmacological activities including antioxidative activity [12], cancer prevention [13,14], antibacterial activity [15], reduction of blood cholesterol [16], antiangiogenesis activity [17], inhibition of fibrillogenesis of amyloid β-peptide [18,19], and anti-HIV replication activity [20].Tea catechins show promise as an intervention for clinically preventing influenza infection. A prospective clinical study exhibited that gargling with tea catechin extracts was effective in preventing influenza infection in elderly nursing home residents [21].The incidence of influenza infection was significantly lower in the tea catechin group (1.3%) than in the control group (10%).
Fig. 1. Chemical structures of tea polyphenols and their derivatives.
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A clinical trial of randomized, double-blind, placebo-controlled study reported that the consumption of green tea extracts can enhance systemic immunity and prevent the occurrence of upper respiratory tract infection and influenza symptoms in health adults [22]. Another randomized control trial of 200 healthcare workers reported that taking green tea catechins is effective prophylaxis for influenza infection [23]. Moreover, in the year of 2006, the U.S. FDA has approved the first new botanical drug, called VeregenTM which is principally made from green tea catechins, to be used as a topical treatment of external genital and anal warts caused by human papilloma viruses in adults [24]. A recent in vitro experimental study demonstrated that green tea extract exhibited inhibitory effects on a broad range of viruses including six enveloped viruses (including influenza virus H3N2 and H5N3) and three non-enveloped viruses [25]. In addition, EGCG has been found to exhibit inhibitory effects against influenza virus [26,27]. Recently, three gallate derivatives of theaflavin and an extract containing 80% theaflavin have been reported to show potent inhibitory effects against influenza virus in vitro [9]. Theaflavin-3,3′-digallate displayed the highest inhibitory activity against neuraminidase (NA) among three gallate derivatives of theaflavin, with an IC50 of 10.67 μM. These results suggest that tea polyphenols have significant potential for treatment of influenza virus infection. However, antivirus activities of other tea polyphenols, such as theaflavin, procyanidin B-2, and kaempferol, remain unclear. To the best of our knowledge, no comparative study has been carried out on the inhibition of influenza virus by the various tea polyphenols. Consequently, the structure–activity relationship of tea polyphenols in terms of antivirus activity is yet to be established. In the present study, we used the cytopathic effect (CPE) inhibition assay on Madin–Darby canine kidney (MDCK) cells to evaluate the antivirus effect of a series of tea polyphenols, including six catechins, two proanthocyanidins, two theaflavins, three flavonols, and one structurally modified derivative of epigallocatechin, toward a variety of influenza strains in vitro. In light of our results, the structure–activity relationship of their antiviral activity is summarized. 2. Materials and methods 2.1. Tea polyphenols, cells, reagents, and viruses Procyanidin B-2 and procyanidin B-2 3,3′–O-digallate were purified from grape seed extract (JF-Natural, Tian-Jin, China) by silica gel column chromatography using chloroform/methanol/ water from 7:3:0.5 to 5:5:1 as the mobile phase. Theaflavin and theaflavin-3,3′–O-digallate were purified from a commercial 80% theaflavins extract (Hangzhou Easily Biotechnology, China) by gel filtration chromatography (sephadex LH-20) and an elution gradient from 50 to 90% aqueous methanol. (−)-EC, (−)-EGC, (−)-ECG, and (−)-EGCG were purified from green tea extracts (JF-Natural) by silica gel column chromatography (chloroform/ methanol/water from 7:3:0.5 to 6:4:1 as mobile phase) and octadecylsilane (ODS) column chromatography (20 to 60% aqueous methanol as mobile phase). Kaempferol and quercetin were isolated and purified from flowers of Sophora japonica. These polyphenols were characterized by high-resolution mass spectrometry, 1H and 13C nuclear magnetic resonance (NMR) spectroscopy. (±)-Catechin was obtained from Sigma-Aldrich
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(USA). Myricetin was purchased from Shanghai Winherb Medical Science (Shanghai, China). (−)-Methylated epigallocatechin was prepared according to the literature [28]. The purity of all tested compounds exceeded 98% according to analysis by ultra-performance liquid chromatography–time-of-flight mass spectrometry. MDCK cells, human epidermis larynx carcinoma cells (HEp-2), influenza virus A/PR/8/34 (H1N1), and respiratory syncytial virus (RSV long strain) were purchased from the American Tissue Culture Collection (ATCC). Influenza B virus and adenovirus type 3 (ADV3) were isolated from clinical specimens in our laboratory. The influenza viruses were propagated and passaged in MDCK cells. HEp-2 cells were used as the host for RSV and ADV3. All cells were grown in Dulbecco's modified Eagle's medium (DMEM) with 10% heat-inactivated fetal calf serum (FCS). 2.2. Cytotoxicity assay Cells in 96-well plates were left untreated or treated with the indicated amounts of agents and cultured at 37 °C for 48 h. The viability was measured with the MTT assay as previously described [29]. The 50% toxic concentration (TC50) was calculated by Reed–Muench analysis [30]. 2.3. Cytopathic effect inhibition assay MDCK cells (1.0 × 104) were prepared in 96-well plastic plates and cultured at 37 °C for 24 h. To assess the anti-influenza activity, cells were washed with phosphate-buffered saline (PBS) and infected with 100 TCID50 (median tissue culture infective dose) of influenza virus at 37 °C for 2 h, followed by removal of the medium and addition of the indicated tea polyphenols at different concentrations with dilution (two fold) in serumfree Minimum Essential Medium supplemented with 2 μg/ml TPCK-trypsin. After incubation for 48 h at 34 °C, the CPE caused by the influenza virus was measured microscopically. The concentration required for 50% inhibition of the virus CPE (IC50) was calculated by the Reed–Muench method. The selection index (SI) was calculated by the ratio of TC50/IC50 [31]. 2.4. Activities against RSV and ADV3 The antiviral effects of tea polyphenols against RSV and ADV3 were performed by using a CPE inhibition assay [32,33]. Briefly, HEp-2 cells were washed with PBS and challenged with the virus. Following washing with serum-free MEM, HEp-2 cells were treated with tea polyphenols in the indicated concentrations. The CPEs were observed microscopically for 3–5 days, and the IC50 were determined as previously described [30]. 3. Results 3.1. Cytotoxicity of tea polyphenols to MDCK cells 12 tea polyphenols (1–5, 7–13) and methylated EGC (6) were examined for their cytotoxicity in confluent cultures of MDCK cells before determining the anti-influenza virus activity of these compounds. As shown in Table 1, no significant cellular
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toxic effect was observed within the concentration in the range of 38–204 μg/ml of these tea polyphenols.
3.2. Anti-influenza activity of tea polyphenols Tea polyphenols were assayed for inhibition of influenza virus in vitro by the CPE inhibition assay, as described above. MDCK cells were infected with influenza virus (100TCID50), and then compounds were added at various concentrations. After treatment for 48 h, the antiviral effect of each compound was evaluated. As shown in Table 1, the IC50value for the five polyphenols theaflavin (9), procyanidin B-2 (7), procyanidin B-2 digallate (8), EGC (3), and EGCG (5) against A/PR/8/34 virus were 16.21, 50.85, 35.32, 30.94, and 56.49 μg/ml, respectively. Theaflavin (9) demonstrated the most effective anti-influenza A virus activity with an SI value of 8.44. In addition, kaempferol (11), EGCG (5), procyanidin B-2 (7), EGC (3), methylated EGC (6), and theaflavin (9) exhibited anti-influenza B virus activity, with IC50 values ranging from 8.98 to 49.72 μg/ml. Kaempferol (11) displayed the most potent effect against influenza B virus with an SI value of 19.01. It was reported that tea polyphenols most likely bind to the glycoproteins of the viral envelope to inhibit influenza viral infection. The block of hemagglutinin (HA) domains by tea polyphenols binding inhibits the influenza virions to bind to and consequently enter host cells [24]. In order to explore the specificity of these tea polyphenols for the enveloped influenza viruses and get some insights into the preference of tea polyphenols between enveloped and non-enveloped viruses, RSV and ADV3 were respectively used as a representative of both enveloped and non-enveloped viruses to further examine inhibitory effects of these tea polyphenols on non-influenza viruses. Of all the tea polyphenols, theaflavin (9) showed the strongest anti-ADV3 virus activity with an SI value of 5.02, while kaempferol (11) exhibited the most potent anti-RSV virus effect with an SI value of 5.92. Moreover, theaflavin digallate (10) showed inhibitory effects against ADV and RSV viruses with SI values of 2.75 and 4.93, respectively (see Table 2).
4. Discussion Influenza is a disease with a high mortality rate throughout the world and the effects of vaccines against influenza virus infection are limited because of the frequent variation of viral antigens [27]. Therefore, scientists are expending considerable effort in searching for new antiviral molecules from natural products and synthetic compounds. Tea polyphenols are regarded as a class of biologically active compounds that are believed to be beneficial to human health and are the basis of many claims made about the health benefits of teas, red wine, fruits, vegetables, and some Chinese herbs. We conducted a comprehensive study on the in vitro antiviral activity of thirteen tea polyphenols against both influenza A and B viruses. The results showed that five compounds exhibited anti-influenza A virus activity, and six compounds exhibited anti-influenza B virus activity. Of note, kaempferol (11) showed potent inhibitory activity against influenza B virus with an IC50 value of 8.98 μg/ml (SI value of 19.01). In addition, kaempferol (11) and theaflavin digallate (10) also displayed strong antiviral activity toward RSV with IC50 values of 4.84 (SI value of 5.92) and 33.1 μg/ml (SI value of 4.93), respectively. Moreover, theaflavin (9) and its digallate derivative (10) showed anti-ADV virus activity with IC50 values of 13.62 (SI value of 5.02) and 59.34 μg/ml (SI value of 2.75), respectively. Based on the antiviral results of these tea polyphenols, we found that structures of dimeric flavan-3-ols without a galloyl group (i.e., theaflavin and procyanidin B-2) had potent antiviral activity against both influenza A and B viruses. Thus, the presence of the galloyl group in these dimeric flavanols is unnecessary for anti-influenza virus activity, and it was obvious that the dimers had better antiviral activity than the monomers. It was reported that quercetin showed weak antiviral activity against Japanese encephalitis virus (JEV) [34], and it also inhibits rhinovirus endocytosis and replication in airway epithelial cells [35]. In some previous reports, quercetin and its derivatives have been proved to be effective to lessen the impact of influenza infection in vitro or in vivo, probably partly due to its antioxidant and/or antihypoxant properties [36–39]. However, our results
Table 1 Antivirus effects of polyphenols against influenza A and B viruses. No.
Polyphenols
TC50 (μg/ml) MDCK
IC50 (μg/ml) A/PR/8/34
SI
IC50 (μg/ml) B/Lee/1940
SI
1 2 3 4 5 6 7 8 9 10 11 12 13
(±)-Catechin (−)-EC (−)-EGC (−)-ECG (−)-EGCG Methylated EGC Procyanidin B-2 Procyanidin B-2 3,3′-di-O-gallate Theaflavin Theaflavin-3,3′-di-O-gallate Kaempferol Quercetin Myricetin
144.56 145.09 38.11 55.16 57.55 94.58 203.87 55.156 136.94 54.31 143.79 42.28 79.82
N144.56 N145.09 30.94 N55.16 56.49 N94.58 50.85 35.32 16.21 N54.31 N143.79 N42.28 N79.82
b1 b1 1.23 b1 1.01 b1 4 1.56 8.44 b1 b1 b1 b1
N144.56 N145.09 13.47 N55.16 11.09 33.43 56.02 N55.15 49.72 N54.31 8.98 N42.28 N79.82
b1 b1 2.82 b1 5.18 2.82 3.63 b1 2.75 b1 19.01 b1 b1
a Mean of results from three independent experiments; TC50, the concentration required for 50% cytotoxicity of the polyphenols; IC50, the concentration required for 50% inhibition of the virus CPE; The selection index (SI) was calculated by the ratio of TC50/IC50.
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Table 2 Antivirus effects of polyphenols against non-influenza viruses. No.
Polyphenols
TC50 (μg/ml) HEp-2
IC50 (μg/ml) RSV
SI
IC50 (μg/ml) ADV3
SI
1 2 3 4 5 6 7 8 9 10 11 12 13
(±)-catechin (−)-EC (−)-EGC (−)-ECG (−)-EGCG Methylated EGC Procyanidin B-2 Procyanidin B-2 3,3′-di-O-gallate Theaflavin Theaflavin-3,3′-di-O-gallate Kaempferol Quercetin Myricetin
144.56 145.09 62.45 27.58 57.55 104.49 240.32 47.95 68.47 163.38 28.67 7.97 39.91
N144.56 N145.09 N62.45 8.68 21.8 N104.49 N240.32 N47.95 N68.47 33.1 4.84 N7.97 N39.91
b1 b1 b1 3.17 2.63 b1 b1 b1 b1 4.93 5.92 b1 b1
N144.56 N145.09 N62.45 10.96 N57.55 93.07 N240.32 N47.95 13.62 59.34 N28.67 N7.97 N39.91
b1 b1 b1 2.51 b1 1.12 b1 b1 5.02 2.75 b1 b1 b1
a Mean of results from three independent experiments; TC50, the concentration required for 50% cytotoxicity of the polyphenols; IC50, the concentration required for 50% inhibition of the virus CPE; The selection index (SI) was calculated by the ratio of TC50/IC50.
did not show that quercetin has a direct anti-influenza virus effect in our MDCK cells infection model, which may be probably due to different study design and starting point. Many in vitro studies have shown that tea polyphenols can bind to proteins [40], wherefrom the compounds might combine with the glycoproteins of the viral envelope. It is particularly important that kaempferol (11) can inhibit the enveloped viruses such as hepatitis B, herpes simplex virus (HSV), JEV, and so on [41–43]. Among the tea polyphenols, kaempferol (11) exhibited a significant antiviral activity (EC50 = 15 μM) against HSV-1 [42]. Moreover, the inhibition effect of kaempferol on JEV was due to its binding with the frame-shift site RNA (fsRNA) in the JEV serogroup [43]. In addition, kaempferol (11) has been shown to be the most effective compound against influenza viruses (H1N1 and H9N2) through inhibition of neuraminidase activity [44], but no similar activity was observed in our repeated assays. These contradictory results may be due to different assays and warrant further investigation. 4.1. Structure–activity relationship of tea polyphenols in antiviral activity On the basis of the results of the bioassays of the tea polyphenols, the structure–activity relationships (SAR) are summarized as follows. First, the dimers of tea polyphenols, such as theaflavin (9) and procyanidin B-2 (7), generally display more potent antivirus activity against both influenza A and B viruses than the catechin monomers [i.e., (−)-EC (2) and (±)-catechin (1)]. Second, the galloyl group of both theaflavindigallate (10) and procyanidin B-2 digallate (8) was found to play an uncooperative role in their antivirus effect, probably due to its steric hindrance, as compared to theaflavin (9) and procyanidin B-2 (7). This is in contrast with the result that anti-HIV activity of betulinic acid increased when it forms a gallate conjugate [45]. Third, the highest SI value of 19.01 of kaempferol (11) toward influenza B virus indicated that the more planar flavonol with only one C-4′ phenolic hydroxyl group in the B ring is necessary for the anti-influenza B virus activity of flavonols. Such SAR can also be drawn from the assay of anti-RSV activity of all the tested flavonols. Fourth,
theaflavin (9) showed selectivity for inhibiting influenza A virus, while kaempferol (11) and EGCG (5) exhibited specificity for inhibiting influenza B virus. In addition, the phenolic hydroxyl groups of EGC (3) in the B ring play an important role in anti-influenza A virus activity but not in anti-influenza B virus activity, as compared with methylated EGC (6). 4.2. Hypothesis on the significant SAR of tea polyphenols in anti-influenza virus activity The above strict SAR of tested tea polyphenols is attributed to the chemical differences between two subgroups of flavonols and flavanols. The former is chemically more planar and rigid than the latter due to the presence of a C-2–C-3 double bond and a ketone in C-4. In other words, flavanols are more flexible than flavonols. In vitro neuraminidase (NA) inhibition assay [46] revealed that rigid flavonols (kaempferol, quercetin and myricetin) show much stronger inhibitory effect than flexible flavanols (catechin and epicatechin). The functionality of one hydroxyl at C-4′ position in the B ring, a double bond at C-2–C-3, and a ketone at the C-4 position in kaempferol (11) is essential for inhibiting influenza virus as a NA inhibitor. Addition of one more hydroxyl in the B ring, such as quercetin (12) and myricetin (13), causes steric hindrance and disfavors the anti-influenza effect [46]. Based on the literature report, we summarized that the flavonols are strong NA inhibitors, compared with the flavanols. Kaempferol (11) exerts a significant antiviral effect against influenza B virus most likely by targeting the NA of influenza B virus. We speculated that the striking specificity of kaempferol (11) for influenza B virus but not for A virus is probably due to the discrepancy in the NAs between A and B virus. Kaempferol (11) may possess a high affinity with the NA of influenza B. The reported SAR of kaempferol (11) in NA inhibition assay [46] is in full coincidence with our finding in the cellular level. However, the following two mechanisms might be responsible for the antiviral effects of tea catechins and its dimers on both influenza A and B viruses: one is the blockage of viral binding to the cell receptors in the early stage of a virus infection, and the other is attenuation of viral replication after entry [9,26,27,47,48]. Both theaflavin (9) and procyanidin B-2 (7), two dimers of
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