Characterization of anti-viral PAC derived from A. zerumbet
Previously, the structure of AzPAC (more than 40 DP degree of polymerization) from A. zerumbet was investigated using a DMAC assay and mechanical analyses with MALDI-TOF-MS and 13C-NMR (Hatanaka et al. 2021). The results indicated that AzPAC consisted of an epicatechin unit in a B-type carbon-carbon bond. However, the terminal binding pattern and end unit of AzPAC remain unknown. Here, phloroglucinosis clarified the composition of AzPAC and the binding pattern of flavan 3-ols. Epicatechin-phloroglucinol was detected, and the extension unit of AzPAC was epicatechin (Fig. 1a). The procyanidin B2-phloroglucinol and two unknown procyanidin B-type-dimer-adducts were detected, and the C4-C8 bond occupied over half of the extension pattern. After phloroglucinosis, procyanidins B2 and B5 were detected in 73.1% and 26.5% of cases, respectively, and procyanidin B1 was detected in trace amounts. The results indicated that the C4-C8 inter-flavan bond almost occupied the terminal pattern, followed by the C4-C6 inter-flavan bond. These results showed that more than 99% of the end unit in AzPAC was epicatechin, and the terminal structure of AzPAC showed B2 and B5 linkage patterns (Fig. 1b). ApPAC was formed of only epicatechin in the extension unit and consisted of 75% epicatechin and 25% catechin in the terminal units (Fig. 1c). This result was similar to the ratio of catechin and epicatechin as terminal units in the PAC-rich fraction extracted from apple fruit skin previously (Mendoza-Wilson et al. 2016). Additionally, chlorogenic acid was detected in phloroglucinosis of AzPAC, but this compound originally contained in ApPAC extract (data not shown). The oligomeric PAC extracts isolated from green tea leaves (GtPAC) were predominantly comprised of 2,3-cis stereochemistry flavane-3-ol, which accounted for over 90% of the total terminal units, and epicatechin and epigallocatechin 3-gallate accounted for 20% and 41.8%, respectively (Fig. 1d). The extension units of GtPAC consisted of 40.8% and 53.0% catechin and epicatechin, respectively. The (epi)gallocatechin-phloroglucinols were estimated to be extension units by MS analysis. Previously, Punyasiri et al. (2004) indicated that PAC isolated from tea leaves possessed gallocatechins as extension units. Thus, these results suggested that GtPAC had (epi)gallocatechin as a minor unit in the extension of PAC. In this study, the terminal patterns of ApPAC and GtPAC could not be determined because these PAC extracts contained substantial amounts of procyanidin dimers.
Interaction of Azpac With Viral Membrane Proteins
The affinity between PACs and IAV membrane proteins was analyzed using a quenching assay. Both AzPAC and ApPAC decreased fluorescence from HA and NA in a dose-dependent manner, whereas the addition of GtPAC to the viral proteins caused a gentle decrease in fluorescence (Fig. 2a and c). Additionally, the top peak of the fluorescence of HA shifted toward the red spectrum in the presence of a high concentration 0.16 mg/mL of AzPAC. The blue shifts were clearly confirmed in the NA in the presence of all types of PAC, suggesting a change in the NA conformation. This change in the NA conformation may be related to the anti-IAV activities of AzPAC, ApPAC, and GtPAC that were previously reported by Narusaka et al. (2021). The Ksv values of AzPAC against both HA and NA were significantly higher than those of ApPAC and GtPAC (Fig. 2b and d). These results indicated that AzPAC has a higher affinity for the two viral membrane proteins when compared with ApPAC and GtPAC.
The CD spectrum of the proteins in the presence of PAC was also evaluated to understand the change in the secondary structure of the viral membrane proteins, in addition to the binding affinity of PAC against viral proteins. When AzPAC and ApPAC interacted with HA, the rates of the secondary structures changed, but not GtPAC. In particular, the interaction of AzPAC and ApPAC with the viral proteins preferentially influenced the rate of α-helix and random coil formation. When AzPAC interacted with HA, the rate of α-helix a in the secondary structures of HA decreased by 2.0% and was accompanied by a 2.4% increase in the random coil rate (Table 1 and Fig. 3a). On the other hand, when ApPAC interacted with HA, the rates of α-helix and random coil increased or decreased by 1.8% and 1.1%, respectively. Additionally, the rate of β-sheet antiparallel in HA decreased by 0.9% in the presence of ApPAC. The rate of α-helix and NA decreased or increased by 3.2%, accompanied by a 2.4% increase in the rate random coil in the presence of AzPAC (Table 2 and Fig. 3b). The interaction of ApPAC with NA changed the rate of the α-helix in NA from 82.3–84.5%. An increase of 1.1% in the rate of the α-helix of NA was observed after the addition of GtPAC. The change in the rate of α-helix in NA after the addition of the three PACs may be attributed to the blue shift in NA in the presence of the three PACs in the quenching assay.
Table 1
Hemagglutinin secondary structures in the presence of AzPAC, ApPAC and GtPAC.
Sample | Percentage of the hemagglutinin (HA) secondary structures |
α-helix | β-sheet antiparallel | β-sheet parallel | β-turn | Random coil |
HA free | 39.3 | 8.7 | 6.1 | 17.0 | 20.1 |
HA+AzPAC | 37.3 | 9.2 | 6.6 | 17.1 | 22.5 |
HA+ApPAC | 41.1 | 7.8 | 5.8 | 16.9 | 19.0 |
HA+GtPAC | 39.5 | 8.5 | 6.1 | 17.0 | 20.1 |
AzPAC, Alpinia zerumbet-derived PAC; ApPAC, immature apple fruit-derived PAC; GtPAC, green tea-derived PAC. |
Table 2
Neuraminidase secondary structures in the presence of AzPAC, ApPAC and GtPAC.
Sample | Percentage of the neuraminidase (NA) secondary structures |
α-helix | β-sheet antiparallel | β-sheet parallel | β-turn | Random coil |
NA free | 82.3 | 0.1 | 1.9 | 9.6 | 6.2 |
NA+AzPAC | 79.1 | 0.2 | 2.4 | 9.8 | 8.6 |
NA+ApPAC | 84.5 | 0.1 | 1.8 | 9.1 | 5.8 |
NA+GtPAC | 83.4 | 0.1 | 1.8 | 9.4 | 5.8 |
AzPAC, Alpinia zerumbet-derived PAC; ApPAC, immature apple fruit-derived PAC; GtPAC, green tea-derived PAC. |
Relationship between PAC structure and the inhibition of viral infection
The mean degree of polymerization of the PACs from the kiwifruit pericarps affected both the binding ability with tyrosinase and the efficiency of tyrosinase inhibition (Chai et al. 2014), suggesting that the degree of polymerization is important for bioactivity. This study also suggested that the high polymerization degree of PAC is related to the strength of the affinity against the IAV-derived proteins and the anti-IAV activity, as was clarified by Narusaka et al. (2021). Otherwise, the bioactivity is thought to be affected by the three-dimensional structure determined by the type of flavan 3-ol in the extension and end units of the PAC (Takanashi et al. 2017). Epicatechin pentamer strongly suppressed the gene expression of the fatty acid-binding protein 5, involving cancer-cell invasion, in comparison with the arecatannins A2 and A3, which possess catechin as the end unit. Therefore, the higher anti-IAV activity of AzPAC could be derived from the high degree of polymerization of epicatechin and the conformation that is familiar to the viral proteins. Generally, NA plays an important role in release and spread of the daughter cells from the host cells, but it has been shown that NA helps HA to bind to the sugar chains, thereby increasing the efficiency of infection in a recent report (Sakai et al. 2017). Considering the results of the present study and the recent report concerning virus motility, we have suggested that AzPAC binds strongly to the IAV-derived membrane proteins with changes in the secondary structure of the proteins, and consequently impairs the attachment of the viral particles to the host cells.