Molecular investigation of the virucidal activity of proanthocyanidin from Alpinia zerumbet against the in uenza A virus


 Proanthocyanidins (PACs) have various bioactivities, such as being anti-bacterial, anti-cancer, and anti-oxidant. Consequently, they have been vigorously studied for the development of new natural bioactive compounds. Recently, AzPAC was isolated from the medicinal plant Alpinia zerumbet, and it was found to inhibit the infection of animal viruses, influenza A viruses (IAVs), and porcine epidemic diarrhea virus. The virucidal activity of AzPAC means that it can interact directly with viral particles. However, few studies have investigated the preventive mechanism utilized by AzPAC on influenza virus replication. In this study, the composition of AzPAC and the affinity between AzPAC and IAVs was investigated in detail. We found that AzPAC was composed of an epicatechin monomer, which was linked by inter-flavan bonds between the C4 and C8 positions (B2-type) and the C4 and C6 positions (B5-type) in the terminal units of the PAC. A quenching assay indicated that AzPAC interacted with IAV membrane proteins, hemagglutinin and neuraminidase. Additionally, circular dichroism analysis indicated that AzPAC affected the change in the secondary structure rate of the viral membrane proteins. AzPAC was able to impair the infective process of IAVs via direct interaction with their viral membrane proteins. These results indicate that A. zerumbet is an invaluable bioresource for the development of preventive drugs against IAV infection.


Introduction
The three genera of in uenza viruses A, B, C, and D in the Orthomyxoviridae family cause contagious respiratory problems that lead to annual epidemics and pandemics around the world ( Alpinia zerumbet, namely shell ginger, is a representative medical and herbal plant that grows in tropical and subtropical zones of Asia, including the Okinawa prefecture in Japan. It is used for traditional cuisine and in perfumes, whereas many shell gingers have little industrial use. Many medicinal components have been isolated from the plants in the Alpinia genus, including shell gingers, and they are recognized as traditional folk medicines (Ghosh and Ranga 2013). Narusaka et al. (2020) recently demonstrated that the extract from a mixture of leaves and pseudostems of A. zerumbet possessed antiviral activity against two plant-speci c viruses, tomato mosaic virus (ToMV) and tobacco mosaic virus (TMV). Subsequently, polymetric procyanidin was isolated from an A. zerumbet extract and identi ed as an anti-plant viral molecule as well as an antiviral molecule against IAVs and porcine epidemic diarrhea virus (Hatanaka et al. 2021; Narusaka et al. 2021). However, detailed structures such as the end unit and terminal binding pattern of A. zerumbet-derived PAC (AzPAC), which are involved in the strong bioactivity of PAC, have not yet been determined. Additionally, the mechanism underlying the inhibitory process of IAV infection remains unknown.
The aim of this study was to clarify the compositional units of AzPAC and to investigate the binding a nity of PAC toward the IAV membrane proteins HA and NA. Moreover, the effect of AzPAC on the conformation of HA and NA was also assessed. These results have helped to elucidate the role of AzPAC in the prevention of IAV infection.

Analysis of PAC composition by phloroglucinosis
The leaves and pseudostems from A. zerumbet, polyphenol supplements originating from unripe fruit thinning of apple in the DHC Corporation (Tokyo, Japan), and the leaves of green tea (produced in Okayama, Japan) were used for PAC extraction. The extraction and con rmation of the weight-average mL methanol containing 5 g phloroglucinol and 1 g ascorbic acid) was added. The dissolvent was divided into equal parts (250 µL) into two microtubes. To con rm the presence of free 3-avan-ol monomers, one of the two tubes was immediately neutralized with 250 µL of 200 mM sodium acetate. The other aliquot for the acid-catalyzed cleavage of PA was incubated at 50°C for 20 min. The phloroglucinol reaction was stopped by adding 200 µL of 200 mM sodium acetate. The samples were centrifuged at 15,000 rpm for 10 min at 25°C. The supernatants were ltered using a 0.2 µm membrane centrifugal lter (Merck Millipore, Darmstadt, Germany) for the HPLC system. The LC/MS analysis was performed using a Shimadzu HPLC system (Shimadzu, Tokyo, Japan) connected to the MS spectrum Amazon SL-OP (Bruker, Massachusetts, U.S.A.). The ionization was conducted using an electrospray ionization (ESI) source, and their mass spectra were detected in the negative ion mode. The injection sample (1 µL) was subjected to HPLC using an Atlantis T3 column (2.5 × 100 mm, 3 µm; Waters, Manchester, UK) protected by a guard column containing the same material. The column was maintained at 40°C. The separation was performed using a linear gradient of solvent A (0.1% formic acid in H 2 O, v/v) and solvent B (acetonitrile). At a ow rate of 0.2 mL·min−1. The gradient parameter of the solvent B concentration was as follows: 0-3 min, 1%; 3-5 min, 6%; 5-15 min, 18%; 15-20 min, 55%; 20-20.1 min, 1%; 20.1-22 min, 1%. Additionally, a washing step was performed to prevent carryover as much as possible. The avan-3-ols and phloroglucinol adducts were detected based on the absorption at 280 nm and identi ed by co-chromatography using authentic chemical compounds and an MS spectrum. We identi ed the compounds by comparing their chemical properties with the following commercial standards using HPLC: (+)-catechin (Sigma-Aldrich, Tokyo, Japan), (-)-epicatechin (Nacalai Tesque, Inc., Kyoto, Japan), (-)-epicatechin-3-O-gallate, (-)-epigallocatechin (Tokyo Chemical Industry), (-)epigallocatechin (Tokyo Chemical Industry, Tokyo, Japan), procyanidin B1, procyanidin B2 (Extrasynthese, Genay, France), procyanidin B3 (ChemFaces, Wuhan, China), procyanidin B5 (Planta Analytica, New Milford, USA), chlorogenic acid (Sigma-Aldrich), neochlorogenic acid (Sigma-Aldrich), and 4-O-caffeoylquinic acid (Sigma-Aldrich). Phloroglucinol adducts for the monomeric epicatechin, monomeric catechin, and dimeric epicatechin were prepared from procyanidins B2, B3, and C1, respectively (Additional File 1: Figure S1).

Fluorescence Quenching Assay
The binding ability of PAC to the viral proteins was investigated using uorescence spectroscopy based on the Stern-Volmer equation (Keizer 1983), as follows:

Results And Discussion
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 13 C-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 clari ed the composition of AzPAC and the binding pattern of avan 3-ols. Epicatechinphloroglucinol 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-avan bond almost occupied the terminal pattern, followed by the C4-C6 inter-avan 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 avane-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)

Interaction of Azpac With Viral Membrane Proteins
The a nity between PACs and IAV membrane proteins was analyzed using a quenching assay. Both AzPAC and ApPAC decreased uorescence from HA and NA in a dose-dependent manner, whereas the addition of GtPAC to the viral proteins caused a gentle decrease in uorescence ( Fig. 2a and c).
Additionally, the top peak of the uorescence 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 con rmed 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 K sv values of AzPAC against both HA and NA were signi cantly higher than those of ApPAC and GtPAC ( Fig. 2b and d). These results indicated that AzPAC has a higher a nity 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 a nity 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 in uenced the rate of α-helix and random coil formation. When AzPAC interacted with HA, the rate of αhelix 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 βsheet 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.  AzPAC, Alpinia zerumbet-derived PAC; ApPAC, immature apple fruit-derived PAC; GtPAC, green teaderived 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 e ciency 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 a nity against the IAV-derived proteins and the anti-IAV activity, as was clari ed by Narusaka et al. (2021). Otherwise, the bioactivity is thought to be affected by the three-dimensional structure determined by the type of avan 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 e ciency 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 IAVderived membrane proteins with changes in the secondary structure of the proteins, and consequently impairs the attachment of the viral particles to the host cells.

Conclusions
In this study, we have identi ed the PAC components from A. zerumbet, which possess high virucidal activity against IAVs. AzPAC strongly interacted with the IAV-derived membrane proteins HA and NA. Furthermore, AzPAC changed the secondary structural rate of the α-helix and the random coil of HA and NA, which suggested that there would be a reduction in the infectious activity of the functional protein.
Consequently, AzPAC prevents IAV infection by impairing the attachment of viral membrane proteins to host cells. These results will contribute to the development of drugs for the prevention of IAV infections in the future.