Kunihiro Kaihatsu

Enhanced anti-influenza A virus activity of (−)-epigallocatechin-3-O-gallate fatty acid monoester derivatives: Effect of alkyl chain length

A series of fatty acid monoester derivatives of (-)-epigallocatechin-3-O-gallate (EGCG) were prepared by one-pot lipase-catalyzed transesterification. The introduction of long alkyl chains enhanced anti-influenza A/PR8/34 (H1N1) virus activity 24-fold relative to native EGCG.

Antioxidant and antiviral activities of silybin fatty acid conjugates

Two selective acylation methods for silybin esterification with long-chain fatty acids were developed, yielding a series of silybin 7-O- and 23-O-acyl-derivatives of varying acyl chain lengths. These compounds were tested for their antioxidant (inhibition of lipid peroxidation and DPPH-scavenging) and anti-influenza virus activities. The acyl chain length is an important prerequisite for both biological activities, as they improved with increasing length of the acyl moiety.

Antibacterial and Antifungal Activities of New Acylated Derivatives of Epigallocatechin Gallate

(−)-Epigallocatechin-3-O-gallate (EGCG) has useful antiviral, antimicrobial, antitoxin, and antitumor properties. Previously, Mori et al. (2008) found that addition of long acyl chains (C16–18) to EGCG enhanced its anti-influenza virus activity up to 44-fold. The chemical stability of EGCG against oxidative degradation was also enhanced by acylation. We further evaluated the in vitro activity spectrum of the EGCG derivatives against a wide range of bacteria and fungi. A series of EGCG O-acyl derivatives were synthesized by lipase-catalyzed transesterification. These derivatives exhibited several-fold higher activities than EGCG, particularly against Gram-positive organisms. Antifungal MICs of the derivatives were also two to fourfold lower than those of EGCG. The activities of the EGCG derivatives against Gram-negative bacteria were not distinguishable from those of EGCG. Among the derivatives evaluated, MICs of dioctanoate and palmitate (C16) for 17 Staphylococcus aureus strains were 4–32 μg/ml, although MIC of EGCG for these 17 strains was ≥128 μg/ml. C16 demonstrated rapid bactericidal activity against methicillin-resistant S. aureus (MRSA) ATCC43300 at ≥16 μg/ml. The enhanced activity of C16 against S. aureus was supported by its increased membrane-permeabilizing activity determined by increased SYTOX Green uptake. The EGCG derivatives were exported in Escherichia coli using the efflux pump AcrAB–TolC. The tolC deletion mutant exhibited higher sensitivity to EGCG and the derivatives than wild-type. Addition of long alkyl chains to EGCG significantly enhanced its activities against several bacteria and fungi, particularly against S. aureus including MRSA. C16 might potentially become under specified circumstances an alternative or supplement to antibiotics and disinfectants in the future.

Enhanced antitumor activities of (−)-epigallocatechin-3-O-gallate fatty acid monoester derivatives in vitro and in vivo

(-)-Epigallocatechin-3-O-gallate (EGCG) monoesters modified with butanoyl (EGCG-C4), octanoyl (EGCG-C8), palmitoyl groups (EGCG-C16) were synthesized by a lipase-catalyzed transesterification method and their antitumor activities were investigated in vitro and in vivo. The in vitro antitumor activities of EGCG-monoester derivatives increased in an alkyl chain length-dependent manner. The cytotoxicity of EGCG, EGCG-C4, EGCG-C8 was mainly caused by H(2)O(2) which was generated with their oxidation. On the other hand, EGCG-C16 was more stable than EGCG and it did not generate H(2)O(2) in the cell culture medium. Furthermore, EGCG-C16 inhibited cell proliferation and induced apoptosis in the presence of catalase. EGCG-C16 was found to inhibit the phosphorylation of the epidermal growth factor receptor (EGFR), which is related to various types of tumor growth. EGCG-C16 suppressed tumor growth in vivo in colorectal tumor bearing mice in comparison to an untreated control, vector control (DMSO) and EGCG.

Virus purification and enrichment by hydroxyapatite chromatography on a chip

Abstract The spread of infectious diseases has become a global health concern. In order to diagnose infectious diseases quickly and accurately, next-generation DNA sequencing techniques for genetic analysis of infectious viruses have been developed rapidly. However, it takes a very long time to pretreat clinical samples for genetic analysis using next-generation sequencers. We have therefore developed a microfluidic chromatography chip that can purify and enrich viruses in a sample using hydroxyapatite particles packed in a micro-column. We demonstrated the purification of virus from a mixture of virus and FBS protein, and enrichment of the virus using this novel microfluidic chip.

Label-free single-particle imaging of the influenza virus by objective-type total internal reflection dark-field microscopy.

Here we report label-free optical imaging of single particles of the influenza virus attached on a glass surface with a simple objective-type total internal reflection dark-field microscopy (TIRDFM). The capability of TIRDFM for the imaging of single viral particles was confirmed from fine correlation of the TIRDFM images with fluorescent immunostaining image and scanning electron microscopy image. The density of scattering spots in the TIRDFM images showed a good linearity against the virus concentration, giving the limit of detection as 1.2×104 plaque-forming units per milliliter. Our label-free optical imaging method does require neither elaborated sample preparation nor complex optical systems, offering a good platform for rapid and sensitive counting of viral particles.

Broad and potent anti-influenza virus spectrum of epigallocatechin-3-O-gallate-monopalmitate.

Numerous influenza pandemics in the last century resulted in the death of millions of people worldwide. With the current and future threats of influenza pandemics development of new antiviral compounds remains a great demand. However, currently only a limited number of drugs are available for the treatment and prophylaxis of influenza. A neuraminidase (NA) inhibitor, oseltamivir phosphate, is the most commonly used antiviral drug. However, a number of reports suggest the emergence of oseltamivir phosphate resistance in new seasonal influenza viruses and highly pathogenic avian influenza (H5N1) (for example, de Jong et al, 2005). (–)-Epigallocatechin-3-O-gallate (EGCG: 1), a major component of green tea plant (Camellia sinensis), has been recognized to possess antiviral properties. Reports on the anti-influenza activity of 1 found that it inhibited virus adsorption (Nakayama et al, 1993), as well as acidification of endosomes and lysosomes (Imanishi et al, 2002). Such virus inhibition activity is different from other current NA or proton pump inhibitors, suggesting that 1 can be developed into a new class of antiviral compounds that are effective against current drug resistant influenza strains. However, 1 has not been used as an antiviral compound because of its poor lipid membrane permeability and low chemical stability. It was previously reported that the introduction of long alkyl chain groups to 1 improved its lipid membrane permeability (Tanaka et al, 1998), while protection of its hydroxyl groups with acyl groups increased its chemical stability under physiological conditions (Lam et al, 2004). Recently, we reported a method to synthesize EGCG-fatty acid monoesters using lipase-catalyzed transesterification and demonstrated that EGCG-fatty acids monoesters possessed improved influenza virus inhibitory effect against influenza A/PR8/34/(H1N1) in an alkyl length dependent manner (Mori et al, 2008). Here, we investigated the spectrum of influenza virus inhibition activity of EGCG (1) and EGCG-C-16 (2). As shown in Figures 1a, 2 is a mixture of four regio-isomers and the ratio of each regio-isomer 2a:2b:2c:2d is 38:35:7:20, respectively. Because the B-ring-modified esters (2a and 2b) and D-ring-modified esters (2c and 2d) showed exactly the same antiviral activities (data not shown), we used the mixture of four regio-isomers (2a-d) in the following assays. Figure 1. A. Chemical structure of EGCG (1) and EGCG-C16 (2). 2 is a mixture of four regio-isomers (2a-d). 2a: R2=R3= R4=H, R1= CO(CH)14CH3, 2b: R1=R3=R4=H, R2= CO(CH)14CH3, 2c: R1=R2=R4=H, R3= CO(CH)14CH3, 2d: R1=R2=R3=H, R4= CO(CH)14CH3. B. Avian influenza virus ... A series of human influenza viruses, an experimental strain (A/Puerto Rico/8/34/(H1N1)), vaccine strains (A/Beijing/262/95/(H1N1), A/Panama/2007/99/(H3N2), and B/Yamanashi/166/98/), drug-resistant strains (Yokohama/77/2008/(H1N1) OPR: oseltamivir phosphate-resistant (OPR), Yokohama/63/2007/(H1N1) AR: amantadine-resistant (AR), A/Yokohama/91/2008/(H1N1) OPR/AR: (OPR/AR) and avian pathogenic influenza (A/Duck/Hong Kong/342/78/(H5N2)), were directly incubated with 1 or 2 for 30 min prior to inoculation into MDCK cells. The cells were inoculated with virus, with or without the compounds, for 1hr, and the direct virus inhibitory effects were assessed by a plaque formation assay at 54hr post-incubation. The plaque inhibition activity was calculated relative to no compound. The cytotoxicity of compounds on MDCK cells were evaluated by the MTT cell proliferation assay. Briefly, the cells were incubated with 1 or 2 for 24hr for the MTT assay. The results of the plaque inhibition assay and MTT assay are expressed as mean ± standard error of three independent experiments. We also investigated the virus inhibition activity of 1 and 2 on avian influenza A/Duck/Hong Kong/342/78 (H5N2) virus in ovo using 11-day-old chicken embryonated eggs (n=12) inoculated with compound-treated or untreated viruses. As shown in Table 1, both 1 and 2 showed broad virus inhibitory effects on MDCK cells. The EC50 values of 2 on these viruses were between 10 to 61nM, which were approximately 7.1 to 44-fold lower than those of 1. The CC50 value of 2 was 82μM, which was only 3.1-fold lower than that of 1 (255 μM). Thus, the SI value of 2 was improved 2.2 to 14-fold compared to 1. Table 1. Direct virus inhibitory effect of 1 and 2 based on the plaque formation assay With respect to the avian influenza virus inhibition assay in ovo, virus treated with 1, zanamivir, or oseltamivir phosphate showed a moderate viral inhibitory effect (Figures 1b). However, 2 almost completely inhibited the infection (Figures. 1b), and the efficacy was retained even at 0.1μM (data not shown). In summary, 2 inhibited human and avian influenza A and B viruses, including drug-resistant viruses. 2 was found to be more effective than neuraminidase inhibitors, and strongly inhibited the infection of avian influenza (H5N2) virus in chicken embryonated eggs. This unique viral inhibitory action has the potential to be utilized to effectively control a broad spectrum of influenza viruses.

Potential Anti-Influenza Virus Agents Based on Coffee Ingredients and Natural Flavonols

Natural phenolic compounds have been reported to directly inhibit influenza virus. Nonetheless, there have been no reports on the direct inhibitory effects of coffee ingredients. Here, we fractionated the hydrophobic and hydrophilic components of coffee. The hydrophobic fraction directly inhibited both a seasonal influenza A/Puerto Rico/8/24(H1N1) virus and a neuraminidase-resistant influenza A/Yokohama/77/2008(H1N1) virus infection in a dosedependent manner, while the hydrophilic fraction did not show any inhibitory effects, even at concentrations above 100 μg/ml. The HPLC profile of the hydrophobic fraction indicated that caffeine is the major component. Indeed, caffeine alone showed comparable anti-influenza virus activity. Interestingly, caffeic acid also inhibited viral infection, while chlorogenic acid, which is an ester of caffeic acid and (-)-quinic acid, showed no obvious antiviral effect at less than 2 mg/ml. We previously reported a method for enhancing the anti-influenza virus activity of epigallocatechin gallate, a major tea catechin, by lipase-catalyzed acylation. Using this methodology, we synthesized fatty acid esters of caffeic acid and evaluated their influenza virus-inhibitory effects. It was found that dioctanoyl ester of caffeic acid exhibited approximately 38-fold higher direct antiviral activity. To understand the essential structure required for virus inhibition, we further examined the antiviral activity of natural flavonoids containing either the caffeic acid skeleton or its analogous structure. Flavonols (quercetin, myricetin and morin) and hexahydrobenzophenone containing extended planar pi-conjugated systems efficiently inhibited the virus infection. Flavonoids possessing both radical scavenging activity and cytotoxicity tended to show higher antiviral activity, probably due to their affinity with viral surface factors. On the other hand, there was no apparent correlation between their antiviral activity and antioxidative activity. These findings provide insight into the design of anti-influenza virus agents from natural polyphenols.

Substitution at the C-3 Position of Catechins Has an Influence on the Binding Affinities against Serum Albumin

It is known that catechins interact with the tryptophan (Trp) residue at the drug-binding site of serum albumin. In this study, we used catechin derivatives to investigate which position of the catechin structure strongly influences the binding affinity against bovine serum albumin (BSA) and human serum albumin (HSA). A docking simulation showed that (−)-epigallocatechin gallate (EGCg) interacted with both Trp residues of BSA (one at drug-binding site I and the other on the molecular surface), mainly by π–π stacking. Fluorescence analysis showed that EGCg and substituted EGCg caused a red shift of the peak wavelength of Trp similarly to warfarin (a drug-binding site I-specific compound), while 3-O-acyl-catechins caused a blue shift. To evaluate the binding affinities, the quenching constants were determined by the Stern–Volmer equation. A gallate ester at the C-3 position increased the quenching constants of the catechins. Against BSA, acyl substitution increased the quenching constant proportionally to the carbon chain lengths of the acyl group, whereas methyl substitution decreased the quenching constant. Against HSA, neither acyl nor methyl substitution affected the quenching constant. In conclusion, substitution at the C-3 position of catechins has an important influence on the binding affinity against serum albumin.

Sequence-Specific and Visual Identification of the Influenza Virus NS Gene by Azobenzene-Tethered Bis-Peptide Nucleic Acid

To rapidly and specifically identify highly virulent influenza virus strains, we prepared an azobenzene-tethered hairpin-type peptide nucleic acid, bisPNA-AZO, which has a complementary sequence against a highly conserved genomic RNA sequence within the ribonucleoprotein complex of the 2009 pandemic influenza A virus, H1N1 subtype. bisPNA-AZO recognizes the conserved virus genome sequence in a sequence-specific manner. Immobilization of bisPNA-AZO on a plate allowed capture of the target virus gene and the generation of a visual colour signal.