Anti-Influenza Virus Activity of Citrullus lanatus var. citroides as a Functional Food: A Review.

Influenza is an acute respiratory illness caused by the influenza virus, in response to which vaccines and antiviral drugs are administered. In recent years, the antiviral effects of plants and foods have garnered attention. This review is the first to summarize the therapeutic properties of wild watermelon (Citrullus lanatus var. citroides) against influenza from a phytochemical viewpoint. Wild watermelon is a wild plant with significant potential as a therapeutic candidate in antiviral strategies, when focused on its multiple anti-influenza functionalities. Wild watermelon juice inhibits viral growth, entry, and replication. Hence, we highlight the possibility of utilizing wild watermelon for the prevention and treatment of influenza with stronger antiviral activity. Phytochemicals and phytoestrogen (polyphenol, flavonoids, and prenylated compounds) in wild watermelon juice contribute to this activity and inhibit various stages of viral replication, depending on the molecular structure. Wild plants and foods closely related to the original species contain many natural compounds such as phytochemicals, and exhibit various viral growth inhibitory effects. These natural products provide useful information for future antiviral strategies.

Anti-influenza A virus activity of flavonoids in vitro: a structure-activity relationship.

Secondary plant metabolites from food extracts, namely daidzein, quercetin, and luteolin, exhibit anti-influenza virus effects, with IC50 values of 143.6, 274.8, and 8.0 µM, respectively. The activities of these metabolites differ depending on the functional groups. Therefore, in this study, we focused on members of the flavonoid group, and investigated the anti-influenza viral effects of different flavonoid classes (flavone, isoflavone, flavonol, flavanone, and flavan-3-ol) in vitro. The IC50 values were 4.9-82.8 µM, 143.6 µM, 62.9-477.8 µM, 290.4-881.1 µM, and 22.9-6717.2 µM, respectively, confirming their activity. The modifying group factors (number, position, type) in the flavonoid skeleton may be significantly related to the anti-influenza virus activity. Moreover, time-of-addition assay revealed that the mechanism of inhibition varied for the different classes; for example, flavonoids that inhibit virus adsorption or the early stage of viral growth. Interestingly, all the examined flavonoids inhibited the late stages of viral growth, suggesting that flavonoids mainly inhibit the late events in viral growth before the release of viral particles. Additionally, apigenin might be effective against oseltamivir-resistant strains. Our results may be important in the development of anti-influenza virus therapeutic strategies in the future.

Effect of Structural Differences in Naringenin, Prenylated Naringenin, and Their Derivatives on the Anti-Influenza Virus Activity and Cellular Uptake of Their Flavanones.

Vaccines and antiviral drugs are widely used to treat influenza infection. However, they cannot rapidly respond to drug-resistant viruses. Therefore, new anti-influenza virus strategies are required. Naringenin is a flavonoid with potential for new antiviral strategies. In this study, we evaluated the antiviral effects of naringenin derivatives and examined the relationship between their cellular uptake and antiviral effects. Madin-Darby canine kidney (MDCK) cells were infected with the A/PR/8/34 strain and exposed to the compound-containing medium for 24 h. The amount of virus in the supernatant was calculated using focus-forming reduction assay. Antiviral activity was evaluated using IC50 and CC50 values. Cells were exposed to a constant concentration of naringenin or prenylated naringenin, and intracellular uptake and distribution were evaluated using a fluorescence microscope. Prenylated naringenin showed strong anti-influenza virus effects, and the amount of intracellular uptake was revealed by the strong intracellular fluorescence. In addition, intracellular distribution differed depending on the position of the prenyl group. The steric factor of naringenin is deeply involved in influenza A virus activity, and prenyl groups are desirable. Furthermore, the prenyl group affects cellular affinity, and the uptake mechanism differs depending on its position. These results provide important information on antiviral strategies.

Influenza virus entry and replication inhibited by 8-prenylnaringenin from Citrullus lanatus var. citroides (wild watermelon).

We previously demonstrated the anti-influenza activity of Citrullus lanatus var. citroides (wild watermelon, WWM); however, the active ingredient was unknown. Here, we performed metabolomic analysis to evaluate the ingredients of WWM associated with antiviral activity. Many low-molecular weight compounds were identified, with flavonoids accounting for 35% of all the compounds in WWM juice. Prenylated flavonoids accounted for 30% of the flavonoids. Among the measurable components of phytoestrogens in WWM juice, 8-prenylnaringenin showed the highest antiviral activity. We synthesized 8-prenylnaringenin and used liquid chromatography-mass spectrometry to quantitate the active ingredient in WWM. The antiviral activities of 8-prenylnaringenin were observed against H1N1 and H3N2 influenza A subtypes and influenza B viruses. Moreover, 8-prenylnaringenin was found to inhibit virus adsorption and late-stage virus replication, suggesting that the mechanisms of action of 8-prenylnaringenin may differ from those of amantadine and oseltamivir. We confirmed that 8-prenylnaringenin strongly inhibited the viral entry of all the influenza virus strains that were examined, including those resistant to the anti-influenza drugs oseltamivir and amantadine. This result indicates that 8-prenylnaringenin may activate the host cell's defense mechanisms, rather than directly acting on the influenza virus. Since 8-prenylnaringenin did not inhibit late-stage virus replication of oseltamivir-resistant strains, 8-prenylnaringenin may interact directly with viral neuraminidase. These results are the first report on the anti-influenza virus activity of 8-prenylnaringenin. Our results highlight the potential of WWM and phytoestrogens to develop effective prophylactic and therapeutic approaches to the influenza virus.

Inhibition of influenza virus replication by Apiaceae plants, with special reference to Peucedanum japonicum (Sacna) constituents.

ETHNOPHARMACOLOGICAL RELEVANCE: Apiaceae plants possess various pharmacological properties, such as antimicrobial, antioxidant, hypoglycemic, hypolipidemic, anxiolytic, analgesic, anti-inflammatory, anti-convulsant, and anti-cancer activities; however, data on their antiviral activity are limited. Peucedanum japonicum, also known as Sacna, is a plant used as food and as a traditional folk medicine for treating coughs. However, the active components in the leaves of this plant are yet unexplored. AIM OF THE STUDY: To assess Apiaceae plants, especially Peucedanum japonicum, with anti-viral activity, and the function and antiviral potential of Sacna constituents, considering the emergence of influenza virus strains resistant to the currently available drugs. MATERIALS AND METHODS: We prepared grinds of the freeze-dried leaves and roots of the Apiaceae family and the hot water extracts. The antiviral activities of the extracts were determined by focus formation reduction assay. In the time-of-addition assay, the test medium containing Sacna extract at 2 mg/mL was added at -1 to 0 h (adsorption) or from 0 to 4, 4 to 8, or 0 to 8 h (replication). The Sacna extract was separated by reversed-phase flash column chromatography using an Isolera Spektra system. The antiviral activity of each fraction was then determined using the focus formation reduction assay. The active fraction was analyzed using an LC20ADXR high performance liquid chromatography system equipped with a microTOF-QII quadrupole time-of-flight tandem mass spectrometer. RESULTS: All examined extracts of Apiaceae plants showed anti-influenza activity. Sacna extract most strongly inhibited the replication of influenza viruses. Individual components of Sacna possess antiviral activities against the influenza A/PR/8/34 virus. Sacna was found to inhibit the multiplication of A (H1N1 and H3N2) types and B types of influenza viruses, including amantadine-resistant and oseltamivir-resistant viruses. Sacna also inhibited influenza infection during viral replication. However, Sacna did not inhibit influenza infection during cell adsorption and did not suppress hemagglutination inhibition or cell fusion. Further, our findings suggest that the antiviral compounds in Sacna include flavonoids (quercetin and luteolin) and other polyphenols (caffeic acid, hymecromone, and umbelliferone). Although several effective compounds in Sacna inhibit multiple steps of viral replication, caffeic acid, which was increased by heat treatment at the time of extraction, significantly inhibited only the late period of viral growth, similar to the Sacna extract, indicating that it is the major component responsible for the antiviral activity of Sacna. CONCLUSIONS: Apiaceae plants possess antiviral activity. Caffeic acid is the major component responsible for the antiviral activity of Sacna. To our knowledge, this is the first report regarding the anti-influenza virus activity of Sacna. Overall, these results indicate that Sacna has potential as a novel treatment against influenza A and B viruses.

Difference in Movement between Superficial and Deep Parts of the Infrapatellar Fat Pad during Knee Extension.

(1): The superficial and deep parts of the infrapatellar fat pat (IFP) have different morphological and functional characteristics. Knee pain often occurs during movement, and it is important to clarify the movement of the IFP during knee joint movement. The purpose of this study is to clarify that the movement of the superficial and deep parts of the IFP are different during knee extension in vivo using ultrasonography (US). (2): US was performed on 15 knees of 15 healthy adults. The probe was placed longitudinally at the center of the patellar tendon and the IFP was imaged. Measurements were taken during active extension of the knee from 90 degrees to 10 degrees of knee flexion at a rate of 30 times/min. The captured US videos were analyzed using Flow particle image velocimetry (Flow PIV) fluid measurement software. The region-of-interest (ROI) was set at the superficial part and the deep part of the IFP, and the flow velocity was calculated for each. (3): The flow velocity of the deep part (1.37 ± 0.13 cm/s) of the IFP was significantly faster than that of the superficial part (0.80 ± 0.23 cm/s). (4): Our results show that the flow velocity of the IFP is different between the superficial and deep parts and that US may be a better assessment tool for the movement of the IFP.

Juice of Citrullus lanatus var. citroides (wild watermelon) inhibits the entry and propagation of influenza viruses in vitro and in vivo.

Vaccines and various anti-influenza drugs are clinically used to prevent and treat influenza infections. However, with the antigenic mismatch of vaccines and the emergence of drug-resistant viral strains, new approaches for treating influenza are warranted. This study focused on natural foods as potential candidates for the development of new treatment options for influenza infections. The screening of plants from the Cucurbitaceae family revealed that the juice of Citrullus lanatus var. citroides (wild watermelon) had the strongest ability to inhibit the replication of influenza virus in Madin-Darby canine kidney cells. The results of a time-of-addition assay indicated that wild watermelon juice (WWMJ) inhibits the adsorption and late stages of viral replication, suggesting that WWMJ contains multiple constituents with effective anti-influenza activity. A viral adsorption analysis showed that WWMJ reduces the amount of viral RNA in the cells at 37°C but not at 4°C, confirming that WWMJ inhibits viral entry into the host cells at 37°C. These results suggest that a mechanism other than the inhibition of viral attachment is involved in the anti-influenza action of WWMJ, which is perhaps responsible for a reduction in internalization of the virus. Administration of WWMJ into the nasal mucosa of BALB/c mice infected with the A/PR/8/34 mouse-adapted influenza virus was seen to significantly improve the survival rate. The findings of this study, therefore, demonstrate the anti-influenza potential of WWMJ in vitro and in vivo, thereby suggesting the candidature of WWMJ as a functional food product that can be used to develop anti-influenza agents and drugs.

Induction of a 5-lipoxygenase product by daidzein is involved in the regulation of influenza virus replication.

This study was conducted to evaluate the regulation mechanism of influenza virus replication following treatment of Madin-Darby canine kidney cells with the soy isoflavone daidzein. We performed comparative qualitative and quantitative analyses of lipid peroxide between mock-infected and virus-infected cells treated with or without daidzein, as it had been reported that daidzein was an antioxidant and lipid peroxide levels increased upon virus infection. Contrary to our belief, lipid peroxides were not elevated in virus-infected cells and no decrease in lipid peroxides was observed in daidzein-treated cells. In daidzein-treated cells, 5-hydroxyeicosatetraenoic acid, the 5-lipoxygenase product derived from arachidonate, was significantly elevated compared to other lipid peroxides. Zileuton (5-lipoxygenase inhibitor) and 5-lipoxygenase knockdown reduced the daidzein-induced antiviral effect. Moreover, virus replication was regulated by treatment with 5-hydroperoxyeicosatetraenoic acid, a precursor of 5-hydroxyeicosatetraenoic acid and 5-lipoxygenase primary product. These results suggest that daidzein regulates virus replication via signal transduction through 5-lipoxygenase products.

Anti-Influenza Virus Activity of Adlay Tea Components.

Our previous study showed anti-influenza virus activity in adlay tea prepared from adlay seeds, naked barley seeds, soybean, and cassia seeds. In this study, we evaluated the anti-influenza virus activity of each component of this tea and analyzed their active ingredients. Each component was roasted and extracted in hot water; the extracts were tested for antiviral activity and their mechanisms of action were studied. All the tea components showed antiviral activity against the H1N1 and H3N2 influenza subtypes and against influenza B. The viral stages inhibited by the components were virus adsorption and replication in proliferative process, suggesting that the action mechanisms of the components might differ from those of oseltamivir acid. Of the tea components, soybean showed the strongest activity. Therefore, we analyzed its active ingredients by liquid chromatography quadruple time-of-flight mass spectrometry (LC/qTOF-MS) and daidzein and glycitein were detected as active ingredients. Here, anti-influenza virus action of glycitein was the first report.

Inhibition of influenza virus replication by adlay tea.

BACKGROUND: The present study was conducted aiming to examine the antiviral activity of adlay tea and its components against influenza viruses. We further aimed to clarify the mechanism by which these components regulate virus replication. RESULTS: Adlay tea at a concentration suitable for drinking inhibited the multiplication of influenza viruses. Moreover, our results suggest that individual components of the tea had antiviral activities against the influenza A/PR/8/34 virus. Adlay tea inhibited multiplication of the H1N1, H3N2 and B types of influenza virus, including oseltamivir-resistant viruses. In addition, adlay tea inhibited influenza infection during the periods of virus adsorption to the cell and virus replication. Adlay tea did not suppress hemagglutination inhibition or cell fusion, although it slightly inhibited virus binding to Malin Darby canine kidney cells. Furthermore, our findings suggest that the antiviral compounds included in adlay tea were ingredients other than polyphenols and that there were several types of effective compounds in adlay tea inhibiting several steps of viral replication. CONCLUSION: The results of the present study demonstrate that adlay tea had antiviral effects against influenza viruses. Our findings with respect to adlay tea suggest that the polyphenols might have a small influence on its antiviral activity and that other ingredients might have more influence. © 2017 Society of Chemical Industry.