Anti-influenza virus activity of biflavonoids - Ginkgetin

[Mingus]

http://www.sciencedirect.com/scienc...=1072191&md5=c22a77de93335c072b72f60b95359cdc



Anti-influenza virus activity of biflavonoids


Kazuhiko Miki^a, Takayuki Nagai^b^,^c, Kazushige Suzuki^a, Ryo Tsujimura^a, Kiyotaka Koyama^a, Kaoru Kino****a^a, Kimio Furuhata^d, Haruki Yamada^b^,^c and Kunio Takahashi^a^,^,

^aMeiji Pharmaceutical University, Noshio 2-522-1, Kiyose-shi, Tokyo 204-8588, Japan
^bKitasato Institute for Life Sciences and Graduate School of Infection Control Sciences, Kitasato University, Shirokane 5-9-1, Minato-ku, Tokyo 108-8641, Japan
^cOriental Medicine Research Center, The Kitasato Institute, Shirokane 5-9-1, Minato-ku, Tokyo 108-8642, Japan
^dSchool of Pharmacy, Kitasato University, Shirokane 5-9-1, Minato-ku, Tokyo 108-8641, Japan

Received 24 July 2006; revised 4 October 2006; accepted 25 October 2006. Available online 28 October 2006.



Abstract

Ginkgetin was found to inhibit the influenza virus sialidase. Ginkgetin-sialic acid conjugates showed a significant survival effect in the influenza-virus-infected mice.



Graphical abstract

Ginkgetin was found to inhibit the influenza virus sialidase. Ginkgetin-sialic acid conjugates showed a significant survival effect in the influenza-virus-infected mice.

Keywords: Influenza; Ginkgetin; Sialic acid; Ginkgo biloba; Biflavonoid; Sialidase

Article Outline

<dl><dt>References</dt></dl>

5,7,4′-Trihydroxy-8-methoxyflavone (F36) was surveyed as an influenza virus sialidase inhibitory compound among many flavonoids.¹ It also showed an anti-influenza virus activity in Madin?Darby canine kidney (MDCK) cells and in the allantoic sac of embryonated egg.¹^,²^and³ In the influenza-virus-infected mice, F36 revealed a distinct survival effect.³^,⁴^and⁵ Compound F36 was characterized as an 8-methoxy-flavone derivative, which does not frequently occur in nature.⁶ On the other hand, biflavonoids including amentoflavone-type compounds were reported to show the inhibitory activity against respiratory syncytial,⁷ herpes⁸, and measles viruses.⁸
Accordingly, some naturally occurring biflavonoids were screened for the inhibitory activities against influenza virus sialidase.
As the result, it was demonstrated that ginkgetin isolated from Ginkgo biloba L. and Cephalotaxus harringtonia K. Koch showed a potent inhibitory activity against influenza virus sialidase.
On the other hand, influenza A and B viruses are known to bind themselves on the host cells via sialic acid residue of glycoconjugate receptors at the first stage of infection. It may suggest that synthetic sialic acid-aglycone conjugates are important to inhibit the proliferation of viruses. Therefore, ginkgetin-sialic acid conjugates were synthesized herewith.
N-Acetylneuraminic acid 1 is widely distributed internally in animals playing crucial roles in various biological events such as infection, cell adhesion, immune response, and neural function. The glycosylation of the arylic hydroxyl group to sialic acid is performed by Williamson?s method.⁹^and¹⁰ The synthetic route for ginkgetin-neuraminoside is outlined in Scheme 1 and Scheme 2. In the treatment of 1 with strong cation-exchange resin (Dowex-50W-X2), MeOH afforded 2.¹¹ Followed by acetylation of 2 with Ac₂O, DMAP, and pyridine,⁹^and¹⁰ compound 4 was prepared from 3 by AcCl and HCl gas.⁹ The glycosides of 6?7 were prepared from 4 and ginkgetin 5 with NaH, DMF.⁹^,¹⁰^and¹² When N-acetylneuraminic acid was combined with C-7″ position, it caused a stereochemical obstruction to yield atropic isomers (R and S formation). These atropisomeric compounds, 6R and 6S, 7R and 7S, were separated by HPLC, respectively. Compounds 8R, 8S, 9R, and 9S were prepared from 6R, 6S, 7R, and 7S by hydrolysis of the acetyl group with NaOMe, MeOH, and H₂O, respectively.

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[SIZE=-1]Scheme 1. Reagents and conditions: (a) Dowex-50 W-X2, dry MeOH, rt, under Ar, 24 h (92%); (b) Ac₂O, DMAP, dry pyridine, rt, under Ar, 24 h (83%); (c) AcCl, HCl gas, 0 ?C to rt, 24 h (98%). [/SIZE]
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[/SIZE]
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[SIZE=-1]
(52K)[/SIZE][SIZE=-1]Scheme 2. Reagents and conditions: (a) 4, NaH, dry DMF, rt, under Ar, 24 h (5% for 6R, 4% for 6S, 12% for 7R, 8% for 7S); (b) NaOMe, dry MeOH, H₂O, rt, 24 h (6R → 8R, 96%; 6S → 8S, 76%; 7R → 9R, 80%; 7S → 9S, 69%). [/SIZE]
[SIZE=-1]

[/SIZE]
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The influenza A and B virus sialidase inhibitory activities¹³ of ginkgetin, ginkgetin-sialic acid conjugates 6R?9S, are shown in Table 1, and F36 was used as a positive standard. The IC₅₀ values of ginkgetin against A/PR/8/34 (H1N1) and A/Guizhou/54/89 (H3N2) sialidases were 55.00 and 9.78 μg/mL, respectively. 6R, 6S, 7R, and 7S, which are ginkgetin acetylated neuraminosides, showed lower inhibitory activities against A/PR/8/34 (H1N1) and B/Ibaraki/2/85 sialidases comparing with F36. However, 6S and 7R showed the increase in the activity against A/Guizhou/54/89 (H3N2) (Table 1).

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Table 1. [/SIZE]
[SIZE=-1]Inhibition of influenza A and B virus sialidases by ginkgetin-sialic acid conjugates <table border="1" cellpadding="6" cellspacing="0" frame="hsides" rules="groups"> <colgroup span="4"> <col><col><col><col></colgroup><thead> <tr> <th align="left" valign="center">[SIZE=-1]Compound[/SIZE]</th><th colspan="3" id="col2" align="center" valign="center">[SIZE=-1]Sialidase inhibitory activity [IC₅₀ (μg/mL)][/SIZE]</th> </tr><tr> <th headers="col1">
</th><th align="left" valign="center">[SIZE=-1]A/PR/8/34 (H1N1)[/SIZE]</th><th align="left" valign="center">[SIZE=-1]A/Guizhou/54/89 (H3N2)[/SIZE]</th><th align="left" valign="center">[SIZE=-1]B/Ibaraki/2/85[/SIZE]</th> </tr></thead> <tbody> <tr> <td class="nowrap" align="left" valign="center">[SIZE=-1]F36[/SIZE]</td><td class="nowrap" valign="center">[SIZE=-1]9.78[/SIZE]</td><td class="nowrap" valign="center">[SIZE=-1]8.95[/SIZE]</td><td class="nowrap" valign="center">[SIZE=-1]6.58[/SIZE]</td> </tr><tr> <td class="nowrap" align="left" valign="center">[SIZE=-1]Ginkgetin[/SIZE]</td><td class="nowrap" valign="center">[SIZE=-1]55.00[/SIZE]</td><td class="nowrap" valign="center">[SIZE=-1]9.78[/SIZE]</td><td class="nowrap" align="left" valign="center">[SIZE=-1]>100.00[/SIZE]</td> </tr><tr> <td class="nowrap" align="left" valign="center">[SIZE=-1]6R[/SIZE]</td><td class="nowrap" valign="center">[SIZE=-1]79.20[/SIZE]</td><td class="nowrap" align="left" valign="center">[SIZE=-1]>10.00[/SIZE]</td><td class="nowrap" align="left" valign="center">[SIZE=-1]>100.00[/SIZE]</td> </tr><tr> <td class="nowrap" align="left" valign="center">[SIZE=-1]6S[/SIZE]</td><td class="nowrap" align="left" valign="center">[SIZE=-1]>100.00[/SIZE]</td><td class="nowrap" valign="center">[SIZE=-1]1.10[/SIZE]</td><td class="nowrap" valign="center">[SIZE=-1]>100.00[/SIZE]</td> </tr><tr> <td class="nowrap" align="left" valign="center">[SIZE=-1]7R[/SIZE]</td><td class="nowrap" align="left" valign="center">[SIZE=-1]>100.00[/SIZE]</td><td class="nowrap" valign="center">[SIZE=-1]1.35[/SIZE]</td><td class="nowrap" valign="center">[SIZE=-1]>100.00[/SIZE]</td> </tr><tr> <td class="nowrap" align="left" valign="center">[SIZE=-1]7S[/SIZE]</td><td class="nowrap" align="left" valign="center">[SIZE=-1]>100.00[/SIZE]</td><td class="nowrap" align="left" valign="center">[SIZE=-1]>10.00[/SIZE]</td><td class="nowrap" align="left" valign="center">[SIZE=-1]>100.00[/SIZE]</td> </tr><tr> <td class="nowrap" align="left" valign="center">[SIZE=-1]8R[/SIZE]</td><td class="nowrap" valign="center">[SIZE=-1]5.50[/SIZE]</td><td class="nowrap" valign="center">[SIZE=-1]0.82[/SIZE]</td><td class="nowrap" valign="center">[SIZE=-1]9.11[/SIZE]</td> </tr><tr> <td class="nowrap" align="left" valign="center">[SIZE=-1]8S[/SIZE]</td><td class="nowrap" valign="center">[SIZE=-1]6.35[/SIZE]</td><td class="nowrap" valign="center">[SIZE=-1]0.99[/SIZE]</td><td class="nowrap" valign="center">[SIZE=-1]8.64[/SIZE]</td> </tr><tr> <td class="nowrap" align="left" valign="center">[SIZE=-1]9R[/SIZE]</td><td class="nowrap" valign="center">[SIZE=-1]6.61[/SIZE]</td><td class="nowrap" valign="center">[SIZE=-1]6.57[/SIZE]</td><td class="nowrap" valign="center">[SIZE=-1]9.05[/SIZE]</td> </tr><tr> <td class="nowrap" align="left" valign="center">[SIZE=-1]9S[/SIZE]</td><td class="nowrap" valign="center">[SIZE=-1]6.99[/SIZE]</td><td class="nowrap" valign="center">[SIZE=-1]0.94[/SIZE]</td><td class="nowrap" valign="center">[SIZE=-1]9.34[/SIZE]</td> </tr></tbody> </table> [/SIZE]
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[/SIZE]
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The atropic isomers of ginkgetin mononeuraminoside (8R, 8S) and bisneuraminoside (9R, 9S), respectively, inhibited both A/PR/8/34 (H1N1) and A/Guizhou/54/89 (H3N2) sialidases more potently than F36.
Especially, 8R showed the most potent inhibitory activity whose IC₅₀ value against A/PR/8/34 (H1N1) sialidase was 5.50 μg/mL, and that against A/Guizhou/54/89 (H3N2) sialidase was 0.82 μg/mL. On the other hand, 8R, 8S, 9R, and 9S showed lower activities than F36 against B/Ibaraki/2/85 sialidase (Fig. 1).

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[SIZE=-1]Figure 1. [/SIZE]
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Next, proliferation of influenza virus and the cytotoxicities were tested for compounds 6R?9S by using MDCK cells infected with A/PR/8/34 (H1N1) (Fig. 2). The assay was carried out by the described method.¹⁴ It means that the prevention of virus proliferation reflects the decrease of the sialidase activity, and the damage of MDCK cells treated by compounds causes to decrease formasan formation. Ginkgetin, which was found as a sialidase inhibitor in the former experiment, showed a cytotoxicity, whereas its neuraminoside derivatives have no cytotoxic effects to MDCK cells. Among these compounds 6R?9S, O-acetylation of sialic acid moiety did not affect the inhibitory activity of virus proliferation. Mononeuraminoside, 8R and 8S, significantly showed the inhibitory activity of virus proliferation in comparison to bisneuraminosides, 9R and 9S, at least 3- and 8-fold, respectively. These results revealed that neuraminosylation of ginkgetin lowered the cytotoxicity and enhanced inhibitory activity against influenza virus sialidase of aglycone.

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[SIZE=-1]Figure 2. Anti-influenza virus activity of ginkgetin-sialic acid conjugates on MDCK cells (concentration of samples was 12.5 μg/mL). [/SIZE]
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[/SIZE]
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The efficacies of intranasally administered compounds, 8R?9S and F36, were tested in the influenza virus A/PR/8/34 (H1N1)-infected mice on the basis of the survival days (Table 2).¹⁵

[SIZE=-1]

Table 2. [/SIZE]
[SIZE=-1]Effects of ginkgetin-sialic acid conjugates on survival rates of influenza virus-infected mice^a <table border="1" cellpadding="6" cellspacing="0" frame="hsides" rules="groups"> <colgroup span="3"> <col><col><col></colgroup><thead> <tr> <th align="left" valign="center">[SIZE=-1]Compound^b[/SIZE]</th><th colspan="2" id="col2" align="center" valign="center">[SIZE=-1]No. of survivors/total no. of mice[/SIZE]</th> </tr><tr> <th align="left" valign="center">
</th><th align="left" valign="center">[SIZE=-1]10 days after infection[/SIZE]</th><th align="left" valign="center">[SIZE=-1]21 days after infection[/SIZE]</th> </tr></thead> <tbody> <tr> <td class="nowrap" align="left" valign="center">[SIZE=-1]Control (saline)[/SIZE]</td><td class="nowrap" valign="center">[SIZE=-1]3/9[/SIZE]</td><td class="nowrap" valign="center">[SIZE=-1]2/9[/SIZE]</td> </tr><tr> <td class="nowrap" align="left" valign="center">[SIZE=-1]F36[/SIZE]</td><td class="nowrap" valign="center">[SIZE=-1]5/9[/SIZE]</td><td class="nowrap" valign="center">[SIZE=-1]2/9[/SIZE]</td> </tr><tr> <td class="nowrap" align="left" valign="center">[SIZE=-1]8R[/SIZE]</td><td class="nowrap" valign="center">[SIZE=-1]6/8[/SIZE]</td><td class="nowrap" valign="center">[SIZE=-1]5/8[/SIZE]</td> </tr><tr> <td class="nowrap" align="left" valign="center">[SIZE=-1]8S[/SIZE]</td><td class="nowrap" valign="center">[SIZE=-1]7/9[/SIZE]</td><td class="nowrap" valign="center">[SIZE=-1]5/9[/SIZE]</td> </tr><tr> <td class="nowrap" align="left" valign="center">[SIZE=-1]9R[/SIZE]</td><td class="nowrap" valign="center">[SIZE=-1]5/9[/SIZE]</td><td class="nowrap" valign="center">[SIZE=-1]4/9[/SIZE]</td> </tr><tr> <td class="nowrap" align="left" valign="center">[SIZE=-1]9S[/SIZE]</td><td class="nowrap" valign="center">[SIZE=-1]8/9[/SIZE]</td><td class="nowrap" valign="center">[SIZE=-1]4/9[/SIZE]</td> </tr></tbody> </table> ^a Mice were dosed with compound and then infected with influenza virus A/PR/8/34 (H1N1).
^b A single dose of ginkgetin-sialic acid conjugates and F36 were administered intranasally at 0.5 mg/kg.[/SIZE]
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[/SIZE]
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The control mice began to die from day 8 after the virus infection, and the survival rate was 22% at day 21. F36 as a positive control gave the similar result in comparison to the control. On the other hand, compounds 8R, 8S, 9R, and 9S prolonged the survival days. Especially, compound 8R exhibited the highest survival rates, 75% at day 10 and 62.5% at day 21 after the A/PR/8/34 (H1N1) virus infection (p = 0.0385, Logrank test, Kaplan?Meier method), while 8S showed almost the same results, 78% at day 10 and 56% at day 21 (p = 0.0714), compared with those of 8R. Bisneuraminoside 9R and 9S exhibited slightly weak results in comparison to mononeuraminosides, 8R and 8S, respectively.
In summary, we presented new influenza virus sialidase inhibitors, which were prepared by the conjugation of biflavonoid ginkgetin from the plant source and sialic acid from the animal origin. We also revealed that the ginkgetin-sialic acid conjugates remarkably presented potent anti-influenza virus activities in vivo.

References and notes

1 T. Nagai, Y. Miyaichi, T. Tomimori, Y. Suzuki and H. Yamada, Chem. Pharm. Bull. 38 (1990), p. 1329. Abstract-MEDLINE | Abstract-EMBASE | Abstract + References in Scopus | Cited By in Scopus
2 T. Nagai, R. Moriguchi, Y. Suzuki, T. Tomimori and H. Yamada, Antiviral Res. 26 (1995), p. 11. SummaryPlus | Full Text + Links | PDF (825 K) | Abstract + References in Scopus | Cited By in Scopus
3 T. Nagai, Y. Suzuki, T. Tomimori and H. Yamada, Biol. Pharm. Bull. 18 (1995), p. 295. Abstract-MEDLINE | Abstract-EMBASE | Abstract + References in Scopus | Cited By in Scopus
4 T. Nagai, Y. Miyaichi, T. Tomimori, Y. Suzuki and H. Yamada, Antiviral Res. 19 (1992), p. 207. Abstract | Abstract + References in Scopus | Cited By in Scopus
5 T. Nagai, Y. Nishibe, Y. Makino, T. Tomimori and H. Yamada, Biol. Pharm. Bull. 20 (1997), p. 1082. Abstract-MEDLINE | Abstract-EMBASE | Abstract + References in Scopus | Cited By in Scopus
6 T. Tomimori, Y. Miyaichi and H. Kizu, Yakugaku Zasshi 102 (1982), p. 388 (Journal written in Japanese). Abstract-MEDLINE | Abstract-EMBASE | Abstract + References in Scopus | Cited By in Scopus
7 S.-C. Ma, P. P.-H. But, V. E.-C. Ooi, Y.-H. He, S. H.-S. Lee, S.-F. Lee and R.-C. Lin, Biol. Pharm. Bull. 24 (2001), p. 311. Abstract-EMBASE | Abstract-MEDLINE | Full Text via CrossRef | Abstract + References in Scopus | Cited By in Scopus
8 Y.-M. Lin, M.T. Flavin, R. Schure, F.-C. Chen, R. Sidwell, D.L. Barnard, J.H. Huffman and E.R. Kern, Planta Med. 65 (1999), p. 120. Abstract-EMBASE | Abstract-MEDLINE | Full Text via CrossRef | Abstract + References in Scopus | Cited By in Scopus
9 R.W. Myers, R.T. Lee, Y.C. Lee, G.H. Thomas, L.W. Reynolds and Y. Uchida, Anal. Biochem. 101 (1980), p. 166. Abstract | Abstract + References in Scopus | Cited By in Scopus
10 K. Furuhata, Trends Glycosci. Glycotechnol. 16 (2004), p. 143. Abstract + References in Scopus | Cited By in Scopus
11 R. Kuhn, P. Lutz and D.L. MacDonald, Chem. Ber. 99 (1966), p. 611. Abstract-MEDLINE | Abstract + References in Scopus | Cited By in Scopus
12 K. Furuhata, K. Komiyama, H. Ogura and T. Hata, Chem. Pharm. Bull. 39 (1991), p. 255. Abstract-EMBASE | Abstract-MEDLINE | Abstract + References in Scopus | Cited By in Scopus
13
Influenza virus sialidase inhibition activity (A/PR/8/34, H1N1; A/Guizhou/54/89, H3N2; B/Ibaraki/2/85) was determined in a fluorometric assay. The reaction mixture containing 0.1 mM 4-MU-NeuAc (50 μl), sample solution (10 μl), influenza vaccine (10 μl), and buffer was incubated at 37 ?C for 10 min in 96-well microtiter plate.
14
In vitro anti-influenza virus activity was assayed by using MDCK cells. Influenza virus A/PR/8/34 was inoculated into confluent monolayers of MDCK cells in 96-well culture plate, and then sample solutions were put into the well. The plate was cultured at 37 ?C for 3 days under a 5% CO₂ atmosphere. Inhibition of proliferation of influenza virus was determined in a fluorometric assay by measuring sialidase activity and the viable cells were determined by MTT assay.
15
In vivo anti-influenza virus activity was assayed in the influenza virus A/PR/8/34-infected mice. BALB/c mice (female, 6-week-old) were anesthetized by an intraperitoneal injection of amobarbital sodium. Test samples were dissolved in saline and 10 μl of the solution was administered to the mouse intranasally. Then 20 μl mouse-adapted influenza virus A/PR/8/34 suspension in 0.1% BSA in PBS was infected by intranasal inoculation. The survival rates were observed for 21 days.

Corresponding author. Tel.: +81 424 95 8912; fax: +81 424 95 8912.

 

[Mingus]

Re: Anti-influenza virus activity of biflavonoids

Re: Anti-influenza virus activity of biflavonoids

Ginkgetin is a substance include in Ginkgo biloba.

 

[AlaskaDenise]

Re: Anti-influenza virus activity of biflavonoids - Ginkgetin

Some sources also say it's an anti-inflammatory.

See http://lib.bioinfo.pl/pmid:16327145

<TABLE id=all_16327145 style="PADDING-BOTTOM: 8px" width="100%"><TBODY><TR><TD class=reference noWrap>Biol Pharm Bull. 2005 Dec ;28:2181-4 <TD noWrap align=right>[Pubmed] [Scholar] [Select] [Drop] [Hide] [Show] <TR><TD colSpan=2><!-- google_ad_section_start -->Ginkgetin, a Biflavone from Ginko biloba Leaves, Inhibits Cyclooxygenases-2 and 5-Lipoxygenase in Mouse Bone Marrow-Derived Mast Cells.<!-- google_ad_section_end --><TR><TD colSpan=2>Jong Keun Son, Min Jung Son, Eunkyung Lee, Tae Chul Moon, Kun Ho Son, Cheorl-Ho Kim, Hyun Pyo Kim, Sam Sik Kang, Hyeun Wook Chang


<TR><TD colSpan=2>Ginkgetin, a biflavone from Ginkgo biloba leaves, was previously reported to be a phospholipase A(2) inhibitor and this compound showed the potent antiarthritic activity in rat adjuvant-induced arthritis as well as analgesic activity. This investigation was carried out to find effects on cyclooxygenase-2 (COX-2) in vitro effect. Ginkgetin inhibits COX-2 dependent phases of prostaglandin D(2) (PGD(2)) generation in bone marrow-derived mast cells (BMMC) in a concentration-dependent manner with IC(50) values of 0.75 muM. Western blotting probed with specific anti-COX-2 antibodies showed that the decrease in quantity of the PGD(2) product was accompanied by a decrease in the COX-2 protein level. In addition, this compound consistently inhibited the production of leukotriene C(4) (LTC(4)) in a dose dependent manner, with an IC(50) value of 0.33 muM. These results demonstrate that ginkgetin has a dual cyclooxygenase-2/5-lipoxygenase inhibitory activity. Furthermore, this compound also inhibited degranulation reaction in a dose dependent manner, with an IC(50) value of 6.52 muM. Therefore, this compound might provide a basis for novel anti-inflammatory
agents.

</TD></TR></TBODY></TABLE>

 

[LMonty]

Re: Anti-influenza virus activity of biflavonoids - Ginkgetin

any idea if the compound is water or alcohol soluble? the article appears to use the pure compounds and not an available preparation.

 

[Mingus]

Re: Anti-influenza virus activity of biflavonoids - Ginkgetin

http://www.sciencedirect.com/cache/M...=dGLzVlz-zSkzV

From the chemical formula model, I would say It should be more soluble un alcohol because of the many aromatic cycle inside the molecule. There is some polar group but not enaugh to make the molecule very soluble in water, I think.

It's a theoretical assesment, I don't have empirical data to back it up.

 

[fredness]

Re: Anti-influenza virus activity of biflavonoids - Ginkgetin

A short search on Google came up with these two patents...

http://www.freepatentsonline.com/20070004649.html
Anti-influenza virus compound comprising biflavonoid-sialic acid glycoside
The compounds of the present invention comprise biflavonoid-sialic acid conjugates. Such biflavonoid-sialic acid conjugates show anti-influenza virus activities, not only in in vitro systems using cultured cells, but also in in vivo systems using mice. Thus, they are useful as preventive or therapeutic agents for influenza, and moreover, are useful in food and drink products for preventing or treating influenza.
<TABLE width="100%" border=0><TBODY><TR><TD class=textreg11>Claims:</TD></TR><TR><TD class=textreg11>1. A biflavonoid-sialic acid conjugate.

2. The biflavonoid-sialic acid conjugate of claim 1, wherein the biflavonoidsialic acid conjugate is a compound of any of formula (I), (II), or (III), or a salt thereof: where in formula (I), R.sup.1, R.sup.2, R.sup.3, R.sup.4, R.sup.5, R.sup.6, R.sup.7, and R.sup.8 are each independently a hydrogen atom, methyl group, hydroxy group, acetyloxy group, methyloxy group, ethyloxy group, n-propyloxy group; n-butyloxy group, n-octyloxy group, benzyloxy group, allyloxy group, or glycosyloxy group, R.sup.9 is a hydrogen atom, sodium atom, potassium atom, ammonium group, or methyl group; R.sup.10, R.sup.11, R.sup.12, and R.sup.13 are each independently a hydrogen atom, sulfate group, or acetyl group; X is an acetamino group, glycolylamino group, hydroxy group, or acetyloxy group; and A is an oxygen atom or sulfur atom; where in formula (II), R.sup.1, R.sup.2, R.sup.3, R.sup.4, R.sup.6, R.sup.7, and R.sup.8 are each independently a hydrogen atom, methyl group, hydroxy group, acetyloxy group, methyloxy group, ethyloxy group, n-propyloxy group, n-butyloxy group, n-octyloxy group, benzyloxy group, allyloxy group, or glycosyloxy group; R.sup.9 and R.sup.14 are each independently a hydrogen atom, sodium atom, potassium atom, ammonium group, or methyl group; R.sup.10, R.sup.11, R.sup.12, R.sup.13, R.sup.15, R.sup.16, R.sup.17, and R.sup.18 are each independently a hydrogen atom, sulfate group, or acetyl group; X and Y are each independently an acetamino group, glycolylamino group, hydroxy group, or acetyloxy group; and A and B are each independently an oxygen atom or sulfur atom; and where in formula (III), R.sup.1, R.sup.2, R.sup.3, R.sup.4, R.sup.6, R.sup.7, R.sup.8, and R.sup.19 are each independently a hydrogen atom, methyl group, hydroxy group, acetyloxy group, methyloxy group, ethyloxy group, n-propyloxy group, n-butyloxy group, n-octyloxy group, benzyloxy group, allyloxy group, or glycosyloxy group; R.sup.14 is a hydrogen atom, sodium atom, potassium atom, ammonium group, or methyl group; R.sup.15, R.sup.16, R.sup.17, and R.sup.18 are each independently a hydrogen atom, sulfate group, or acetyl group; Y is an acetamino group, glycolylamino group, hydroxy group, or acetyloxy group; and B is an oxygen atom or sulfur atom.

3. The biflavonoid-sialic acid conjugate of claim 2, where in formulae (I) to (III), R.sup.9, R.sup.10, R.sup.11, R.sup.12, R.sup.13, R.sup.14, R.sup.15, R.sup.16, R.sup.17, and R.sup.18 are hydrogen atoms.

4. A pharmaceutical agent comprising the biflavonoid-sialic acid conjugate of claim 1 as an active ingredient.

5. A preventive agent or a therapeutic agent for influenza, comprising the biflavonoid-sialic acid conjugate of claim 1 as an active ingredient.

6. A food or drink product comprising the biflavonoid-sialic acid conjugate of claim 1 as an active ingredient.

7. A food or drink product for preventing or treating influenza comprising the biflavonoid-sialic acid conjugate of claim 1 as an active ingredient.

8. A pharmaceutical agent comprising the biflavonoid-sialic acid conjugate of claim 2 as an active ingredient.

9. A pharmaceutical agent comprising the biflavonoid-sialic acid conjugate of claim 3 as an active ingredient.

10. A preventive agent or a therapeutic agent for influenza comprising the biflavonoid-sialic acid conjugate of claim 2 as an active ingredient.

11. A preventive agent or a therapeutic agent for influenza comprising the biflavonoid-sialic acid conjugate of claim 3 as an active ingredient.

12. A food or drink product comprising the biflavonoid-sialic acid conjugate of claim 2 as an active ingredient.

13. A food or drink product comprising the biflavonoid-sialic acid conjugate of claim 3 as an active ingredient.

14. A food or drink product for preventing or treating influenza comprising the biflavonoid-sialic acid conjugate of claim 2 as an active ingredient.

15. A food or drink product for preventing or treating influenza comprising the biflavonoid-sialic acid conjugate of claim 3 as an active ingredient.
</TD></TR></TBODY></TABLE>


http://www.patentstorm.us/patents/5773462-fulltext.html
Biflavanoids and derivatives thereof as antiviral agents

US Patent Issued on June 30, 1998
Abstract

Substantially purified antiviral biflavanoids robustaflavone, hinokiflavone, amentoflavone, agathisflavone, volkensiflavone, morelloflavone, rhusflavanone, succedaneaflavanone, GB-1a, and GB-2a are provided. Antiviral biflavanoid derivatives and salt forms thereof, e.g., robustaflavone tetrasulfate potassium salt, and methods for preparing the same are also disclosed. Pharmaceutical compositions which include the antiviral biflavanoids, derivatives or salts thereof are also provided. Also disclosed is an improved method for obtaining substantially pure robustaflavone from plant material. The biflavanoid compounds, derivatives or salts thereof of the invention may be used in a method for treating and/or preventing viral infections caused by viral agents such as influenza, e.g., influenza A and B; hepatitis, e.g., hepatitis B; human immunodeficiency virus, e.g., HIV-1; Herpes viruses (HSV-1 and HSV-2); Varicella Zoster virus (VZV); and measles.

What we claim:

1. A method for treating an influenza infection in a mammal which comprises administering to said mammal an effective therapeutic amount of a biflavanoid selected from the group consisting of robustaflavone, amentoflavone, and derivative or salt thereof.

2. The method according to claim 1, wherein said derivative or salt comprises a robustaflavone or amentoflavone alkyl ether, ester, acid adduct, amine or sulfate.

3. The method according to claim 2, wherein said biflavonoid derivative or salt is robustaflavone tetrasulfate potassium salt.

4. A method for treating a hepatitis B viral infection in a mammal which comprises administering to said mammal an effective therapeutic amount of robustaflavone, or derivative or salt thereof.

5. The method according to claim 4, wherein said derivative or salt comprises a robustaflavone alkyl ether, ester, acid adduct, amine or sulfate.

6. The method according to claim 5, wherein said derivative or salt is robustaflavone tetrasulfate potassium salt.

7. A pharmaceutical composition comprising a therapeutically effective amount of purified robustaflavone or derivative or salt thereof and a pharmaceutically acceptable carrier therefor.

8. The composition according to claim 7, wherein said derivative or salt comprises robustaflavone alkyl ether, ester, acid adduct, amine or sulfate.

9. The composition according to claim 7, wherein said derivative or salt is robustaflavone tetrasulfate potassium salt.

10. Robustaflavone tetrasulfate potassium salt.

 

[sharon sanders]

Re: Anti-influenza virus activity of biflavonoids - Ginkgetin

Thank you Fredness.

 

[gsgs]

Re: Anti-influenza virus activity of biflavonoids - Ginkgetin

is it available ? what does it cost ? how likely will it prevent/reduce
H5N1-illness ?