U.S. patent application number 12/935473 was filed with the patent office on 2011-02-03 for antiviral agent and antiviral composition.
This patent application is currently assigned to HIROSHIMA UNIVERSITY. Invention is credited to Yoshiaki Nakai, Yasushi Okinaka, Takemasa Sakaguchi, Tadashi Shimamoto, Toru Tsuji.
Application Number | 20110027399 12/935473 |
Document ID | / |
Family ID | 41135554 |
Filed Date | 2011-02-03 |
United States Patent
Application |
20110027399 |
Kind Code |
A1 |
Shimamoto; Tadashi ; et
al. |
February 3, 2011 |
Antiviral Agent and Antiviral Composition
Abstract
An antiviral agent is provided which has an excellent effect on
a non-enveloped virus and which is highly safe to the human body,
and an antiviral composition which comprises the antiviral agent
and which is useful for disinfection of the virus or prevention of
infection of the virus. The antiviral agent comprises as an active
ingredient an extract from a plant of the genus Diospyros
containing tannin, a catechin, wattle tannin, pentagalloyl glucose,
coffee tannin, an alkyl gallate (excluding that in the
above-described persimmon extract), pyrogallol, or an organic acid
and/or a salt thereof (excluding that in the above-described
persimmon extract). The antiviral composition is characterized by
comprising at least the antiviral agent and an alcohol, a
surfactant, a microbicide, a humectant, or a cosmetic fat and oil,
and preferably further comprises citric acid and/or a salt thereof
or vitamin C.
Inventors: |
Shimamoto; Tadashi;
(Higashi-Hiroshima-shi, JP) ; Okinaka; Yasushi;
(Higashi-Hiroshima-shi, JP) ; Sakaguchi; Takemasa;
(Hiroshima-shi, JP) ; Tsuji; Toru; (Tokyo, JP)
; Nakai; Yoshiaki; (Tokyo, JP) |
Correspondence
Address: |
THE WEBB LAW FIRM, P.C.
700 KOPPERS BUILDING, 436 SEVENTH AVENUE
PITTSBURGH
PA
15219
US
|
Assignee: |
HIROSHIMA UNIVERSITY
Higashi-Hiroshima-shi
JP
ALTAN CO., LTD.
Tokyo
JP
|
Family ID: |
41135554 |
Appl. No.: |
12/935473 |
Filed: |
March 31, 2009 |
PCT Filed: |
March 31, 2009 |
PCT NO: |
PCT/JP2009/056635 |
371 Date: |
September 29, 2010 |
Current U.S.
Class: |
424/777 |
Current CPC
Class: |
A61K 31/235 20130101;
A61K 31/60 20130101; A61K 31/194 20130101; A61K 45/06 20130101;
A61K 36/74 20130101; A61K 31/05 20130101; A61P 31/12 20180101; A61K
36/44 20130101; A61K 31/7024 20130101; A61P 31/02 20180101; A01N
65/08 20130101; A61P 31/14 20180101; A61P 17/00 20180101; Y02A
50/30 20180101; Y02A 50/465 20180101; A61K 31/19 20130101; A61P
31/20 20180101; A61P 3/00 20180101; A61K 31/353 20130101; A61K
31/05 20130101; A61K 2300/00 20130101; A61K 31/19 20130101; A61K
2300/00 20130101; A61K 31/194 20130101; A61K 2300/00 20130101; A61K
31/235 20130101; A61K 2300/00 20130101; A61K 31/353 20130101; A61K
2300/00 20130101; A61K 31/60 20130101; A61K 2300/00 20130101; A61K
31/7024 20130101; A61K 2300/00 20130101; A61K 36/44 20130101; A61K
2300/00 20130101; A61K 36/74 20130101; A61K 2300/00 20130101 |
Class at
Publication: |
424/777 |
International
Class: |
A61K 36/00 20060101
A61K036/00; A61P 31/12 20060101 A61P031/12 |
Foreign Application Data
Date |
Code |
Application Number |
Mar 31, 2008 |
JP |
2008-091602 |
Aug 5, 2008 |
JP |
2008-201841 |
Claims
29. An antiviral agent against a non-enveloped virus comprising, as
an active ingredient, a treated persimmon extract obtained by
heating a squeezed juice or an extract from a fruit of a plant of
the genus Diospyros containing tannin or treating the squeezed
juice or the extract with an alcohol in order to inactive an enzyme
contained therein and derived from the plant of the genus
Diospyros, wherein the non-enveloped virus is a non-enveloped virus
belonging to the genus Betanodavirus, Aquavirnavirus, Ranavirus,
Enterovirus, Mastadenovirus, Vesivirus, or Rotavirus.
30. The antiviral agent according to claim 29, wherein the treated
persimmon extract contains at least condensed tannin.
31. The antiviral agent according to claim 29, wherein the
persimmon extract is obtained from Diospyros kaki.
32. The antiviral agent according to claim 29, further comprising,
as an active ingredient, at least one selected from the group
consisting of a catechin, wattle tannin, pentagalloyl glucose,
coffee tannin, an alkyl gallate (excluding that in the treated
persimmon extract), pyrogallol, and an organic acid and/or a salt
thereof (excluding that in the treated persimmon extract).
33. The antiviral agent according to claim 29, wherein the
non-enveloped virus is nervous necrosis virus, infectious
pancreatic necrosis virus, red seabream iridovius, Coxsackie virus,
adenovirus, feline calicivirus, or rotavirus.
34. An antiviral alcohol preparation comprising at least the
antiviral agent according to claim 29 and an alcohol.
35. An antiviral washing composition comprising at least the
antiviral agent according to claim 29.
36. An antiviral disinfectant composition comprising at least the
antiviral agent according to claim 29 and an antimicrobial agent
(excluding ethanol, an organic acid, and a salt thereof).
37. The antiviral composition according to claim 34, wherein the
antiviral agent contains the treated persimmon extract at a ratio
of 0.01% to 5% by weight (in terms of solid content) based on the
total composition.
38. The antiviral composition according to claim 34, further
comprising vitamin C.
39. The antiviral agent according to claim 30, wherein the
persimmon extract is obtained from Diospyros kaki.
40. The antiviral agent according to claim 30, further comprising,
as an active ingredient, at least one selected from the group
consisting of a catechin, wattle tannin, pentagalloyl glucose,
coffee tannin, an alkyl gallate (excluding that in the treated
persimmon extract), pyrogallol, and an organic acid and/or a salt
thereof (excluding that in the treated persimmon extract).
41. The antiviral agent according to claim 31, further comprising,
as an active ingredient, at least one selected from the group
consisting of a catechin, wattle tannin, pentagalloyl glucose,
coffee tannin, an alkyl gallate (excluding that in the treated
persimmon extract), pyrogallol, and an organic acid and/or a salt
thereof (excluding that in the treated persimmon extract).
42. An antiviral alcohol preparation comprising at least the
antiviral agent according to claim 30 and an alcohol.
43. An antiviral alcohol preparation comprising at least the
antiviral agent according to claim 31 and an alcohol.
44. An antiviral alcohol preparation comprising at least the
antiviral agent according to claim 32 and an alcohol.
45. An antiviral alcohol preparation comprising at least the
antiviral agent according to claim 33 and an alcohol.
46. An antiviral washing composition comprising at least the
antiviral agent according to claim 30.
47. An antiviral disinfectant composition comprising at least the
antiviral agent according to claim 30 and an antimicrobial agent
(excluding ethanol, an organic acid, and a salt thereof).
48. The antiviral composition according to claim 35, wherein the
antiviral agent contains the treated persimmon extract at a ratio
of 0.01% to 5% by weight (in terms of solid content) based on the
total composition.
Description
TECHNICAL FIELD
[0001] The present invention relates to an antiviral agent that has
an excellent effect on non-enveloped viruses (e.g., fish disease
viruses such as viral nervous necrosis virus (NNV), aquavirnavirus
(ABV) and red seabream iridovius (RSIV); feline calicivirus which
is an infectious pathogen of cats; human pathogenic viruses such as
Coxsackie virus and adenovirus), that is highly safe for the human
body, and that contains as an active ingredient at least one
selected from the group consisting of extracts of plants of the
genus Diospyros containing tannin, catechins, wattle tannin,
pentagalloyl glucose, coffee tannin, alkyl gallates, pyrogallol,
organic acids and/or salts thereof, and also relates to an
antiviral composition containing the antiviral agent and useful for
disinfection and infection control against the viruses.
BACKGROUND ART
[0002] In general, viral diseases are difficult to cure because
antibiotics have no effect thereon and thus become a major problem
for humans and animals such as domestic animals. It is still fresh
in our minds that SARS and avian influenza have recently attracted
interest, and viral diseases also become a major problem for
cultured fishes. However, there is no effective curing method for
non-enveloped viruses, while surfactants and alcohols are effective
for enveloped viruses, and a main measure against the non-enveloped
viruses is prevention by vaccines.
[0003] "The astringent juice of the persimmon" produced by
fermenting an extract from a persimmon has been used as Chinese
herbal medicine, such as an antihypertensive agent, in China for a
long time and is also familiar to Japanese as folk medicine. The
astringent juice of the persimmon is rich in tannin and is believed
to have astringency (the property of contracting a tissue by
combining to a protein or another substance), antimicrobial action,
and deodorizing action. The structure and a purification method of
tannin (condensed tannin) contained in Diospyros kaki can be found
in the following two papers (Matsuo and Itoo, 1981a/1981b:
Non-patent Literatures 1 and 2).
[0004] As a composition containing the astringent juice of the
persimmon or tannin of persimmon, for example, there has been
proposed an antimicrobial dental composition (JP 2005-232043 A:
Patent Literature 1) prepared by covering an extract from the
astringent juice of the persimmon with cyclodextrin to further
improve handleability and antimicrobial properties, and an
anticancer agent and an antipneumonic viral agent (JP 2004-331641
A: Patent Literature 2) prepared by adding and mixing the
astringent juice of the persimmon with a component such as a
peptide polysaccharide contained in Reishi spore.
[0005] JP 2006-206558 A (Patent Literature 3) discloses an
antimicrobial composition containing a tannin substance, a fatty
acid ester, and a chelating agent (claim 1). Examples of the tannin
substance include tannic acid, pyrocatechin, gallic acid, persimmon
tannin, tea tannin, and gall tannin (claim 4). However, the
efficacy of the antimicrobial composition is demonstrated only for
tannic acid in Escherichia coli and Staphylococcus aureus
(Examples), and Patent Literature 3 does not specifically disclose
a technical idea that persimmon tannin among the tannin substances
has marked antiviral properties for non-enveloped viruses and thus
persimmon tannin is very important as a component of an antiviral
composition.
[0006] JP 2006-306836 A (Patent Literature 4) discloses that
specified phenol derivatives are effective as inactivators for
various viruses and also discloses various gallates as the phenol
derivatives. However, Patent Literature 4 does not disclose viruses
of the genera Vesivirus and Rotavirus and only n-octyl gallate is
used in examples.
[0007] In addition, the inventors have proposed an anti-norovirus
agent for norovirus that causes food poisoning and infectious
gastroenteritis, the agent using a persimmon extract, and an
anti-norovirus composition containing the anti-norovirus agent
(International Publication No. 08/153,077 pamphlet: Patent
Literature 5).
[0008] Patent Literature 1: JP 2005-232043 A
[0009] Patent Literature 2: JP 2004-331641 A
[0010] Patent Literature 3: JP 2006-206558 A
[0011] Patent Literature 4: JP 2006-306836 A
[0012] Patent Literature 5: International Publication No.
08/153,077 pamphlet
[0013] Non-patent Literature 1: MATSUO, Tomoaki and ITOO, Saburo
(1981a): Comparative Studies of Condensed Tannins from Several
Young Fruits. J. Japan. Soc. Hort. Sci., 50(2), 262-269.
[0014] Non-patent Literature 2: MATSUO, Tomoaki and ITOO, Saburo
(1981b): A Simple and Rapid Purification Method of Condensed
Tannins from Several Young Fruits. Agric. Biol. Chem., 45(8),
1885-1887.
DISCLOSURE OF INVENTION
Technical Problem
[0015] It is an object of the present invention to provide an
antiviral agent that has an excellent inactivating action on
non-enveloped viruses and that is highly safe for the human body,
and an antiviral composition containing the antiviral agent and
useful for disinfection for the viruses and prevention of infection
of the viruses.
Solution to Problem
[0016] To solve the problems described above, the present inventors
searched substances and plant components approved as food or food
additives, that is, materials safe to eat, for a substance having
an antiviral activity, and completed the present invention by
finding that an extract from a plant of the genus Diospyros
containing tannin has marked antiviral activity for a wide range of
non-enveloped viruses and that other tannin substances or
tannin-like substances have antiviral activity for non-enveloped
viruses.
[0017] The gist of the present invention is as follows.
[0018] [1] An antiviral agent against an non-enveloped virus
comprising, as an active ingredient, at least one selected from the
group consisting of an extract of a plant of the genus Diospyros
containing tannin (hereinafter referred to as a "persimmon
extract"), a catechin, wattle tannin, pentagalloyl glucose, coffee
tannin, an alkyl gallate (excluding that in the persimmon extract),
pyrogallol, an organic acid and/or a salt thereof (excluding that
in the persimmon extract), the antiviral agent satisfying at least
one selected from the group consisting of the following
requirements [a], [b], [c], [d], and [e]:
[0019] [a] A persimmon extract is used as the active ingredient for
non-enveloped viruses other than viruses of the genera
Mastadenovirus, Vesivirus, Rotavirus, Betanodavirus,
Aquavirnavirus, and Ranavirus.
[0020] [b] When the non-enveloped virus is a virus of the genus
Mastadenovirus, at least one selected the group consisting of a
persimmon extract, a catechin, and wattle tannin is used as the
active ingredient.
[0021] [c] When the non-enveloped virus is a virus of the genus
Vesivirus, at least one selected the group consisting of a
persimmon extract, a catechin, an alkyl gallate (excluding that in
the persimmon extract), pyrogallol, an organic acid and/or a salt
thereof (excluding that in the persimmon extract) is used as the
active ingredient.
[0022] [d] When the non-enveloped virus is a virus of the genus
Rotavirus, at least one selected the group consisting of a
persimmon extract, a catechin, pentagalloyl glucose, coffee tannin,
an alkyl gallate (excluding that in the persimmon extract),
pyrogallol, an organic acid and/or a salt thereof (excluding that
in the persimmon extract) is used as the active ingredient.
[0023] [e] When the non-enveloped virus is a virus of the genus
Betanodavirus, Aquavirnavirus, and/or Ranavirus, at least one
selected the group consisting of a persimmon extract, wattle
tannin, pentagalloyl glucose, and a catechin is used as the active
ingredient.
[0024] [2] The antiviral agent according to [1], wherein the
persimmon extract is prepared by heating a squeezed juice or an
extract from a fruit of a plant of the genus Diospyros or treating
the squeezed juice or the extract with an alcohol in order to
inactivate an enzyme contained therein and derived from the plant
of the genus Diospyros.
[0025] [3] The antiviral agent according to [1] or [2], wherein the
persimmon extract contains at least condensed tannin.
[0026] [4] The antiviral agent according to any one of [1] to [3],
wherein the persimmon extract is obtained from Diospyros kaki.
[0027] [5] The antiviral agent according to [1], wherein the
catechin is at least one selected from the group consisting of
catechin, epicatechin, epigallocatechin, epicatechin gallate, and
epigallocatechin gallate.
[0028] [6] The antiviral agent according to [1], wherein the alkyl
gallate is propyl gallate.
[0029] [7] The antiviral agent according to [1], wherein the
organic acid and/or the salt thereof is an organic acid having 2 to
10 carbon atoms and/or a salt thereof.
[0030] [8] The antiviral agent according to [7], wherein the
organic acid and/or the salt thereof is a hydroxyl-containing
organic acid having 2 to 10 carbon atoms and/or a salt thereof.
[0031] [9] The antiviral agent according to [7] or [8], wherein the
organic acid and/or the salt thereof is at least one organic acid
selected from the group consisting of lactic acid, malic acid,
citric acid, tartaric acid, salicylic acid, succinic acid, fumaric
acid, and itaconic acid and/or a salt thereof.
[0032] [10] The antiviral agent according to any one of [1] to [9],
wherein the antiviral agent includes an aqueous solution containing
an organic acid and/or a salt thereof and having a pH of 4.5 or
less.
[0033] [11] The antiviral agent according to any one of [1] to
[10], wherein the non-enveloped virus is a non-enveloped virus
belonging to the genus Betanodavirus, Aquavirnavirus, Ranavirus,
Enterovirus, Mastadenovirus, Vesivirus, or Rotavirus.
[0034] [12] The antiviral agent according to [11], wherein the
non-enveloped virus is nervous necrosis virus, infectious
pancreatic necrosis virus, red seabream iridovius, Coxsackie virus,
adenovirus, feline calicivirus, or rotavirus.
[0035] [13] An antiviral alcohol preparation comprising at least
the antiviral agent according to any one of [1] to [12] and an
alcohol.
[0036] [14] The antiviral alcohol preparation according to [13],
wherein the alcohol is ethanol and/or isopropanol.
[0037] [15] An antiviral washing composition comprising at least
the antiviral agent according to any one of [1] to [12] and a
surfactant.
[0038] [16] The antiviral washing composition according to [15],
wherein the surfactant is an anionic surfactant and/or a nonionic
surfactant.
[0039] [17] The antiviral washing composition according to [16],
wherein the anionic surfactant and/or the nonionic surfactant is at
least one surfactant selected from the group consisting of glycerin
fatty acid partial esters, sorbitan fatty acid partial esters, and
sucrose fatty acid partial esters.
[0040] [18] An antiviral disinfectant composition comprising at
least the antiviral agent according to any one of [1] to [12] and
an antimicrobial agent (excluding ethanol, an organic acid, and a
salt thereof).
[0041] [19] The antiviral disinfectant composition according to
[18], wherein the antimicrobial agent is at least one synthetic
antimicrobial agent selected from the group consisting of
isopropylmethylphenol, butyl p-hydroxybenzoate, and triclosan.
[0042] [20] A cosmetic composition comprising at least the
antiviral agent according to any one of [1] to [12] and a humectant
and/or a cosmetic fat and oil.
[0043] [21] The cosmetic composition according to [20], wherein the
humectant is at least one humectant selected from the group
consisting of propylene glycol, sorbitol, polyethylene glycol,
hyaluronic acid, sodium chondroitin sulfate, ceramide, and aloe
extract.
[0044] [22] The cosmetic composition according to [20], wherein
beeswax is contained as the cosmetic fat and oil.
[0045] [23] An antiviral composition comprising at least the
antiviral agent according to any one of [1] to [12], wherein the
antiviral agent contains the persimmon extract at a ratio of 0.01
to 5% by weight (in terms of a solid content) based on the total
composition.
[0046] [24] An antiviral composition comprising at least the
antiviral agent according to any one of [1] to [12], the antiviral
agent not containing an organic acid and/or a salt thereof, wherein
the antiviral composition further contains an organic acid and/or a
salt thereof.
[0047] [25] An antiviral composition comprising at least the
antiviral agent according to any one of [1] to [12], wherein the
antiviral composition contains an organic acid and/or a salt
thereof and has a pH of 4.5 to 2.0.
[0048] [26] The antiviral composition according to any one of [23]
to [25] further comprising vitamin C.
[0049] [27] A method for disinfection or infection control against
a non-enveloped virus characterized by using an antiviral
composition comprising the antiviral agent according to any one of
[1] to [12].
[0050] [28] A therapeutic or prophylactic agent for an infectious
disease caused by a non-enveloped virus, characterized by
comprising the antiviral agent according to any one of [1] to [12]
as an active ingredient.
[0051] The persimmon extract constituting the antiviral agent
according to the present invention is preferably prepared by
heating squeezed juice or an extract from a fruit of a plant of the
genus Diospyros or treating the squeezed juice or the extract with
an alcohol in order to inactivate an enzyme contained therein and
derived from the plant of the genus Diospyros. The persimmon
extract preferably contains at least condensed tannin and is, for
example, an extract from Diospyros kaki.
[0052] In the antiviral agent according to the present invention,
besides the persimmon extract, a catechin (catechin, epicatechin,
epigallocatechin, epicatechin gallate, and epigallocatechin
gallate, or the like), wattle tannin, pentagalloyl glucose, coffee
tannin, an alkyl gallate (propyl gallate or the like), pyrogallol,
an organic acid (citric acid or the like) and/or a salt thereof can
be used as an active ingredient according to the intended
non-enveloped virus.
[0053] Such an antiviral agent is preferably used as an active
ingredient for the non-enveloped virus in a composition in
combination with an alcohol, a surfactant, a bactericide, a
humectant, a cosmetic fat and oil, or the like.
[0054] That is, an antiviral composition according to the present
invention is characterized by containing at least the antiviral
agent, and an alcohol, a surfactant, a bactericide, a humectant, or
a cosmetic fat and oil. Such an antiviral composition preferably
comprises the antiviral agent at a ratio of 0.01% to 5% by weight
(in terms of solid content) based on the total composition.
Preferably, the antiviral composition further comprises an organic
acid and/or a salt thereof and further comprises vitamin C.
[0055] In one aspect of the present invention, the antiviral
composition is provided as an antiviral alcohol preparation
comprising at least the antiviral agent and an alcohol. The alcohol
is preferably ethanol and/or isopropanol.
[0056] In one aspect of the present invention, the antiviral
composition is provided as an antiviral washing composition (e.g.,
a hand soap) comprising at least the antiviral agent and a
surfactant. The surfactant is preferably an anionic surfactant
and/or a nonionic surfactant, and more preferably at least one
surfactant selected from the group consisting of glycerin fatty
acid partial esters, sorbitan fatty acid partial esters, and
sucrose fatty acid partial esters.
[0057] In one aspect of the present invention, the antiviral
composition is provided as an antiviral disinfectant composition
comprising at least the antiviral agent and an antimicrobial agent.
The antimicrobial agent is preferably at least one synthetic
antimicrobial agent selected from the group consisting of
isopropylmethylphenol, butyl p-hydroxybenzoate, and triclosan.
[0058] In one aspect of the present invention, the antiviral
composition is provided as a cosmetic composition (a lotion, a
milky lotion, a cream, or the like) comprising at least the
antiviral agent, a humectant and/or a cosmetic fat and oil. The
humectant is preferably aloe extract, and the cosmetic fat and oil
is preferably beeswax.
[0059] The antiviral agent according to the present invention can
also be used as an active ingredient of a therapeutic or
prophylactic agent for infectious diseases caused by non-enveloped
viruses. That is, a therapeutic or prophylactic agent for
infectious diseases caused by non-enveloped viruses according to
the present invention is characterized by comprising the antiviral
agent as an active ingredient. In other words, in one aspect of the
present invention, there is provided a method using the antiviral
agent or the antiviral composition for disinfection and prevention
of infection of the non-enveloped viruses.
ADVANTAGEOUS EFFECTS OF INVENTION
[0060] A persimmon extract, a catechin, wattle tannin, pentagalloyl
glucose, coffee tannin, an alkyl gallate, pyrogallol, an organic
acid and/or salt thereof used as an antiviral agent according to
the present invention has much excellent effect on non-enveloped
viruses than conventional bactericides, and is capable of killing
99% or more of non-enveloped viruses. Such an antiviral agent is
not only directly used alone for disinfection or prevention of
infection of the non-enveloped viruses but also very useful as an
active ingredient of a composition, such as an alcohol preparation,
a washing composition, a hand soap, a disinfectant composition, a
lotion, a milky lotion, or a cream, or a pharmaceutical agent for
the non-enveloped viruses.
[0061] In addition, since a persimmon extract (persimmon tannin), a
catechin, an alkyl gallate (propyl gallate or the like), an organic
acid and/or salt thereof (sodium citrate or the like) is approved
as a food additive, all the components of the antiviral composition
according to the present invention can be composed of food or food
additives to produce a composition that causes no problem even if
the composition adhering to food or dishes is ate or drunk. Use of
the antiviral composition according to the present invention that
contains such a persimmon extract, a catechin, an alkyl gallate, an
organic acid and/or salt thereof as an active ingredient allows
efficient disinfection and infection control against the
non-enveloped viruses under circumstances where food is handled or
in medical institutions, holding promise of greatly decreasing the
incidence of diseases caused by the viruses.
BRIEF DESCRIPTION OF DRAWINGS
[0062] FIG. 1 is a graph showing the results of measurement of
viral infectivity of Coxsackie virus in Example 2.
[0063] FIG. 2 is a fluorescent staining photograph of
adenovirus-infected cells with anti-adenovirus antibody in Example
3.
[0064] FIG. 3 is a graph showing the antiviral effect of each of
reagents on adenovirus in Example 3.
[0065] FIG. 4 is a drawing showing an example of a plate after
fixing and staining in a feline calicivirus test in Example 4.
[0066] FIG. 5 is a graph showing the antiviral effect of each of
reagents on feline calicivirus in Example 4.
[0067] FIG. 6 is a fluorescent staining photograph of
rotavirus-infected cells with anti-rotavirus antibody in Example
5.
DESCRIPTION OF EMBODIMENTS
Non-Enveloped Virus
[0068] An antiviral agent and an antiviral composition according to
the present invention can be applied over a wide range of
non-enveloped viruses.
[0069] Known non-enveloped viruses include viruses of the families
Iridoviridae, Adenoviridae, Polyomaviridae, and Papillomaviridae
(having dsDNA); Circoviridae and Parvoviridae (having ssDNA);
Reoviridae and Birnaviridae (having dsRNA); Picornaviridae,
Caliciviridae, Hepaviridae, Astroviridae, and Nodaviridae (having
positive sense strand ssRNA); and the like. The present invention
can be intended for these non-enveloped viruses.
[0070] More specifically, examples of the non-enveloped viruses
include non-enveloped viruses known as fish disease viruses
belonging to the genera Betanodavirus (viral nervous necrosis virus
(NNV) and the like), Aquavirnavirus (infectious pancreatic necrosis
virus and the like), Aquareovirus, and Ranavirus (red seabream
iridovirus (RSBI) and the like), the families Parvoviridae
(Infectious hypodermal and hematopoietic necrosis virus (IHHNV) and
the like) and Dicistroviridae (trout strawberry disease virus,
Taura syndrome virus, and the like).
[0071] Other intended non-enveloped viruses include non-enveloped
viruses known as human pathogenic viruses belong to the genera
Enterovirus (Coxsackie virus, enterovirus, poliovirus, echovirus,
and the like), Hepatovirus (hepatitis A virus and the like),
Rhinovirus (rhinovirus and the like), Sapovirus (sapovirus and the
like), Mamastrovirus (human astrovirus and the like),
Papillomavirus (papillomavirus and the like), Polyomavirus
(polyomavirus and the like), and Mastadenovirus (human adenovirus
and the like), and Rotavirus (rotavirus and the like); feline
calicivirus of the genus Vesivirus; and the like.
--Antiviral Agent--
Adaptability of Antiviral Agent to Intended Virus
[0072] With respect to adaptability of the antiviral agent
according to the present invention to the intended non-enveloped
viruses, at least one requirement selected from the group
consisting of the following [a], [b], [c], [d], and [e] is
satisfied.
[0073] [a]A persimmon extract is used as an active ingredient for a
non-enveloped virus other than viruses of the genera
Mastadenovirus, Vesivirus, Rotavirus, Betanodavirus,
Aquavirnavirus, and Ranavirus.
[0074] [b] When the non-enveloped virus is a virus of the genus
Mastadenovirus (human adenovirus or the like), at least one
selected from the group consisting of a persimmon extract, a
catechin, and wattle tannin is used as an active ingredient.
[0075] [c] When the non-enveloped virus is a virus of the genus
Vesivirus (feline calicivirus or the like), at least one selected
from the group consisting of a persimmon extract, a catechin, an
alkyl gallate, pyrogallol, and an organic acid and/or a salt
thereof is used as an active ingredient.
[0076] [d] When the non-enveloped virus is a virus of the genus
Rotavirus, at least one selected from the group consisting of a
persimmon extract, a catechin, pentagalloyl glucose, coffee tannin,
alkyl gallates, pyrogallol, and an organic acid and/or a salt
thereof is used as an active ingredient.
[0077] [e] When the non-enveloped virus is a virus of the genus
Betanodavirus, Aquavirnavirus, and/or Ranavirus, at least one
selected from the group consisting of a persimmon extract, wattle
tannin, pentagalloyl glucose, and a catechin is used as an active
ingredient.
Persimmon Extract
[0078] In the present invention, an extract from a plant of the
genus Diospyros containing tannin, that is, a persimmon extract
(also referred to as persimmon tannin), is used as the active
ingredient of the antivirus agent. Not only a purified product of a
substance derived from a plant of the genus Diospyros containing
tannin, but also a mixture with a solvent used for extracting the
substance, an alcohol used for alcohol treatment, or the like may
be used as the persimmon extract.
[0079] Although the raw material for the persimmon extract is not
particularly limited, use of an immature fruit of an astringent
persimmon (e.g., Diospyros kaki Hachiya, Hiratanenashi, or the
like) rich in persimmon tannin (particularly condensed tannin) is
efficient and economical.
[0080] A portion of a plant of the genus Diospyros other than
fruit, such as leaves or bark, may be used as a raw material,
provided that a persimmon extract composed of the same components
can be produced.
[0081] Examples of a method for preparing a persimmon extract from
such a raw material include, but not limited to, a method in which
an astringent persimmon from which a calyx is removed is ground and
squeezed to recover squeezed juice, a method in which an astringent
persimmon from which a calyx is removed is cut into a suitable
size, is processed into a liquid with a blender, and is centrifuged
to recover the supernatant liquid, and a method for recovering an
extract using water or an aqueous solvent from an astringent
persimmon from which a calyx is removed.
Heating, Alcohol Treatment, and Other Treatments
[0082] Although a squeezed juice or an extract from the fruit of a
plant of the genus Diospyros can be directly used as the persimmon
extract according to the present invention, the squeezed juice or
the extract is preferably treated by heating or with an alcohol in
order to inactivate an enzyme contained therein and derived from
the plant of the genus Diospyros (hereinafter also referred to as a
"treated persimmon extract"). Heating or alcohol treatment can
further improve the antiviral properties of the persimmon extract
and prevent the verification measurement of antiviral properties
from being interfered. Such a treated persimmon extract has the
advantages of being relatively easily prepared and substantially
free from color and odor issues when used as a component of the
composition. The treated persimmon extract is therefore
industrially easier to use than the astringent juice of the
persimmon (described below) which takes time to ferment and is
difficult to use in terms of color and odor.
[0083] The temperature and time conditions of the heat treatment
may be such that an enzyme derived from a plant of the genus
Diospyros contained in squeezed juice or the like can be
inactivated and may be general conditions under which the enzyme
can be inactivated. More specifically, the heat treatment may be
generally performed at a temperature in the range of 60.degree. C.
to 130.degree. C. for a time period in the range of 5 seconds to 30
minutes, for example, at 120.degree. C. to 130.degree. C. for 5 to
10 seconds or at approximately 85.degree. C. for 5 to 15 minutes.
Examples of the heat-treatment process include, but not limited to,
heating in a process for sterilizing squeezed juice, heating in a
powdering process, and heating before fermentation for producing
the astringent juice of the persimmon (described in detail
below).
[0084] On the other hand, the alcohol treatment may be generally
performed with 30% to 100%, preferably 50% to 100%, alcohol, such
as ethanol or the like. For example, persimmon squeezed juice is
treated with substantially the same amount of 95 v/v % ethanol, and
then the persimmon juice is preferably preserved in an airtight
light-resistant container (usually resulting in a light-brown
solution). The alcohol treatment process is also not limited to a
particular process. For example, an extraction process using an
alcohol solvent, such as ethanol or the like, corresponds to the
alcohol treatment process. Typically, as a process for producing an
alcohol preparation as an antiviral composition according to the
present invention, an alcohol treatment process can be performed by
adding an alcohol to a persimmon extract, which is, if necessary,
heat-treated as described above. Such an alcohol treatment also has
a sterilization effect on saprophytic bacteria in a persimmon
extract.
[0085] Alternatively or in addition, the persimmon extract
according to the present invention may be subjected to
concentration, solidification by drying or lyophilization, and
powdering by grinding (the color of a lyophilized powder is
generally light yellow), or purification using an ion-exchange
resin, if necessary, without compromising the advantages of the
present invention. Since the persimmon extract contains much
polyphenol, a liquid persimmon extract is liable to undergo
deterioration, such as coloring or the like. The persimmon extract
is therefore preferably preserved in a solid state in frozen
storage by lyophilization. Preferably, these procedures are
performed under mild conditions such that tannin and other
components in the persimmon extract are not decomposed.
[0086] The persimmon extract in the present invention may be "the
astringent juice of the persimmon" which is a liquid produced by
fermenting and aging squeezed juice of an immature astringent
persimmon for a long period of time (approximately 1 to 3 years)
and which contains several percent solid (persimmon tannin) and an
organic acid produced by the fermentation. The astringent juice of
the persimmon has been used as folk medicine or a paint and is
commercially available as a commodity, such as "Kakishibu" (Toyama
Kakishibu Seizojo). Alternatively, a fermented product prepared by
adding a yeast culture solution derived from a persimmon to
persimmon juice and fermenting it at a temperature in the range of
20.degree. C. to 25.degree. C. for 1 to 3 months may be used (a
reddish-brown liquid is usually produced). Then, the fermented
product is preferably preserved in an airtight container.
[0087] An additive described as "persimmon tannin"
(Name/Synonym=The astringent juice of the persimmon, a persimmon
extract. Origin/Preparation/Nature=Squeezed juice of the fruit of
Diospyros kaki THUNB., or a water or ethanol extract. The main
component is tannin or tannic acid.) in "List of Existing Food
Additives" based on the Food Sanitation Act in Japan may also be
used as the persimmon extract in the present invention.
Persimmon Tannin
[0088] A plant of the genus Diospyros, particularly its fruit, is
rich in substances having certain properties, such as astringency
and affinity for metal ions, and responsible for astringency, that
is, compounds collectively referred to as tannin. In many
instances, persimmon tannin characteristically contains, as a major
component, "persimmon condensed tannin" having the following
probable structural formula (1), which is composed of catechin,
gallocatechin, and gallic acid esters thereof. For example,
persimmon condensed tannin contained in the fruit of Diospyros
kaki, which is a plant of the genus Diospyros native to China and
is cultivated worldwide, including Japan, is a high-molecular
compound in which catechin, catechin gallate, gallocatechin, and
gallocatechin gallate are condensed via carbon-carbon bonds at a
ratio of approximately 1:1:2:2 (Matsuo & Itoo (1981): see
Non-patent Literature 1 cited above).
##STR00001##
[0089] In addition to the "persimmon condensed tannin" described
above, persimmon tannin sometimes contains another tannin compound,
such as catechin or hydrolyzable tannin.
[0090] Hydrolyzable tannin is an ester between an alcohol (such as
glucose) and a carboxylic acid (such as gallic acid) or an oligomer
thereof and is depolymerized by hydrolysis. In contrast, persimmon
condensed tannin is not depolymerized by hydrolysis (the basis
carbon-carbon skeleton of the polymer is not hydrolyzed). The
persimmon condensed tannin can therefore be differentiated from the
hydrolyzable tannin and can be purified and quantified by, for
example, a method described in Matsuo & Itoo (1981b)
(Non-patent Literature 2 cited above).
[0091] To develop antiviral properties for non-enveloped viruses in
the present invention, although it is assumed that a persimmon
extract preferably contains at least the persimmon condensed
tannin, possible contribution of another component in the persimmon
extract is not excluded. In general, the persimmon extract prepared
by a method described below using the fruit of Diospyros kaki
spontaneously contains persimmon condensed tannin. Thus, it is
assumed that the antiviral agent according to the present invention
is preferably produced using such a persimmon extract as a raw
material.
Catechin
[0092] When the antiviral agent according to the present invention
is intended for a non-enveloped virus of the genus Betanodavirus,
Aquavirnavirus, Ranavirus, Mastadenovirus, Vesivirus, or Rotavirus,
catechin contained in a plant, such as catechu (also referred to as
"gambir", botanical name "Acacia Catechu") which is a shrub of the
leguminous genus Acacia indigenous to India, a tea tree (botanical
name "Camellia Sinensis"), or the like, may be used as the active
ingredient.
[0093] "Catechins" is a generic name of
3,5,7,3',4'-pentahydroxyflavan (catechin in a narrow sense),
3,5,7,3',4',5'-hexahydroxyflavan (gallocatechin), and 3-galloyl
derivatives thereof, and includes stereoisomers thereof.
[0094] For example, (+)-catechin isolated from catechu has the
following structural formula (2).
##STR00002##
[0095] Catechins (also referred to as "green tea tannin") contained
in a tea tree contain as major components epicatechin (structural
formula (3)) and its hydroxy derivative, epigallocatechin
(structural formula (4)), and gallates thereof, i.e., epicatechin
gallate (structural formula (5)) and epigallocatechin gallate
(structural formula (6)). These compounds may be isomerized by heat
treatment.
##STR00003##
[0096] Catechins are also contained in other plants. In the present
invention, these compounds isolated from a plant containing
catechins may be used or an extract from a plant containing
catechins may be used. Any one of catechins may be used or a
combination of two or more may be used. Catechins contained in a
tea tree are most preferably used.
Wattle Tannin
[0097] When the antiviral agent according to the present invention
is intended for a non-enveloped virus of the genus Betanodavirus,
Aquavirnavirus, Ranavirus, Mastadenovirus, or Rotavirus, wattle
tannin can be further used as the active ingredient.
[0098] Wattle tannin is tannin extracted from bark, leaves,
seedpods of a plant of the genus Acacia, for example, derived from
Acacia pycnantha (Golden Wattle), Acacia decurrens (Tan Wattle),
Acacia dealbata (Silver Wattle, also referred to as "Mimosa
Wattle"), Acacia mearnsii (Black Wattle), or the like.
Pentagalloyl Glucose
[0099] When the antiviral agent according to the present invention
is intended for a non-enveloped virus of the genus Betanodavirus,
Aquavirnavirus, Ranavirus, or Rotavirus, pentagalloyl glucose can
be further used as the active ingredient.
[0100] Pentagalloyl glucose is a compound in which gallic acid is
ester-bonded to hydroxyl groups (1-, 2-, 3-, 4-, and 6-positions)
of glucose and is produced by, for example, decomposing and
purifying gallnut tannin used as a raw material.
Coffee Tannin
[0101] When the antiviral agent according to the present invention
is intended for a non-enveloped virus of the genus Rotavirus,
coffee tannin can be further used as the active ingredient.
[0102] Coffee tannin is tannin (or a tannin-like substance)
extracted from coffee beans or the like and mainly contains
chlorogenic acid (compound in which coffeic acid is ester-bonded to
a hydroxyl group (any one or more of the 3-, 4-, and 5-positions)
of quinic acid).
Alkyl Gallate
[0103] When the antiviral agent according to the present invention
is intended for a non-enveloped virus of the genus Vesivirus or
Rotavirus, an alkyl gallate (an ester compound of gallic acid
(3,4,5-trihydroxybenzenecarboxylic acid) and an alcohol, excluding
the above-described pentagalloyl glucose and epicatechin gallate
and epigallocatechin gallate included in the catechins) can be
further used as the active ingredient (excluding a case in which a
persimmon extract originally containing an alkyl gallate is
used).
[0104] Examples of the alkyl gallate include ethyl gallate,
n-propyl gallate, n-butyl gallate, n-pentyl gallate, n-octyl
gallate, n-nonyl gallate, n-decyl gallate, isobutyl gallate,
isoamyl gallate, and the like. For example, n-butyl gallate is
preferred. In addition, alkyl gallates (n-propyl gallate and the
like) are also used as an antioxidant (food additive).
Pyrogallol
[0105] When the antiviral agent according to the present invention
is intended for a non-enveloped virus of the genus Vesivirus or
Rotavirus, pyrogallol (1,2,3-trihydroxybenzene) can be further used
as an active ingredient.
Organic Acid and/or Salt Thereof
[0106] When the antiviral agent according to the present invention
is intended for a non-enveloped virus of the genus Vesivirus or
Rotavirus, an organic acid and/or a salt thereof can be further
used as an active ingredient (excluding a case in which the
persimmon extract (particularly the astringent juice of the
persimmon) originally containing an organic acid and/or a salt
thereof is used).
[0107] The organic acid and/or salt thereof is preferably an
organic acid having 2 to 10 carbon atoms and/or a salt thereof, and
more preferably a hydroxyl-containing organic acid having 2 to 10
carbon atoms and/or a salt thereof. More specifically, at least one
organic acid selected from the group consisting of lactic acid,
malic acid, citric acid, tartaric acid, salicylic acid, succinic
acid, fumaric acid, and itaconic acid and/or a salt thereof is
preferred, and particularly citric acid or a salt thereof is
preferred. A salt of an organic acid is preferably a sodium salt, a
potassium salt, or the like of the organic acid.
[0108] When the antiviral agent according to the present invention
is intended for a non-enveloped virus of the genus Vesivirus or
Rotavirus, a solution containing an organic acid and/or a salt
thereof used as an active ingredient preferably has a pH of 4.0 or
less and more preferably 2.0 to 4.0. The pH of an aqueous solution
of the antiviral agent can be measured with a general pH meter
(e.g., a pH meter manufactured by Beckmann Coulter Inc.). When the
antiviral agent is used singly, the pH of the antiviral agent is
preferably adjusted within the above-described range. However, when
the antiviral agent is mixed (diluted) in an antiviral composition,
the pH of the antiviral agent may be previously adjusted within a
range lower than the above-described range according to demand so
that the final pH of the composition is in the preferred range.
--Antiviral Composition--
[0109] The antiviral composition according to the present invention
comprises the antiviral agent as an active ingredient for the
non-enveloped virus, and at least one component selected from
alcohols, surfactants, bactericides, humectants, and cosmetic fats
and oils and, if necessary, further comprises citric acid and/or a
salt thereof or vitamin C. Although the antiviral composition
according to the present invention is not limited to a particular
aspect, representative aspects are as follows:
[0110] An alcohol preparation that comprises at least the antiviral
agent and an alcohol.
[0111] A washing composition that comprises at least the antiviral
agent and a surfactant.
[0112] A disinfectant composition that comprises at least the
antiviral agent and an antimicrobial agent.
[0113] A cosmetic composition (a lotion, a milky lotion, a cream,
or the like) that comprises at least the antiviral agent and a
humectant and/or a cosmetic fat and oil.
[0114] The washing composition according to one aspect of the
present invention can remove contamination of food, dishes, cooking
utensils, breeding instruments and breeding-water tanks for fishes,
fingers of workers, and clothes and kill non-enveloped viruses. For
example, the washing composition is provided as a liquid or solid
detergent. The alcohol preparation and the disinfectant composition
according to other aspects of the present invention are used to
inactivate the non-enveloped viruses and bacteria deposited on
food, dishes, cooking utensils, breeding instruments and breeding
water tanks for fishes, fingers of workers, and instruments with
which the excreta of patients are treated. For example, like
conventional ethanol preparations, the alcohol preparation and the
disinfectant composition are provided as sprays. The washing
composition or the disinfectant composition according to another
aspect of the present invention can be used for killing viruses
which infect eggs of fishes, young and larval fishes, or parent
fishes. The cosmetic composition (a lotion, a milky lotion, a
cream, or the like) according to a still another aspect of the
present invention can be applied to fingers of workers that tend to
be chapped from washing to improve the condition of the skin and
can kill the non-enveloped viruses (basic skin-care
preparation).
[0115] It goes without saying that an alcohol, a surfactant, a
bactericide, a humectant, and a cosmetic fat and oil may be used in
combination in the antiviral composition according to the present
invention. For example, the alcohol preparation preferably further
comprises a surfactant, such as a fatty acid ester or the like, to
improve antimicrobial properties. According to other aspects of the
present invention, the washing composition can be a hand soap that
comprises a bactericide or an alcohol, as well as a surfactant, and
the cream can contain an antimicrobial agent or an alcohol for
keeping the skin clean, as well as a component for protecting the
skin.
[0116] In addition to the components specifically described herein,
the antiviral composition according to the present invention may
appropriately comprise a component for imparting a desired
capability to improve the quality of the composition, for example,
a thickener (such as xanthan gum, locust bean gum, sodium
polyacrylate, or the like), an antioxidant, a perfume, and/or a
dye, and cosmetics, such as a lotion and the like, may
appropriately comprise a barrier cream and/or an antiphlogistic
agent.
Amount of Antiviral Agent
[0117] The amount of the antiviral agent in the antiviral
composition according to the present invention can be appropriately
adjusted in a manner that depends on the component ratios of the
composition and a method for using the composition, provided that
the composition has antiviral properties for non-enveloped viruses,
and may be such that the amount of the active ingredient, such as
the persimmon extract, catechin, or the like, in the antiviral
agent preferably ranges from 0.01% to 5% by weight, more preferably
0.1% to 2% by weight, still more preferably 0.2% to 2% by weight,
most preferably 0.5% to 2% by weight, of the total amount of the
antiviral composition.
[0118] The amount of the antiviral agent is based on a "solid". For
example, when a liquid substance, such as a persimmon fruit
extract, an extract from a plant containing catechins such as a tea
tree, or the like, is used as a raw material, the amount of the
liquid substance is controlled such that the weight of a solid (a
powder produced by drying or lyophilization) in the liquid
substance is in the range described above. In general, squeezed
juice of a persimmon fruit contains approximately 5% to 10%
solid.
Organic Acid and/or Salt Thereof
[0119] When a persimmon extract (persimmon tannin), catechin,
wattle tannin, pentagalloyl glucose, coffee tannin, an alkyl
gallate, or pyrogallol is used as the active ingredient of the
antiviral agent, but an organic acid and/or a salt thereof is not
used, the antiviral composition according to the present invention
preferably comprises an organic acid and/or a salt thereof in
addition to the antiviral agent. An organic acid and/or a salt
thereof independently has no antiviral properties depending on the
type of the non-enveloped virus. However, in combination with the
persimmon extract, the organic acid and/or a salt thereof may
further increase the efficacy of the persimmon extract and the
like. In this case, usable organic acids and/or salts thereof are
the same as those described above as an active ingredient for the
specified non-enveloped viruses. In particular, citric acid and/or
a salt thereof is preferred.
[0120] Organic acids and/or salts thereof are substances excellent
in antimicrobial activity against bacteria, are approved as food
additives, and function to enhance the solubility of tannin and
function as chelating agents for preventing coloring of tannin in
contact with iron.
[0121] The amount of the organic acid and/or a salt thereof added
preferably ranges from 0.05% to 5.0% by weight, more preferably
0.1% to 2.0% by weight, of the total amount of the antiviral
composition (including a solvent). When the organic acid and/or a
salt thereof is used for the above-described purpose in an aqueous
solution composition, not as an active ingredient for non-enveloped
viruses of the genus Vesivirus or Rotavirus, the amount of organic
acid and/or a salt thereof is preferably such that the pH ranges
from 2.0 to 6.0, preferably 2.0 to 4.5, more preferably 2.0 to
4.0.
Vitamin C
[0122] In addition to the antiviral agent described above, the
antiviral composition according to the present invention preferably
comprises vitamin C, which is known as an antioxidant to be added
to food. "Vitamin C" is a generic name and includes DL-ascorbic
acid and ascorbic acid esters (such as palmitate), as well as
L-ascorbic acid which is generally referred to as vitamin C.
Vitamin C can prevent oxidation of the persimmon extract
(particularly persimmon tannin), ensure stable and persistent
efficacy against non-enveloped viruses, and prevent reddening due
to oxidation.
[0123] The amount of vitamin C added preferably ranges from 0.01%
to 3.0% by weight, more preferably 0.05% to 1.0% by weight, of the
total amount of the antiviral composition (including a
solvent).
Alcohol
[0124] The alcohol may be an alcohol used in general alcohol
preparations and is preferably ethanol and/or propanol, which has
an excellent antimicrobial activity against bacteria and is
approved as a food additive. The concentration of the alcohol may
be substantially the same as in general alcohol preparations, can
be adjusted in consideration of antimicrobial activity, and
preferably ranges from approximately 20% to 80% of the total amount
of alcohol preparation. An alcohol may be used as a solvent in a
composition other than alcohol preparations and may be contained in
cosmetics as a component for imparting astringency to the skin or
an antiseptic property.
Surfactant
[0125] Surfactants include cationic, anionic, amphoteric, and
nonionic surfactants. Taking the chemical properties of persimmon
condensed tannin (polyphenol), catechin, tannic acid, gallates,
pyrogallol, or an organic acid and/or a salt thereof into account,
an anionic surfactant and/or a nonionic surfactant is preferably
used in the present invention.
[0126] Examples of the anionic surfactant include soap (alkali
salts of higher fatty acids), monoalkyl sulfates, alkyl
polyoxyethylene sulfates, alkylbenzene sulfonates, and monoalkyl
phosphates.
[0127] Examples of the nonionic surfactant include polyoxyethylene
alkyl ethers, polyoxyethylene fatty acid esters, fatty acid partial
esters of polyhydric alcohols (such as glycerin and sugar alcohol),
and fatty acid diethanolamides.
[0128] Among these surfactants, surfactants approved as food
additives, such as glycerin fatty acid partial esters, sorbitan
fatty acid partial esters, and sucrose fatty acid partial esters,
are preferred in the present invention because these surfactants
present no problem even if they are deposited on food, dishes, or
cooking utensils.
[0129] The surfactants also destroy cell membranes of bacteria or
envelopes of viruses (other than the intended non-enveloped viruses
of the present invention). For example, partial esters of glycerin
and fatty acids having 6 to 18 carbon atoms have high antimicrobial
activity against Escherichia coli and Staphylococcus aureus. Thus,
the alcohol preparation preferably comprises such a surfactant. The
surfactants are also used to mix an oil phase and an aqueous phase
in creams and milky lotions.
Antimicrobial Agent
[0130] Antimicrobial agents (including substances called
bactericides and disinfectants) other than ethanol and citric acid
described above for use in the present invention are not
particularly limited. Examples of preferred antimicrobial agents
include antimicrobial agents that are efficacious against
Escherichia coli, Staphylococcus aureus, MRSA, Salmonella, Vibrio
parahaemolyticus, or Pseudomonas aeruginosa, which causes problems
of infection in food processing or nosocomial infection. Examples
of such antimicrobial agents are as follows: [0131] Natural
antimicrobial agents: proteins (such as milt protein and egg-white
lysozyme) and peptides (such as polylysine); [0132] Antibiotics:
penicillin antibiotics, chloramphenicol, streptomycin, tetracycline
antibiotics, and cephalosporin antibiotics; and [0133] Synthetic
antimicrobial agents: chlorine compounds (such as triclosan),
iodine compounds (such as povidone-iodine), zinc compounds (such as
zinc cetylpyridinium), benzenecarboxylic acids (such as benzoic
acid, salicylic acid, isopropylmethylphenol, and butyl
p-hydroxybenzoate (=butylparaben)), organic acid esters (such as
glycerin esters and sucrose esters), aldehydes (such as
glutaraldehyde and formaldehyde), biguanide compounds (such as
chlorhexidine gluconate), and quaternary ammonium salts (such as
benzalkonium chloride and cetylammonium bromide).
[0134] Among these antimicrobial agents, isopropylmethylphenol,
butyl p-hydroxybenzoate, and triclosan are preferred in the present
invention because of strong antimicrobial action and high
compatibility with a persimmon extract.
[0135] A substance that can be used as an antimicrobial agent as
described above may be contained in the ethanol preparation
described above and is sometimes used as an antiseptic in
cosmetics.
Humectant
[0136] Humectants (wetting agents) that can be used in the present
invention are those used in cosmetics such as, general lotions,
milky lotions, and creams, and include glycerin, propylene glycol,
sorbitol, polyethylene glycol, hyaluronic acid, sodium chondroitin
sulfate, ceramide, and an aloe extract. Among these humectants,
humectants approved as food or food additives, such as an aloe
extract, are preferred in the present invention because they cause
no problem even if they adheres to food, dishes, or cooking
utensils.
[0137] Cosmetic Fats and Oils
[0138] Cosmetic fats and oils form a film on the skin to protect
the skin and impart flexibility, smoothness, and gloss to the skin.
Cosmetic fats and oils also impart moderate availability to
cosmetics. In the present invention, cosmetic fats and oils used in
cosmetics, such as general milky lotions and creams, can be used.
Examples of the cosmetic fats and oils are as follows: [0139] Fats
and oils (esters of higher fatty acids and glycerin): vegetable
oils and fats, animal oils and fats, hydrogenated compounds thereof
(such as partially hydrogenated rapeseed oil), and synthetic
triglyceride (such as tri(capryl/capric acid) glyceryl); [0140] Wax
(esters of higher fatty acids and higher alcohols, the esters being
solid at normal temperature): vegetable wax and animal wax (such as
beeswax and lanolin); [0141] Hydrocarbons: mineral hydrocarbons
(such as liquid paraffin, vaseline, and paraffin) and animal
hydrocarbons (such as squalane); [0142] Higher fatty acids: lauric
acid, myristic acid, palmitic acid, stearic acid, oleic acid, and
isostearic acid; [0143] Higher alcohols: cetanol, stearyl alcohol,
and lanolin alcohol; and [0144] Esters (esters of fatty acids and
alcohols other than wax): myristyl myristate, propylene glycol
dioleate, and cetyl lactate.
[0145] Among these, cosmetic fats and oils approved as food or food
additives, such as beeswax, are preferred in the present invention
because these cosmetic fats and oils cause no problem even if they
adheres to food, dishes, or cooking utensils.
Manufacturing Method
[0146] A method for manufacturing the antiviral composition
according to the present invention is the same as a method for
manufacturing a conventional alcohol preparation, detergent,
disinfectant, or a cosmetic (lotion, milky lotion, or cream),
except that the antiviral agent (persimmon extract) is comprised as
a raw material and, if necessary, citric acid and/or a salt thereof
or vitamin C is comprised, and accordingly additional adjustments
are appropriately made. The antiviral composition according to the
present invention can be produced using the antiviral agent, in
addition to general (or, if necessary, finely adjusted) raw
materials of these conventional products by the same (or, if
necessary, finely adjusted) manufacturing process as that for
conventional products. For example, a cream according to one aspect
of the present invention may be produced by adding the antiviral
agent and other components to purified water to prepare an aqueous
phase, preparing an oil phase composed of a cosmetic fat and oil
and other materials, and mixing the aqueous phase and the oil phase
at a predetermined ratio.
[0147] A method for using the antiviral composition according to
the present invention is also the same as a method for using a
conventional alcohol preparation, detergent, disinfectant, or a
cosmetic (lotion, milky lotion, or cream). The antiviral
composition according to the present invention can be
commercialized in accordance with a suitable aspect depending on a
method for using the antiviral composition. For example, a
detergent may be a concentrated detergent, which is diluted before
use; and an alcohol preparation or a disinfectant may be of a spray
type or a wiper in which a nonwoven fabric is impregnated with the
alcohol preparation or the disinfectant.
[0148] When the antiviral agent or the antiviral composition
according to the present invention comprises an organic acid and/or
a salt thereof, the organic acid and/or a salt thereof is
preferably added in such an amount that the pH of an aqueous
solution ranges from 2.0 to 4.5, more preferably 2.0 to 4.0
(particularly for non-enveloped viruses belonging to the genus
Vesivirus or Rotavirus).
Pharmaceutical Agent
[0149] The antiviral agent according to the present invention can
be used as an active ingredient of a therapeutic or prophylactic
agent for an infectious disease caused by the non-enveloped
viruses. The dosage form of such a pharmaceutical agent can be
appropriately selected from, for example, oral ingestion forms,
such as a liquid, a syrup, a tablet, a capsule, a powder, and
granules, and an injection, and can be produced by a general
preparation method, if necessary, in combination with various
additive agents, such as an excipient (for example, lactose or
another saccharide), a binder (for example, starch,
methylcellulose, or poly(vinyl alcohol)), a stabilizer (for
example, ascorbic acid), a preservative (for example,
p-hydroxybenzoate), a sweetener, and/or a solvent. The effective
dose of such a pharmaceutical agent can be appropriately determined
in accordance with the age, the body weight, and the symptom of a
patient, the administration route, the administration schedule, and
the formulation of the pharmaceutical agent, and the inhibitory
activity of materials. The amount of antiviral agent in a
pharmaceutical agent may be adjusted to these conditions.
[0150] The antiviral agent according to the present invention and a
pharmaceutical agent comprising the antiviral agent can take not
only an administration form for humans but also an administration
form for animals other than humans, such as mammals and fishes
which are possibly infected with non-enveloped viruses. Examples of
a method which can be applied to fishes include a method of orally
administrating a mixture containing a bait and the antiviral agent
or a pharmaceutical agent comprising the antiviral agent, a method
of forcibly orally administering the antiviral agent or a
pharmaceutical agent comprising the antiviral agent using a
syringe, or a method using a "medicated bath" in which fishes are
bred for a certain time or constantly in breeding water containing
the antiviral agent.
EXAMPLES
Preparation of Persimmon Extract
[0151] (a) Persimmon juice fermented liquid (for HA-23): a ground
product of an immature fruit of Diospyros kaki (astringent
persimmon) was charged in a fine-weave cloth bag and was squeezed.
The resultant squeezed juice was charged in a glass vessel and was
naturally fermented for approximately one year. A solid was then
filtered off to prepare a reddish-brown solution. The solution
contained approximately 10% solid component (a persimmon extract),
and approximately half (approximately 5%) of the solid component
was persimmon tannin.
[0152] (b) Persimmon juice FD powder (for HA-72A/HA-201): an
immature fruit of persimmon from which a calyx was removed and
which was sufficiently sterilized (NaClO), washed, and treated
(vitamin C) for preventing coloring was diced and crushed. The
resultant fruit/fruit juice solution was passed through a 200-mesh
sieve, centrifuged, sterilized at a high temperature (120.degree.
C. to 130.degree. C., 7 to 10 seconds), and then freeze-dried to
prepare a powdery persimmon extract.
Preparation of Reagent
[0153] Reagents according to the component ratios shown in Table 1
were prepared and used for verification of the effect on fish
disease viruses or human pathogenic viruses described below
(Examples 1 to 5).
TABLE-US-00001 TABLE 1 Component Ratios of Reagent (the remainder
is water) Glycerin Persimmon extract/ Citric Trisodium mono-
Control Ethanol acid citrate caprate*.sup.6 HA-23 Persimmon juice
10% 50% 1.6% 0.5% 0.5% fermented liquid (solid content 1%) HA-72A
Persimmon juice FD powder 0.3% 50% 1.6% 0.5% 0.5% HS-201 Persimmon
juice FD powder 1% 50% -- -- -- HA-2*.sup.1 Gallnut tannin*.sup.2
0.5% 51% 1.6% 0.5% 0.6% HA-16 Pentagalloyl glucose 1% 50% 1.6% 0.5%
0.5% HA-17 Wattle tannin*.sup.3 1% 50% 1.6% 0.5% 0.5% HA-18 Coffee
tannin*.sup.4 1% 50% 1.6% 0.5% 0.5% HA-19 Propyl gallate 1% 50%
1.6% 0.5% 0.5% HA-20 Green tea tannin*.sup.5 1% 50% 1.6% 0.5% 0.5%
HA-21 Pyrogallol 1% 50% 1.6% 0.5% 0.5% HA-22 Gallic acid 1% 50%
1.6% 0.5% 0.5% HA-202 -- -- 50% 1.6% 0.5% 0.3% *.sup.1HA-2: further
comprising 0.15% glycerol (glycerin). *.sup.2Gallnut tannin: trade
name "Tannic acid AL" (Fuji Chemical Industry Co., Ltd.), tannin
derived from gallnut (insect gall of sumac). *.sup.3Wattle tannin:
trade name "Tannic acid ME-S" (Fuji Chemical Industry Co., Ltd.),
tannin derived from mimosa acacia. *.sup.4Coffee tannin: trade name
"Cafenol" (Fuji Chemical Industry Co., Ltd.), tannin derived from
green coffee beans. *.sup.5Green tea tannin: trade name "Catechin
FP95" (Fuji Chemical Industry Co., Ltd.), tannin derived from green
tea. *.sup.6Glycerin monocaprate: trade name "Poem M-200" (Riken
Vitamin Ltd.).
Example 1
Verification of Effect on Fish Disease Virus
[0154] (1) Viruses used
[0155] The characteristics of the viruses used are shown in Table
2. Viral nervous necrosis caused by viral nervous necrosis virus
(NNV) (Schneemann et al., 2005) is a disease which occurs in
various marine fishes at a very high death rate. Aquavirnavirus
(ABV) (Nguyen et al., 1994) is a spherical virus which has a genome
composed of double-stranded DNA and which causes infectious
pancreatic necrosis in fishes, such as brook trout, rainbow trout,
eel, flounder, and the like. Red seabream iridovirus (RSIV)
(Kazuhiro NAKAJIMA, Jun KURITA, 2005) causes red seabream
iridovirus disease, but this disease occurs in not only red
seabream but also various fishes such as yellowtail, amberjack, see
bass, and the like. This disease is one of the diseases with the
largest number of reported victims among the fish diseases in
Japan.
TABLE-US-00002 TABLE 2 Characteristics of Viruses and Fish Cultured
Cells Used Nucleic acid Cultured cell Virus Particle shape Envelope
(polarity) (biological origin) NNV Spherical No ssRNA (+) E-11
(Ophicephalus striatus) ABV Spherical No dsRNA (+/-) RTG-2
(Oncorhynchus mykiss) RSIV Spherical No dsDNA (+/-) GF (Haemulon
sciurus)
(2) Mixing of Virus and Test Reagent
[0156] Among the above-described reagents prepared according to the
compositions, each of the reagents shown in Table 3 was diluted
with Hanks' balanced salt solution (Nissui) (HBSS) and adjusted so
that the concentration of a persimmon extract or a control was 0.3%
or 0.03%. Fifty microliters of each virus solution was mixed with
50 .mu.L each of the reagents (the final concentration of the
persimmon extract or the control was 0.15% or 0.015%), and the
resultant mixture was allowed to stand at room temperature for 2
minutes. Then, 9.9 mL of HBSS was added to the mixture to terminate
reaction, and a 10-fold dilution series of the sample was prepared
using HBSS.
(3) Test of Measuring Viral Titer Using Fish Cultured Cells
[0157] The fish cultured cells shown in Table 2 were inoculated in
a 96-well plate (IWAKI) to be 80% confluent, and 90 .mu.L of a
medium was added to each well, followed by culture for 24 hours.
The medium used was L-15 (for E-11), MEM (for RTG-2), or BME (for
GF). Then, 10 L of the dilution series solution previously prepared
was inoculated in each well, and culture was further performed for
10 days to confirm the appearance of a cytopathic effect (index for
viral infection). Then, a limit dilution point where the cytopathic
effect was not observed was confirmed, and a viral titer was
measured according to a Reed and Muench method (1938). A ratio of
the Viral titer in a test area to the viral titer in a control area
(100%) was calculated to evaluate the antiviral effect.
(4) Experiment Result
[0158] The antiviral action of each of the reagents was as shown in
Table 3.
TABLE-US-00003 TABLE 3 Antiviral Effect of Test Reagent* NNV ABV
RSIV Test reagent 0.15% 0.15% 0.15% 0.015% HA-72A (persimmon juice
1.0 0.5 0.32 0.32 FD powder) HA-23 (persimmon juice 10.0 0.5 10 32
fermented liquid) HA-16 (pentagalloyl glucose) 3.9 17.0 10 32 HA-17
(wattle tannin) 1.0 3.0 0.21 0.32 HA-18 (coffee tannin) 143.1 17.0
100 100 HA-19 (propyl gallate) 39.2 36.3 32 100 HA-20 (green tea
tannin) 10.9 5.4 6.6 10 HA-21 (pyrogallol) 83.8 17.0 32 100 HA-22
(gallic acid) 143.1 79.4 65.8 100 HA-2 (gallnut tannin) 65.8 10.7
100 100 *Residual viral titer based on 100% of a control without
treatment of virus. For RSIV, an area of 0.015% treatment
concentration was provided.
Example 2
Verification of Effect on Human Pathogenic Virus (Coxsackie
Virus)
(1) Test Virus
[0159] Coxsackie virus group B, type 5
(non-enveloped virus, clinical isolate, 2 passages in FL cells
derived from aseptic meningitis of a 2-year-old girl)
(2) Method for Measuring Viral Infectivity
[0160] The FL cells were infected with the virus, and a 50%
infection dose (unit: 50% tissue culture infectious dose [TCID50])
was calculated.
[0161] FL cells: Cell line contaminated with HeLa cells (derived
from human cervical cancer epithelial cells).
(3) Test Method
[0162] To eliminate the influence of serum contained in original
viruses, a virus solution was previously 50 times diluted with PBS.
Then, equal amounts (100 .mu.L) of the diluted virus solution, each
of the reagents shown in Table 4 among the reagents prepared
according to the above component ratios, PBS, and 50% (v/v) ethanol
were mixed, followed by reaction at room temperature for 3 minutes.
A 10-fold dilution series was formed using a diluent and inoculated
in cells in a 96-well plate. Several days after, extension of CPE
was observed to measure viral infectivity.
[0163] Diluent: Dulbecco's modified Eagle's minimum essential
medium, penicillin G, streptomycin, amphotericin B (serum-free)
[0164] The results were as shown in Table 4 and FIG. 1.
[0165] The Coxsackie virus infectivity was suppressed to 1/30,000
to 1/50,000 or more with HA-72A and HA-201 (persimmon extract), and
thus strong antiviral activity was observed. On the other hands,
such antiviral activity was not observed with the other reagents.
It is thus considered that the persimmon extract mainly contributes
to antiviral activity, while citric acid and ethanol have no
anti-Coxsackie virus activity. Although viral infectivity appears
to remain after treatment even with HA-72A and HA-201, this is the
level of cytotoxicity due to the reagents but is not necessarily
due to viruses. It is consistent to think that the viruses are
substantially completely inactivated.
TABLE-US-00004 TABLE 4 Infectivity Effect Reagent Infectivity ratio
(times) Name Concentration (TCID50/ml) (to PBS) (to PBS) PBS
1.times. 1.8E+07 1.00000 1 HA-72A Stock solution 3.2E+02 0.00002
56234 HA-201 Stock solution 5.6E+02 0.00003 31623 HA-202 Stock
solution 3.2E+07 1.77828 1 HA-16 Stock solution 1.0E+07 0.56234 2
HA-17 Stock solution 3.2E+06 0.17783 6 HA-18 Stock solution 3.2E+07
1.77828 1 HA-19 Stock solution 1.8E+07 1.00000 1 HA-20 Stock
solution 1.8E+06 0.10000 10 HA-21 Stock solution 5.6E+07 3.16228 0
Ethanol 50% (v/v) 3.2E+07 1.77828 1
Example 3
Verification of Effect on Human Pathogenic Virus (Adenovirus)
(1) Test Virus
[0166] Adenovirus (non-enveloped virus, nonproliferating adenovirus
that expresses adenovirus type-5-derived herpesvirus thymidine
kinase)
(2) Test Method
[0167] Equal amounts (3.5 .mu.L) of a virus solution and a reagent
were mixed, followed by reaction at room temperature for 3 minutes.
A 10-fold dilution series was formed using a diluent and inoculated
in FL cells on a cover slip to measure viral infectivity.
[0168] Diluent: Eagle's minimum essential medium, penicillin G,
streptomycin (serum-free)
(3) Method for Measuring Viral Infectivity
[0169] Monolayer FL cells (cell line contaminated with HeLa cells
(derived from human cervical cancer epithelial cells)) on the cover
slip (9.times.28 mm) were washed with PBS one time and then
infected with a 50 .mu.L of virus solution. After adsorption for 1
hour, the cells were washed with PBS one time, and then a
maintenance solution (the same as the diluent) was added, followed
by culture.
[0170] Two days after, fluorescent staining was performed with
anti-adenovirus antibody (monoclonal antibody: Chemicon MAB805) and
Alexa Fluor 488-labeled anti-mouse IgG antibody (SantaCruz
Biotechnology), and positive cells were counted in a field of view
under a fluorescent microscope (400 times) (average of 20 fields of
view was calculated). The number of positive cells per cover slip
was calculated on the basis of the area of the field of view, and
further infectivity per mL of virus solution was calculated (unit:
CIU (cell infecting units)/ml; Kiyotani et al., Virology 177:65-74,
1990).
[0171] The adenovirus used synthesizes virus proteins in the FL
cells after first infection, but does not produce virus progeny and
not spread to neighbor cells and surrounding cells. Therefore, each
positive cell corresponds to at least one infectious virus. Thus,
the infectivity of the initial virus solution can be measured. FIG.
2 shows an actual fluorescent microscope image. This virus is
capable of multistage proliferation in 293T cells, and spread to
neighbor cells can be observed.
(4) Experiment Result
[0172] The antiviral action of each reagent was as shown in Table 5
and FIG. 3.
TABLE-US-00005 TABLE 5 Residual infectivity Infectivity Effect
after ratio (times) Reagent treatment (to PBS) (to PBS) PBS 3.3E+08
1 1 HA-72A persimmon extract 2.0E+02 0.0000006 1662498 50% (v/v)
ethanol 7.7E+07 0.2 4 HA-201 persimmon tannin 5.0E+02 0.000002
664999 HA-202 citric acid 3.1E+07 0.09 11 HA-16 pentagalloyl
glucose 9.9E+06 0.03 33 HA-17 wattle tannin 2.0E+02 0.0000006
1662498 HA-18 coffee tannin 1.7E+07 0.05 19 HA-19 propyl gallate
7.2E+06 0.02 46 HA-20 green tea tannin 1.0E+03 0.000003 332500
HA-21 pyrogallol 5.3E+06 0.02 63
[0173] HA-72A has high inactivation activity for adenovirus.
Although 50% ethanol and citric acid+50% ethanol (HA-202) have no
inactivation ability, 1% persimmon tannin +50% ethanol (HA-201)
suppresses adenovirus infectivity. Thus it is thought that
persimmon tannin mainly contributes to antiviral activity for
adenovirus. Other reagents, such as tannic acid and catechin, have
the same high inactivation activity for adenovirus.
[0174] In this experiment, gene defective nonproliferating
adenovirus was used in place of adenovirus, but it is supposed that
although the gene is partially defective, the particle structure
and the adsorption and penetration process of viruses are the same
as general viruses. Therefore, the results of the experiment are
considered to apply to usual adenovirus.
[0175] The results shown in Table 5 and FIG. 3 indicate that
persimmon tannin, tannic acid, and catechin have remarked
anti-adenovirus effect.
Example 4
Verification of Effect on Feline Calicivirus
(1) Test Virus
[0176] Feline calicivirus (non-enveloped virus, 2 passages in CRFK
cells, distributed from National Institute of Health Sciences,
previous isolation and passage history unknown)
(2) Cells
[0177] CRFK cells: cultured cells derived from a cat kidney,
distributed from National Institute of Health Sciences (supposed to
be the same as ATCCCCL-94).
(3) Method for Measuring Viral Infectivity
[0178] To eliminate the influence of serum contained in the
original virus, a virus solution was previously 5 times diluted
with a diluent (Eagle's minimum essential medium, penicillin G,
streptomycin). Equal amounts (35 .mu.L) of the diluted virus
solution and each reagent were mixed and allowed to react at room
temperature for 3 minutes. Then, a ten-fold dilution series was
prepared using the diluent.
[0179] Monolayer CRFK cells on a 96-well plate were washed with PBS
one time and then the diluted virus solution was inoculated (50
.mu.l/well). After incubation with a CO.sub.2 incubator for
adsorption and penetration of the virus for 1 hour, the cells were
washed with PBS one time, and then a maintenance solution (the same
as the diluent) was added (100 .mu.L/well), followed by culture.
Three days after, the cells were fixed and stained after extension
of CPE, and a 50% infectious dose (unit: 50% tissue Culture
infectious dose [TCID50]) was calculated using a Behrens-Kaerber
method to measure viral infectivity.
[0180] FIG. 4 shows an example of a plate after fixing and
staining, and the calculated infectivity is described below (in a
preliminary experiment, the effect was investigated by adding serum
to the maintenance solution).
(4) Experiment Result
[0181] The antiviral action of each reagent was as shown in Table 6
and FIG. 5.
TABLE-US-00006 TABLE 6 Infectivity Effect Infectivity ratio (times)
Reagent (TCID50/ml) (to PBS) (to PBS) PBS 1.5E+07 1 1 HA-72A
persimmon extract 3.2E+02 0.005 208 HA-201 persimmon tannin 4.4E+02
0.005 208 HA-202 citric acid 6.6E+07 0.007 150 HA-16 pentagalloyl
glucose 7.8E+06 0.5 2 HA-17 wattle tannin 3.2E+06 0.05 21 HA-18
coffee tannin 3.2E+07 0.2 7 HA-19 propyl gallate 3.7E+07 0.005 208
HA-20 green tea tannin 1.7E+07 0.005 208 HA-21 pyrogallol 7.8E+07
0.009 117 50% (v/v) ethanol 3.2E+07 0.09 12
[0182] In this experiment, the inoculation time of the virus
solution was only 1 hour in order to minimize the toxic influence
of a medicine on cells, but the cells were damaged by the medicine
at 10.sup.-1 dilution. Assuming that a damage at 10.sup.-1 dilution
is due to viral infection, the damage corresponds to an infectivity
of 3.2.times.101 TCID50/ml. It is impossible to determine whether
results lower than this value are due to the medicine or the virus,
and thus the experiment is meaningless. Therefore, a graph was
formed using this value as a lower limit.
[0183] HA-72A and HA-201 (persimmon tannin) showed high suppression
of infectivity, and thus it was confirmed that persimmon tannin has
an inactivation effect on feline calicivirus. On the other hand, a
viral inhibitory effect was also observed with HA-202 (citric
acid), HA-19 (propyl gallate), HA-20 (green tea tannin), and HA-21
(pyrogallol).
[0184] The virus was inactivated with HA-202 (citric acid), while
the virus was not inactivated with HA-16. Also, the experiment was
repeated to confirm reproducibility. As a result of measurement of
pH of the reagents, a small difference was found between pH 4.0 of
HA-202 and pH 4.20 of HA-16 (Table 7).
TABLE-US-00007 TABLE 7 pH meter pH test paper (Beckmann) (1 pH
increments) HA-72A persimmon extract 4.05 4 HA-201 persimmon tannin
5.41 5 HA-202 citric acid 4.00 4 HA-16 pentagalloyl glucose 4.20
4
[0185] Such a difference in pH is possibly significant for
inactivation of feline calicivirus. It is generally considered that
feline calicivirus is a respiratory virus and has high sensitivity
to an acid.
Example 5
Verification of Effect on Rotavirus
[0186] (1) Virus used
[0187] Rotavirus Wa strain
[0188] This strain was distributed from Okayama Prefectural
Institute for Environmental Science and Public Health by Mitsutaka
KUZUYA. In Okayama Prefectural Institute for Environmental Science
and Public Health, a strain distributed from Mitsuaki OSETO, Ehime
Prefectural Institute of Public Health Environmental Science was
subjected to 88 passages. Rotavirus is a non-enveloped virus
(diameter 60 to 80 nm) belonging to the genus Rotavirus of the
family Reoviridae and having a gene consisting of segmented
double-stranded RNA and causes acute gastroenteritis. In
particular, rotavirus causes winter diarrhea in infancy (referred
to as "white diarrhea" because feces is like a white-rice washing
liquid).
(2) Cells
[0189] MA104 cells (derived from African green monkey fetal
kidneys, epithelioid) were distributed from Mitsutaka KUZUYA,
Okayama Prefectural Institute for Environmental Science and Public
Health, which had been subjected to 55 passages there. The cells
were cultured in Eagle's minimum essential medium (MEM) containing
10% fetal bovine serum (FBS).
(3) Method for Measuring Viral Infectivity
[0190] Equal amounts (35 .mu.L) of a virus solution and a reagent
were mixed, followed by reaction at room temperature for 3 minutes.
A 10-fold dilution series was formed using a diluent (MEM) and the
MA104 cells on a cover slip (9.times.28 mm) were infected with 50
.mu.L of the virus solution. After adsorption for 1 hour, the cells
were washed with PBS one time and a growth medium (10% PBS-MEM) was
added, followed by culture.
[0191] One day after, fluorescent staining was performed with
anti-rotavirus antibody (monoclonal antibody: Abgene REMEI-1) and
Alexa Fluor 488-labeled anti-mouse IgG antibody (SantaCruz
Biotechnology), and positive cells were counted in a field of view
under a fluorescent microscope (400 times) (average area of 20
fields of view). The number of positive cells per cover slip was
calculated on the basis of the area of the field of view, and
further infectivity per mL of virus solution was calculated (unit:
CIU (cell infecting units)/ml; Kiyotani et al., Virology 177:65-74,
1990). When the number of positive cells was small, the positive
cells were measured by observing the whole of the cover slip.
[0192] Rotavirus requires trypsin for multistage proliferation. In
this measurement, trypsin was not added, and thus viruses produced
from the first infected cell did not infect neighbor cells.
Therefore, each positive cell corresponds to almost one infectious
virus. Thus, the infectivity of the initial virus solution can be
measured. FIG. 6 shows an actual fluorescent microscope image.
(4) Experiment Result
[0193] The antiviral action of each reagent was as shown in Table
8. Rotavirus was inactivated with any one of persimmon tannin,
citric acid, pentagalloyl glucose, wattle tannin, coffee tannin,
propyl gallate, green tea tannin, and pyrogallol (each mixed with
50% ethanol). In addition, imperfect inactivation was exhibited
only with 50% (v/v) ethanol, but substantially perfect inactivation
was achieved by increasing the ethanol concentration to 80%
(v/v).
TABLE-US-00008 TABLE 8 Residual infectivity Infectivity ratio
Reagent after treatment (to PBS) PBS 1.1E+07 1 HA-72A persimmon
extract 0 0 50% (v/v) ethanol 6.2E+05 0.06 80% (v/v) ethanol 0 0
HA-201 persimmon tannin 8.0E+01 0.000007 HA-202 citric acid 0 0
HA-16 pentagalloyl glucose 8.0E+01 0.000007 HA-17 wattle tannin
4.0E+01 0.000004 HA-18 coffee tannin 0 0 HA-19 propyl gallate 0 0
HA-20 green tea tannin 0 0 HA-21 pyrogallol 0 0
[0194] The antiviral effects of each of the reagents on Coxsackie
virus, adenovirus, feline calicivirus, and rotavirus in Examples 2
to 5 described above are summarized in the table below.
TABLE-US-00009 TABLE 9 Coxsackie Feline Adeno- Rota- Reagent pH
virus calicivirus virus virus HA-72A persimmon 4.05 .largecircle.
.largecircle. .largecircle. .largecircle. extract HA-201 persimmon
5.41 .largecircle. .largecircle. .largecircle. .largecircle. tannin
HA-202 citric acid 4.00 X .largecircle. X .largecircle. 50% (v/v)
ethanol 5.09 X X X X HA-16 pentagalloyl 4.20 X X X .largecircle.
glucose HA-17 wattle tannin X X .largecircle. .largecircle. HA-18
coffee tannin X X X .largecircle. HA-19 propyl gallate X
.largecircle. X .largecircle. HA-20 green tea tannin X
.largecircle. .largecircle. .largecircle. HA-21 pyrogallol X
.largecircle. X .largecircle. .largecircle.: Having antiviral
effect, X: Having no antiviral effect
* * * * *