U.S. patent application number 17/376727 was filed with the patent office on 2021-11-04 for use of at least one glycyrrhiza plant-based preparation, an antidote made from same, and the use of said antidote.
This patent application is currently assigned to Erber Aktiengesellschaft. The applicant listed for this patent is Erber Aktiengesellschaft. Invention is credited to Ursula HOFSTATTER-SCHAEHS, Elisabeth MAYER, Barbara NOVAK, Gerd SCHATZMAYR, Christina SCHWAB-ANDICS.
Application Number | 20210338758 17/376727 |
Document ID | / |
Family ID | 1000005712575 |
Filed Date | 2021-11-04 |
United States Patent
Application |
20210338758 |
Kind Code |
A1 |
MAYER; Elisabeth ; et
al. |
November 4, 2021 |
USE OF AT LEAST ONE GLYCYRRHIZA PLANT-BASED PREPARATION, AN
ANTIDOTE MADE FROM SAME, AND THE USE OF SAID ANTIDOTE
Abstract
The use of at least one Glycyrrhiza plant preparation selected
from the group of flour of a whole, dried Glycyrrhiza plant, flour
of the leaves of the dried Glycyrrhiza plant, flour of roots of the
dried Glycyrrhiza plant, aqueous dry extract of the Glycyrrhiza
plant, aqueous/ethanolic dry extract of the Glycyrrhiza plant,
aqueous extract of the Glycyrrhiza plant, optionally together with
at least one excipient, for reducing the toxic effect of at least
one polypeptide fungitoxin in agrarian products.
Inventors: |
MAYER; Elisabeth; (Fels am
Wagram, AT) ; NOVAK; Barbara; (Tulln an der Donau,
AT) ; SCHWAB-ANDICS; Christina; (Vienna, AT) ;
HOFSTATTER-SCHAEHS; Ursula; (Vienna, AT) ;
SCHATZMAYR; Gerd; (Tulln, AT) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Erber Aktiengesellschaft |
Getzersdorf bei Traismauer |
|
AT |
|
|
Assignee: |
Erber Aktiengesellschaft
Getzersdorf bei Traismauer
AT
|
Family ID: |
1000005712575 |
Appl. No.: |
17/376727 |
Filed: |
July 15, 2021 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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16473643 |
Jun 26, 2019 |
11096980 |
|
|
PCT/EP2017/001426 |
Dec 14, 2017 |
|
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17376727 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A23K 10/30 20160501;
A23K 20/163 20160501; A23K 50/75 20160501; A61K 36/484 20130101;
A61K 9/0053 20130101; A23K 50/30 20160501; A61K 31/704
20130101 |
International
Class: |
A61K 36/484 20060101
A61K036/484; A23K 20/163 20060101 A23K020/163; A23K 10/30 20060101
A23K010/30; A61K 31/704 20060101 A61K031/704 |
Foreign Application Data
Date |
Code |
Application Number |
Dec 28, 2016 |
EP |
16450032.4 |
Claims
1-15. (canceled)
16. An antidote for oral application for reducing the toxic effect
of at least one polypeptide fungitoxin selected from the group of
enniatins, namely enniatin A, enniatin A1, enniatin B, enniatin B1,
enniatin B2 or enniatin B3; beauvericin and apicidin, comprising at
least one Glycyrrhiza plant preparation selected from the group of
flour, aqueous extract, aqueous/ethanolic extract, aqueous dry
extract and aqueous/ethanolic dry extract of a whole Glycyrrhiza
plant or of roots, together with at least one excipient.
17. The antidote according to claim 16, wherein the Glycyrrhiza
plant is selected from the group consisting of Glycyrrhiza glabra
and Glycyrrhiza uralensis.
18. The antidote according to claim 16, wherein the aqueous dry
extract comprises 4% (w/w) to 10% (w/w) glycyrrhizic acid.
19. An antidote according to claim 18, wherein 1 mg to 100 mg, of
the aqueous dry extract is used per kilogram food or feed, or an
amount of at least one of the other Glycyrrhiza plant preparations
consisting of Glycyrrhiza glabra and Glycyrrhiza uralensis with an
equivalent amount of glycyrrhizic acid.
20. The antidote according to claim 16, wherein it comprises more
than 50% (w/w), of the at least one Glycyrrhiza plant
preparation.
21. The antidote according to claim 16, wherein the at least one
excipient is selected from the group consisting of inert carriers,
vitamins, minerals, phytogenic substances, enzymes and further
components for detoxifying mycotoxins, namely mycotoxin-degrading
enzymes, aflatoxin oxidases, ergotamine hydrolases, ergotamine
amidases, zearalenone esterases, zearalenone lactonases,
zearalenone hydrolases, ochratoxin amidases, fumonisin
aminotransferases, fumonisin carboxyltransferases, aminopolyol
aminoxidases, deoxynivalenol epoxide hydrolases, deoxynivalenol
dehydrogenases, deoxynivalenol oxidases, trichothecene
dehydrogenases, trichothecene oxidases; and a
mycotoxin-transforming microorganisms DSM 11798; and
mycotoxin-binding substances, selected from microbial cell walls or
bentonite.
Description
[0001] This is a divisional application of application Ser. No.
16/473,643, filed Jun. 26, 2019, which in turn is a U.S. national
stage application of PCT/EP2017/001426, filed Dec. 14, 2017,
claiming the priority benefit of European Patent Application No.
16450032.4, filed Dec. 28, 2016, all of which are hereby
incorporated by reference.
BACKGROUND OF THE INVENTION
[0002] The present invention relates to the use of at least one
Glycyrrhiza plant extract, and to an antidote for oral application,
for reducing the toxic effect of at least one polypeptide
fungitoxin, and the use of said antidote.
[0003] Glycyrrhiza or licorice plant preparations have been used as
remedies since ancient times and are also mentioned in the
traditional Chinese medicine as one of the 50 fundamental herbs.
Glycyrrhiza is a term from Greek already describing the main
property of the root of this plant, namely that it tastes sweet and
that it is, moreover, the root, "glycos" meaning sweet and "rhize"
meaning root. Among all Glycyrrhiza species, Glycyrrhiza glabra is
probably the most relevant one, constituting the most important
representative of licorices besides Glycyrrhiza uralensis, which is
primarily used in the traditional Chinese medicine. The substances
contained in the root of licorice inter alia have been known for
their anti-inflammatory and mucolytic actions for a long time, a
main component among the effectiveness-determining ingredients of
the licorice root being, above all, glycyrrhizic acid, in
particular 18-beta-glycyrrhizic acid. Glycyrrhizic acid contents
vary as a function of the treatment of the licorice root and its
origin. Thus, different contents of glycyrrhizic acid can be
obtained from one and the same root depending on its treatment. As
further important ingredients of the licorice root, about 9%
nitrogen-containing substances, up to 3.5% fat, minor contents of
tannins, up to more than 30% starch, essential oils, L-asparagine,
up to 10% bitterns, and about 4% resins, malic acid and oxalic acid
have been identified.
[0004] An important component of the licorice root, glycyrrhizic
acid, was inter alia investigated by Liu et al. (Zhongguo Zhong Yao
Za Zhi, 2014, 39(19), 3841 et seq.) in regard to its effect on
lipopolysaccharide-(LPS)-induced cytokine expression in macrophages
and found to exhibit anti-inflammatory activity.
[0005] Fungitoxins are toxic secondary metabolic products formed by
mold fungi. Depending on their type and concentration in feeds,
they have negative effects on the performance and health of farm
animals in that they may inter alia cause fungitoxin toxicoses.
These negative effects include loss of performance, nausea,
diarrhea, reduced fertility, weakening of the immune system, the
development of cancer, and damage to the nervous system.
Fungitoxins thus constitute a health risk, and consequently an
economic risk that is at least as large.
[0006] It will even be intensified by mold fungi being able to
simultaneously form different fungitoxins and secondary
metabolites. Therefore, modern analytical methods, which are highly
precise, in most cases have detected several fungitoxins and
secondary metabolites in raw materials and hence also in feed
samples. Streit et al., Toxins, 2013, 5, 504, et seq. found
fungitoxins and secondary metabolites in over 90% of the tested raw
materials and feed samples. Detected were 7 to 69 metabolites per
sample. Due to the presence of synergistic effects not investigated
in detail, already very low concentrations of simultaneously
occurring fungitoxins may have adverse effects on farm animals.
[0007] Different species of mold fungi produce fungitoxins harmful
to agriculture, e.g. Aspergillus, Fusarium and Penicillium (Frisvad
et al, Adv. Exp. Med. Biol., 2006, 571, 3, et seq.). The best known
and most widely examined mold toxins in the field of animal
nutrition include aflatoxins, e.g. aflatoxin B1, trichothecenes,
e.g. deoxynivalenol, zearalenone, ochratoxin A and fumonisins, e.g.
fumonisin B1.
[0008] Document CN10512673A (D1) describes a sweet-wood containing
tea product which can be used to inhibit and prevent toxin loads or
effects of toxins.
[0009] The document RASHIN MOHSENI ET AL: "Antitoxin characteristic
of licorice extractr: the inhibitory effect on aflatoxin production
in Aspergillus parasiticus", JOURNAL OF FOOD SAFETY,
WILEY-BLACKWELL PUBLISHING, INC, UNITED STATES, Bd. 34, No. 2, Jan.
1, 2014 (2014-01-01) states that Glycyrrhiza glabra extracts have
antitoxin activity and are capable of reducing the production of
aflatoxins.
[0010] By now, more than 500 different fungitoxins and secondary
metabolites thereof are known, beauvericin (CAS-No: 26048-05-5),
enniatins (CAS-NO: 11113-62-5) such as enniatin A (CAS-No:
2503-13-1), A1 (CAS-No: 4530-21-6), B (CAS-No: 917-13-5), B1
(CAS-No: 19914-20-6), B2, B3 and apicidin (CAS-No: 183506-66-3)
being important representatives. In the paper by Streit et al.
(Toxins, 2012, 4, 788 et seq.), beauvericin was found in 89%,
different enniatins in 96% and apicidin in 66%, of the samples.
Enniatins could be detected in 37%, 68% and 76%, respectively, of
the tested food samples (n=4.251), feed samples (n=3.640) and in
141 different unprocessed cereal samples (n=2.647), whereas
beauvericin was detected in 20%, 21% and 54%, respectively, of the
tested food samples (n=732), feed samples (n=861) and 198 different
unprocessed cereal samples (n=554). All samples were collected in
Europe in the period between 2000 and 2013 (EFSA Journal, 2014, 12,
3802).
[0011] Enniatins (ENNs), beauvericin (BEA) and apicidin (API) can
be classified in a common group due to their type of synthesis.
They are formed by peptide biosynthesis and share a polypeptide
structure. Enniatins and beauvericin show symmetric structures and
comprise three peptide bonds with alternating ester and amide
bonds. Apicidin is not symmetrically structured, comprising four
peptide bonds which are amide bonds. In the following, enniatins,
beauvericin and apicidin are commonly referred to as "polypeptide
fungitoxins" as a subgroup of fungitoxins. The disorders and
diseases caused by polypeptide fungitoxins in subjects such as
humans and animals are herein referred to as "polypeptide
fungitoxin toxicoses". By contrast, deoxynivalenol and aflotoxin B1
are prepared by a completely different path, i.e. by isobutyl
biosynthesis, and have a polyisoprene structure.
[0012] Polypeptide fungitoxin toxicoses caused by enniatins,
beauvericin and apicidin:
[0013] Enniatins are known to exhibit cytotoxic effects on
mammalian cell lines in in vitro assays. High enniatin contents
could be detected in the jejunum, the liver and the fatty tissue of
rats, from which it could be concluded that the highest absorption
occurs in the jejunum or small intestine. McKee et al. (J. Nat.
Prod, 1997, 60, 431 et seq.) demonstrated that high doses of
enniatins were lethal to test mice in a study, even low doses
having led to losses of weight.
[0014] Beauvericin, at least in poultry, is linked with an increase
in the heart weight. Already low concentrations of beauvericin
cause toxic effects in in vitro assays.
[0015] Park et al. (Appl. Environ. Microbiol., 1999, 86, 126 et
seq.) showed that apicidin, which is a cyclic histone deacetylase
inhibitor, has toxic effects in rats, noticeable by weight loss,
bleedings in the abdomen, intestines and bladder, followed by
death. Moreover, antiproliferative and cytotoxic effects could be
detected in mammalian cell lines.
[0016] General signs of polypeptide fungitoxin toxicoses in farm
animals, in particular swine and poultry, include lack of appetite
and diarrhea, which have negative effects on performance parameters
such as live weight, feed conversion ratio, or egg weight.
[0017] The first step for avoiding harmful toxins, i.e. both
fungitoxins and polypeptide fungitoxins, is the application of
suitable agricultural practices and good storage conditions of
agrarian products. The analyses of feed samples have, however,
demonstrated that these measures are insufficient. Thus, feed
additives have been used to protect animals from the adverse
effects of fungitoxins on their health and performance. They
comprise different ingredients.
[0018] Efficient feed additives have already been applied in the
event of fungitoxin contaminations of feeds with aflatoxins,
zearalenones, trichothecenes, ochratoxin A and fumonisines.
However, so far no feed additives acting on the polypeptide
fungitoxins: beauvericin, enniatins and/or apicidin have been
known.
[0019] There is thus a substantial need to reduce the contents of
polypeptide fungitoxins in foods and feeds to the largest extent
possible, and repress as far as possible any polypeptide
fungitoxins contained therein, by the use of feed additives and/or
substances or substance groups absorbing or degrading or rendering
harmless such polypeptide fungitoxins.
SUMMARY OF THE INVENTION
[0020] To solve this object, at least one Glycyrrhiza plant
preparation selected from the group of flour, aqueous extract,
aqueous/ethanolic extract, aqueous dry extract and
aqueous/ethanolic dry extract of a whole Glycyrrhiza plant or of
roots of the Glycyrrhiza plant, optionally together with at least
one excipient for oral use, is used according to the present
invention for reducing the toxic effect of at least one polypeptide
fungitoxin selected from the group of enniatins, in particular
enniatin A, enniatin A1, enniatin B, enniatin B1, enniatin B2 or
enniatin B3; beauvericin and apicidin in agrarian products. It has
surprisingly been found that when using a Glycyrrhiza plant
preparation selected from the group of flour, aqueous extract,
aqueous/ethanolic extract, aqueous dry extract and
aqueous/ethanolic dry extract of a whole Glycyrrhiza plant or of
roots of the Glycyrrhiza plant, it is possible to inhibit the toxic
effects of polypeptide fungitoxins selected from the group of
enniatins, in particular enniatin A, enniatin A1, enniatin B,
enniatin B1, enniatin B2 or enniatin B3; beauvericin and apicidin
in agrarian products to such an extent that hazards to a subject
consuming the same, in particular humans or animals, no longer
exist or, in particular, are substantially reduced.
[0021] A nearly complete, in particular substantial, reduction of
the toxic effect of at least one polypeptide fungitoxin selected
from the group of enniatins, apicidin or beauvericin will be
achieved if a Glycyrrhiza plant of the group consisting of
Glycyrrhiza glabra and Glycyrrhiza uralensis is used.
[0022] When using an aqueous dry extract of the Glycyrrhiza plant,
in particular from the roots of the Glycyrrhiza glabra plant, which
contains between 4% (w/w) and 10% (w/w), in particular 7% (w/w), of
glycyrrhizic acid, an even further reduction of the effects of
specific polypeptide fungitoxins, i.e. beauvericin, enniatins and
also apicidin, could surprisingly be achieved, although
glycyrrhizic acid itself is known to not show any activity by
itself, and the used amount of glycyrrhizic acid is therefore just
regarded as an equivalent measure for the active ingredients. By
using at least one Glycyrrhiza plant preparation according to the
present invention, it has become possible to, in particular,
completely eliminate the toxic effect of at least one specific
polypeptide fungitoxin selected from the group of enniatins, in
particular enniatin A, enniatin A1, enniatin B and enniatin B1;
beauvericin and apicidin, or reduce the same to such an extent that
no harmful effects on the animal or even human organism are to be
expected.
[0023] The exact dosage of Glycyrrhiza plant preparations, and
hence the guarantee of a largely complete reduction of the toxic
effects of polypeptide fungitoxins, i.e. enniatins, apicidin and
beauvericin, will be possible if, as in correspondence with a
further development of the invention, the aqueous dry extract, in
particular from the roots of the Glycyrrhiza glabra plant, with
glycyrrhizic acid between 4% (w/w) and 10% (w/w), in particular 7%
(w/w), or an equivalent amount of one of the other Glycyrrhiza
plant preparations according to the present invention, is used in
an amount of at least 1 g, preferably from 1 g to 100 g, in a
particularly preferred manner from 7 g to 50 g for broilers, in a
particularly preferred manner from 5 g to 30 g for laying hens, and
in a particularly preferred manner from 5 g to 30 g for pigs, in
particular breeding piglets, per ton of agrarian product, in
particular feed or food product.
[0024] Equivalence refers to the concentrations of the phytogenic
materials or substances contained in the Glycyrrhiza plant
preparations. A well characterized and easily quantifiable
substance is glycyrrhizic acid, which is found in the roots of
Glycyrrhiza. The fraction of glycyrrhizic acid in Glycyrrhiza plant
preparations serves as an indicator of the concentration of the
totality of phytogenic agents or active substances.
[0025] By equivalent amount of Glycyrrhiza plant preparations,
those amounts at which the overall amount of glycyrrhizic acid is
equal are understood. Thus, 100 g of a dry extract with 4% (w/w)
glycyrrhizic acid is, for instance, equivalent to 50 g of a dry
extract with 8% (w/w) glycyrrhizic acid. If, for instance, 50 g of
an aqueous dry extract with 10% (w/w) glycyrrhizic acid is used per
ton of feed, the administration of 100 g of another Glycyrrhiza
plant preparation, in particular of another aqueous dry extract,
with a content of 5% (w/w) glycyrrhizic acid will be equivalent
thereto.
[0026] The effectiveness of the Glycyrrhiza plant preparations or
Glycyrrhiza antidotes, or antidotes, according to the invention, in
particular their positive effects on the performance parameters of
farm animals suffering from polypeptide fungitoxin toxicoses,
increases with an increasing amount of Glycyrrhiza plant
preparations or Glycyrrhiza antidotes, or antidotes, used. With
polypeptide fungitoxin concentrations in food or feed products of
34 ppb enniatin A, 40 ppb enniatin A1, 510 ppb enniatin B, 392 ppb
enniatin B1, 4 ppb enniatin B2, 0.34 ppb enniatin B3, 717 ppb
beauvericin and 122 ppb apicidin, clear positive effects on the
performance parameters, in particular an increase of the weight and
the egg laying rate, and hence a good effectiveness of the
Glycyrrhiza plant preparations or Glycyrrhiza antidotes, or
antidotes, against polypeptide fungitoxin toxicoses, are
recognizable. The optimum amount of use of the Glycyrrhiza plant
preparations or Glycyrrhiza antidotes, or antidotes, will always
correlate with the concentration of polypeptide fungitoxins. The
more polypeptide fungitoxins in the food or feed, the larger the
required amounts of Glycyrrhiza plant preparations or Glycyrrhiza
antidotes, or antidotes.
[0027] Especially good results will be obtained if the Glycyrrhiza
plant preparations are used such that the agrarian product is
selected from foods or feeds consisting of, or containing at least
one product contaminated with at least one polypeptide fungitoxin
and selected from the group of cereals, corn, rice, soy and other
leguminosa, colza, grasses, herbs.
[0028] In that, as in correspondence with a further development of
the invention, the Glycyrrhiza plant preparations are used such
that the further contained excipient is selected from the group
consisting of inert carriers, vitamins, minerals, phytogenic
substances, enzymes and further components for detoxifying
mycotoxins, such as mycotoxin-degrading enzymes, in particular
aflatoxin oxidases, ergotamine hydrolases, ergotamine amidases,
zearalenone esterases, zearalenone lactonases, zearalenone
hydrolases, ochratoxin amidases, fumonisin aminotrans-ferases,
fumonisin carboxyltransferases, aminopolyol aminoxidases,
deoxynivalenol epoxide hydrolases, deoxynivalenol dehydrogenases,
deoxynivalenol oxidases, trichothecene dehydrogenases,
trichothecene oxidases; and mycotoxin-transforming microorganisms
such as DSM 11798; and mycotoxin-binding substances, e.g. microbial
cell walls or inorganic materials such as bentonite, it has become
possible to also degrade, or render harmless, further polypeptide
fungitoxins partially occurring even in major amounts besides the
polypeptide fungitoxins: enniatins, beauvericin and apicidin.
[0029] In addition to the use of Glycyrrhiza plant preparations for
reducing the toxic effect of polypeptide fungitoxins in foods or
feeds, there has also been the requirement for a product that
counteracts the adverse effects of such polypeptide fungitoxins
after a possible ingestion of the same.
[0030] To solve this object, the present invention further aims at
an antidote for oral application for reducing the toxic effect of
at least one polypeptide fungitoxin, selected from the group of
enniatins, in particular enniatin A, enniatin A1, enniatin B,
enniatin B1, enniatin B2 or enniatin B3; beauvericin and apicidin
comprising at least one Glycyrrhiza plant preparation, selected
from the group of flour, aqueous extract, aqueous/ethanolic
extract, aqueous dry extract and aqueous/ethanolic dry extract of a
whole Glycyrrhiza plant or of roots of the Glycyrrhiza plant,
optionally together with at least one excipient. The oral
application of at least one Glycyrrhiza plant preparation,
optionally together with at least one further excipient, as an
antidote enables the neutralization or inhibition or prevention of
the adverse effects of polypeptide fungitoxins simultaneously with
their ingestion. In that the Glycyrrhiza plant preparation is
selected from group of flour, aqueous extract, aqueous/ethanolic
extract, aqueous dry extract and aqueous/ethanolic dry extract of
whole Glycyrrhiza plant or of roots of the Glycyrrhiza plant it can
be ingested together with the possibly contaminated food or feed
product so as to immediately prevent any harmful effect on the
organism.
[0031] A particularly advantageous effect is provided by the
antidote if, as in correspondence with a further development of the
invention, the Glycyrrhiza plant preparation is selected from the
group of flour, aqueous extract, aqueous/ethanolic extract, aqueous
dry extract and aqueous/ethanolic dry extract of a whole
Glycyrrhiza plant or of roots of the Glycyrrhiza plant, optionally
together with at least one excipient. In such a case, it can be
ingested together with the possibly contaminated food or feed
product so as to immediately prevent any harmful effect on the
organism.
[0032] The antidote will exhibit an especially advantageous and, in
particular, uniformly good activity if the Glycyrrhiza plant is
selected from the group consisting of Glycyrrhiza glabra and
Glycyrrhiza uralensis.
[0033] In that, as in correspondence with a further development of
the invention, the aqueous dry extract containing 4% (w/w) to 10%
(w/w), in particular 7% (w/w), glycyrrhizic acid is used, the
polypeptide fungitoxins contained in food or feed products can be
rendered completely harmless upon digestion, wherein the adverse
effects of glycyrrhizic acid known from the literature, namely
symptoms like hypertension or water retention, will be
prevented.
[0034] According to a further development of the invention, it is
preferably proceeded such that at least 1 g of the aqueous dry
extract with a glycyrrhizic acid concentration of 7% (w/w),
preferably from 1 g to 100 g, in a particularly preferred manner
from 7 g to 50 g for broilers, in a particularly preferred manner
from 5 g to 40 g for laying hens, and in a particularly preferred
manner from 5 g to 30 g for pigs, in particular breeding piglets,
or equivalent amounts are used per ton of agrarian product, in
particular feed or food product. It has become possible by such use
to almost completely render harmless the polypeptide fungitoxins
enniatin, beauvericin and apicidin in food or feed products so as
to safely prevent, or even guard against, the occurrence of adverse
effects such as symptoms of poisoning in humans and animals.
[0035] In that, as in correspondence with a further development of
the invention, the antidote is further devised such that it
contains more than 50% (w/w), preferably more than 90% (w/w), in
particular 100% (w/w), of the at least one Glycyrrhiza plant
preparation, it has become possible to use the synergistic effects
of the ingredients of Glycyrrhiza plant preparations. In doing so,
it has been surprisingly found that the best-known component of
Glycyrrhiza plant preparations, i.e. glycyrrhizic acid itself, does
not exhibit any activity against polypeptide fungitoxins. The
positive action of the Glycyrrhiza plant preparations according to
the invention against polypeptide fungitoxins is, therefore, based
on other ingredients of the licorice root not yet known in detail,
or ingredients of the licorice root not known in detail in terms of
activity, yet contributing to the desired effect, in particular the
prevention of adverse impacts of polypeptide fungitoxins.
[0036] In that the antidote is further developed such that the at
least one excipient is selected from the group consisting of inert
carriers, vitamins, minerals, phytogenic substances, enzymes and
further components for detoxifying mycotoxins, such as
mycotoxin-degrading enzymes, in particular aflatoxin oxidases,
ergotamine hydrolases, ergotamine amidases, zearalenone esterases,
zearalenone lactonases, zearalenone hydrolases, ochratoxin
amidases, fumonisin aminotransferases, fumonisin
carboxyltransferases, aminopolyol aminoxidases, deoxynivalenol
epoxide hydrolases, deoxynivalenol dehydrogenases, deoxynivalenol
oxidases, trichothecene dehydrogenases, trichothecene oxidases; and
mycotoxin-transforming microorganisms such as DSM 11798; and
mycotoxin-binding substances, e.g. microbial cell walls or
inorganic materials such as bentonite, it has become possible to
expand the positive effects to further potentially harmful
substances such as mycotoxins from the group of fumonisins,
aflatoxins, deoxynivalenol, trichothecenes or ochratoxins.
[0037] In this respect, it may preferably be proceeded such that
the antidote is used for the production of a preparation for oral
use for preventing or treating polypeptide fungitoxin toxicoses.
Such a preparation may, in particular, be used for the prophylaxis
and treatment of polypeptide fungitoxin toxicoses caused by
polypeptide fungitoxins selected from the group of enniatins, in
particular enniatin A, enniatin A1, enniatin B, enniatin B1,
enniatin B2 or enniatin B3; beauvericin and apicidin. In doing so,
it is, in particular, possible to prevent or remedy the harmful
effects of polypeptide fungitoxins without involving at the same
time any adverse effects caused by glycyrrhizic acid, such as
hypertension or the like.
[0038] By subjects are to be understood humans and animals, yet in
particular farm animals, preferably swine and poultry such as
broilers or laying hens, turkeys, cattle or calves.
[0039] Polypeptide fungitoxin toxicoses above all can be caused by
contaminated feed or food products, i.e. those contaminated with
polypeptide fungitoxins, polypeptide fungitoxin amounts of as low
as about 500 ppb already showing toxic effects. A polypeptide
fungitoxin toxicosis, in particular in livestock, is herein defined
as a disease triggered by polypeptide fungitoxins selected from the
group of enniatins, in particular enniatin A, enniatin A1, enniatin
B, enniatin B1, enniatin B2 or enniatin B3; beauvericin and
apicidin and leading to a deterioration of at least one performance
parameter by at least 4%, preferably 10%, relative to the positive
control group.
[0040] With broilers, clear external signs of polypeptide
fungitoxin toxicosis, in particular deteriorations of performance
parameters, occur at least from a polypeptide fungitoxin overall
concentration of about 5000 ppb, in particular 4986.34 ppb.
[0041] With laying hens, clear external signs of polypeptide
fungitoxin toxicosis, in particular deteriorations of performance
parameters, occur at least from a polypeptide fungitoxin overall
concentration of about 2000 ppb, in particular 1985 ppb.
[0042] In swine, in particular breeding piglets, clear external
signs of polypeptide fungitoxin toxicosis, in particular
deteriorations of performance parameters, occur at least from a
polypeptide fungitoxin overall concentration of about 7000 ppb, in
particular 7183.6 ppb.
[0043] Any above-described antidote or Glycyrrhiza plant
preparation may be used as Glycyrrhiza antidotes for the treatment
and/or prophylaxis of polypeptide fungitoxin toxicoses, an aqueous
dry extract from the Glycyrrhiza glabra root being preferred.
DESCRIPTION OF THE INVENTION
[0044] The effective amount of the Glycyrrhiza antidote is a
function of the amount of the polypeptide fungitoxins contained in
the contaminated feed or food and may also depend on the subject
concerned. The effective amount of a Glycyrrhiza antidote in the
form of a 100% aqueous dry extract from the Glycyrrhiza glabra root
with a glycyrrhizic acid concentration of 7% (w/w), per kilogram
feed or food approximately amounts to: [0045] at least 1 mg, 1 mg
to 100 mg, preferably 7 mg to 50 mg for broilers; [0046] at least 1
mg, 1 mg to 100 mg, preferably 5 mg to 40 mg for laying hens;
[0047] at least 1 mg, 1 mg to 100 mg, preferably 5 mg to 40 mg for
swine, in particular breeding piglets.
[0048] It will be clear to the skilled artisan that the 100%
aqueous dry extract from the Glycyrrhiza glabra root with a
glycyrrhizic acid concentration of 7% (w/w) may be replaced with
any other herein described Glycyrrhiza antidote as soon as the
latter is used in an equivalent amount.
[0049] This effective amount is sufficient to almost completely
render ineffective the toxic activity of polypeptide fungitoxins,
thus nearly eliminating the adverse effects of polypeptide
fungitoxins on the health of subjects and their performance
parameters.
[0050] In the following, the invention will be explained in more
detail by way of exemplary embodiments.
Example 1: Production of Glycyrrhiza Plant Preparations
[0051] In the following tests and examples, a spray-dried dry
extract containing as end product 7% (w/w) glycyrrhizic acid and
0.003% (w/w) glycyrrhetinic acid was used as Glycyrrhiza plant
preparation. The contents of glycyrrhizic acid and glycyrrhetinic
acid may vary as a function of the starting material and process
operation, as known to the skilled artisan. Such Glycyrrhiza plant
preparations with deviating concentrations of glycyrrhizic acid and
glycyrrhetinic acid are also encompassed by the invention, their
application amounts having to be adapted to the contents of
phytogenic ingredients with a view to ensuring the use of
respectively equivalent amounts, based on the content of
glycyrrhizic acid.
[0052] To produce this extract, it was proceeded as follows: After
the harvest of the 3- to 4-year old Glycyrrhiza glabra root, the
latter was initially disintegrated and triturated with water to a
fine pulp, said pulp having been boiled and concentrated for
several hours, in particular 3 hours. The extract, after having
been filtered and allowed to settle, is further extracted by
aqueous vapor extraction under reduced pressure. Thus, a natural,
concentrated juice is formed, which is subsequently boiled further
under constant stirring and even concentrated further to obtain the
aqueous Glycyrrhiza extract.
[0053] In a further processing and drying step, the aqueous
Glycyrrhiza extract is spray-dried. This high-speed evaporation
technique is based on the drying of small droplets in a tempered,
inert drying gas. In doing so, the liquid formulation is atomized
into fine droplets and uniformly distributed in the drying air of
the spray tower. This leads to an increase in the overall surface
area of the liquid and enables the product to be dried within a
short time. The end product is a dry, free-flowing powder of
relatively uniform particle size, i.e. the Glycyrrhiza dry extract.
After this, the product is qualitatively assessed and can be
redissolved by oral ingestion of subjects, or for carrying out
additional tests (Karasaaslaan and Dalgici, J. Food Sci. Technol.,
2014, 51(11), 3014 et seq.; Bauer et al., Lehrbuch der
Pharmazeutischen Technologie, 2012, ISBN 978-3-8047-2552-2).
Example 2: In Vitro Protective Effect of the Glycyrrhiza Dry
Extract Against Polypeptide Fungitoxins
[0054] In order to test the correlations of polypeptide fungitoxins
on epithelial tissue under controlled conditions, in vitro studies
were performed. Therein, well-characterized test systems based on
cell cultures and described in the literature were employed. For
feed additives, epithelial cells from the respective organ in
question, e.g. in the present case IPEC-J2 cells from the porcine
intestine, were preferred. The used epithelial cells from the
porcine intestine offer the advantage of being non-transformed
cells still having all the important properties of normal
intestinal cells and large physiological similarities such as the
formation of viable tight junction proteins, which are above all
essential for the intestinal barrier, as well as the expression of
characteristic enzymes and transport systems (e.g. P-glycoprotein,
cytochrome P450 3A4, vit. B 12 transporters etc.).
[0055] Such in vitro test systems constitute important and approved
methods for predicting in vivo results, even enabling the
renunciation of bioavailability studies. A human cell line, Caco-2
cells, which are very similar to IPEC-J2 cells in many aspects, was
already published at the end of the 1980s for investigating the
transport of pharmaceutical substances. Later on, good correlations
between the permeability data obtained from cell monolayers and the
oral absorption rate were found. That is why this model found its
way into the pharmaceutical industry. Furthermore, the American
approval agency, FDA, published a directive on this, providing a
context to the so-called Biopharmaceutics Classification System
(BCS), the determination of the intestinal permeability by way of
validated cell culture systems. Based on such in vitro data, the
renouncement of bioavailability studies is possible in certain
cases.
[0056] For the in vitro examinations of a Glycyrrhiza dry extract,
the latter was extracted once again, thus producing a secondary
Glycyrrhiza extract. The secondary Glycyrrhiza extract was prepared
in that 1 g of the Glycyrrhiza dry extract and 9 ml 70% ethanol was
each weighed in, mixed, and shaken at 700 rpm for one hour at room
temperature. After this, the solution was centrifuged, and the
liquid supernatant was sterile filtered using a 0.2 .mu.m filter
and further diluted with IPEC-J2 medium. The secondary Glycyrrhiza
extract was tested in vitro in the following concentrations: 250
.mu.g, 500 .mu.g and 1,000 .mu.g Glycyrrhiza dry extract per ml
medium in the test formulation, in the following also referred to
as 250, 500 and 1,000 .mu.g/ml secondary Glycyrrhiza extract.
[0057] The fact that ethanolic extracts are able to dissolve more
ingredients than do purely aqueous extracts, and thus are closer to
the in vivo situation, in which due to the acid or basic conditions
and various enzymes in the mouth-gastrointestinal tract also more
ingredients are solubilized than by a purely aqueous extraction,
was the key reason for secondary extracts with 70% ethanol having
been prepared for the in vitro assays.
[0058] In the literature, glycyrrhizic acid is considered to be one
of the phytogenic main components of the licorice root. The
glycyrrhizic acid and glycyrrhetinic acid concentrations in the
prepared secondary Glycyrrhiza extracts were determined using
LC-MS/MS (Agilent 1290 Infinity and Sciex 5500 QTrap). To this end,
these extracts were each separated using a Kinetex Biphenyl
(100.times.3 mm) column. Multiple reaction monitoring (MRM) was at
471/105 and 471/119 Da for glycyrrhetinic acid, and 823/453 and
823/647 Da for glycyrrhizic acid. Due to the good solubility of
glycyrrhizic acid and glycyrrhetinic acid, it may be anticipated
that the total amount contained in the aqueous dry extract is also
dissolved in the secondary extract. The prepared Glycyrrhiza dry
extracts contained 7% (w/w) glycyrrhizic acid and 0.03% (w/w)
glycyrrhetinic acid.
[0059] The effectiveness of the dosage form in vivo is always a
function of the bioavailability of the substance, and hence also of
a biokinetic process--this does usually not apply to in vitro
experiments. For this reason, higher concentrations have frequently
to be used in in vitro experiments in order to be able to observe
the same effects as in vivo (Gulden and Seibert, ALTEX 22, Special
Issue 2, 2005). The concentrations of 250 .mu.g, 500 .mu.g and
1,000 .mu.g Glycyrrhiza dry extract per ml medium, which are used
in the present in vitro assays, correspond to in vivo
concentrations of about 250 g to 1 kg Glycyrrhiza dry extract per
ton of feed or food. This is by a factor of 33 to 50 more than the
5 g/t to 30 g/t used in the in vivo tests (cf. Examples 3 to
5).
[0060] The TEER technique described by Geens and Niewold
(Cytotechnology, 2011, 63, 415 et seq.) was used in an adapted
form. In this in vitro model, intestinal porcine epithelial cells
(IPEC-J2, DSMZ No.: ACC 701, Passage 1-15) are cultivated in an
insert on a permeable polyester membrane (1.12 cm.sup.2 surface
area, 0.4 .mu.m pore size, 12 mm membrane diameter) and
differentiated in the incubator for a period of 8 days at
39.degree. C. and 5% CO.sub.2. Per insert, 1.times.10{circumflex
over ( )}.sup.5 cells in 0.5 ml medium are applied to the
respective membrane. During said differentiation period, the used
medium was aspirated every other day and replaced with fresh
medium. The insert was fixed in a 12-well cell culture plate, and
the cells were supplied with 1.5 ml medium from below (basolateral
compartment) and with 0.5 ml medium from above (apical
compartment). These compartments reflect the intestinal side
(apical "A") and the blood side (basolateral "B"). The medium is a
DMEM/Ham's F12 (1:1) medium augmented with 1% 1ST, 2.5 mM Glutamax,
16 mM Hepes, 6 ng/ml EGF, 5% fetal calf serum and 100 IU/ml
penicillin and 100 .mu.g/ml streptomycin.
[0061] Differentiated cells form a cell barrier, being strongly
interconnected by tight junction proteins. These cells are,
moreover, oriented in a polarized manner as in the porcine
intestine and can be used as a representative model. Since the
intestinal barrier constitutes the first line of defense in the
digestive tract against pathogens and toxins, it is important to
health that it remains undamaged. The intactness of the cell
barrier is measured by means of a volt-ohmmeter: The electrical
resistance between the two compartments is referred to as TEER
(transepithelial electrical resistance) value and represented in
kOhm.times.cm.sup.2. The volt-ohmmeter has to be set to "power" and
"R" prior to each measurement. The longer electrode is then
introduced into the basolateral compartment, and the shorter
electrode is introduced into the apical compartment. In doing so,
the cell layer must not be touched.
[0062] The secondary Glycyrrhiza extract with a concentration of
100 mg/ml is diluted with medium to the desired concentration (250
.mu.g/ml, 500 .mu.g/ml or 1,000 .mu.g/ml). The used medium in the
apical compartment is aspirated and supplemented with 250 ml toxin
and 250 .mu.l licorice extract (both doubly concentrated in the
medium) and incubated for further 72 hours at 39.degree. C. and 5%
CO.sub.2. After 24 h, 48 h and 72 h, the TEER measurement is
performed. All measurements are effected in at least three
replicates from which the mean values are taken for further
calculations.
TABLE-US-00001 TABLE 1 Time course of TEER cell culture assay Day 0
Seeding cells into the inserts Day 2, 4, 7 Renewing medium Day 8
Addition of toxins (negativ control); Addition of Glycyrrhiza
extracts (Glycyrrhiza control); No addition (untreated cells; cell
control); Addition of toxins and of Glycyrrhiza extract; Addition
of gycyrrhizic acid Day 9, 10, 11 TEER value measurement Day 11
Cytotoxicity test
[0063] The TEER value was constant with the untreated cells (cell
control) from day 8 to day 11, likewise after the addition of the
secondary Glycyrrhiza extract in concentrations of 250 .mu.g/ml,
500 .mu.g/ml or 1,000 .mu.g/ml (Glycyrrhiza control) on day 8.
[0064] By the addition of the polypeptide fungitoxins, beauvericin,
enniatins and apicidin, in different concentrations (0.2-10 .mu.M)
(negative control) on day 8, the TEER value was, however, lowered,
since the toxins damage the intestinal barrier.
[0065] The simultaneous addition of the polypeptide fungitoxins in
concentrations of 0.438 .mu.M for apicidin, 5 .mu.M for enniatin A,
B, B1, beauvericin, and 10 .mu.M for enniatin A1, each along with
the secondary Glycyrrhiza extract in concentrations of 250
.mu.g/ml, 500 .mu.g/ml or 1,000 .mu.g/ml showed a significantly
smaller or no decrease of the TEER value as compared to the
respective negative control. The addition of the secondary
Glycyrrhiza extract could thus counteract the negative effects of
the polypeptide fungitoxins (cf. Table 2). The calculation of the
protective effects of the secondary Glycyrrhiza extracts relative
to the polypeptide fungitoxins was performed according to the
following formula:
Protective effect (%)=(TEER mean value (toxin+extract)/TEER mean
value (toxin).times.100)-100 [0066] For instance, for enniatin A [5
.mu.M] incubated with 1,000 .mu.g/ml secondary Glycyrrhiza extract,
after 24 hours: [0067] TEER mean value enniatin A: 3.928
kOhm.times. cm.sup.2 [0068] TEER mean value enniatin A+secondary
Glycyrrhiza extract: 6.451 kOhm.times. cm.sup.2 [0069] Protective
effect: (6.451 kOhm.times. cm.sup.2/3.928 kOhm.times.
cm.sup.2).times.100)-100=64.2%
[0070] Following the last TEER measurement on day 11, a
cytotoxicity test (neutral red test) was carried out in order to
exclude a cytotoxic effect of the tested toxin and licorice extract
concentrations. Tests were exclusively carried out in the
non-cytotoxic concentration range with a cell viability of above
99%.
TABLE-US-00002 TABLE 2 Protective effect of 250 .mu.g/ml, 500
.mu.g/ml and 1,000 .mu.g/ml secondary Glycyrrhiza extract against
enniatins A, A1, B, B1, beauvericin and apicidin at three different
measurement times (24, 48 and 72 hours, corresponding to days 9, 10
and 11). Toxin ENN A ENN A1 ENN B ENN B1 BEA API Time 5 .mu.M 10
.mu.M 5 .mu.M 5 .mu.M 5 .mu.M 0.438 .mu.M 250 .mu.g/ml secondary
Glycyrrhiza extract 24 h 11.8 2.6 6.1 2.9 12.8 2.1 48 h 34.8 11.6
4.8 3.6 2.0 3.0 72 h 28.5 6.8 4.6 3.5 1.5 7.7 500 .mu.g/ml
secondary Glycyrrhiza extract 24 h 13.3 11.9 5.5 21.9 26.2 8.4 48 h
36.6 23.5 8.5 16.7 18.4 16.4 72 h 41.4 20.3 15.0 27.3 27.6 19.6
1.000 .mu.g/ml secondary Glycyrrhiza extract 24 h 64.2 28.9 15.3
31.4 0.3 42.4 48 h 45.8 68.5 25.0 27.5 44.7 24.0 72 h 37.6 72.1
21.4 59.8 19.1 17.8
[0071] The strongest protective effects by the secondary
Glycyrrhiza extract were to be seen with enniatin A ([1.000
.mu.g/ml] at 24 h and 48 h, [500 .mu.g/ml] at 72 h) and A1 ([1.000
.mu.g/ml] at 48 h and 72 h). With all toxins, a
concentration-dependent improvement by secondary Glycyrrhiza
extracts could be observed. Good effects were also observed against
enniatins B and B1, the highest concentration of Glycyrrhiza
extract (1.000 .mu.g/ml) having, above all, a positive impact on
the TEER value. Positive effects against beauvericin and apicidin
were observed at 500 .mu.g/ml and 1.000 .mu.g/ml secondary
Glycyrrhiza extract.
Effect of Glycyrrhizic Acid
[0072] In order to investigate a possible effect of glycyrrhizic
acid against enniatins A, A1, B, B1, beauvericin and apicidin,
glycyrrhizic acid instead of the secondary Glycyrrhiza extract was
tested at a concentration of 70 .mu.g/ml (corresponding to 1.000
.mu.g/ml secondary Glycyrrhiza extract) in the TEER test as
described above. Yet, no positive effect could be shown relative to
the negative controls. This clearly indicates in a surprising
manner that the positive effect of Glycyrrhiza plant preparations
does not rely on the best-known phytogenic agent of the Glycyrrhiza
plant, namely glycyrrhizic acid.
Binding Assays
[0073] In order to better understand the mechanism of the positive
or protective effects of Glycyrrhiza plant preparations, binding
assays were performed with the aqueous Glycyrrhiza dry extract and
the polypeptide fungitoxins.
[0074] To this end, 1.000 ml buffer solution (1.36 g sodium acetate
trihydrate and 0.79 g calcium acetate, pH=8.0) was preincubated
with 100 g feed matrix (piglet feed consisting of 50% (w/w) wheat,
10% (w/w) wheat bran, 20% (w/w) soy, 10% (w/w) barley, 10% (w/w)
minerals) (24 h at 4.degree. C.) in order to minimize adsorption
effects of the hydrophobic toxins on the glass. After this, the
solids were centrifuged off, and the clear solution was transferred
into 50-ml glass jars each. To such 50 ml, so much aqueous
Glycyrrhiza dry extract with 7% (w/w) glycyrrhizic acid was each
added that the concentration of the extract in the binding assay
formulation each amounted to 3 mg/I and 3 g/l, respectively. After
this, the toxins, enniatins A, A1, B, B1, beauvericin and apicidin,
were added such that the concentrations in the binding assay each
amounted to 100 ppb. The binding assay formulations were
subsequently incubated at 37.degree. C. under constant stirring for
24 hours. At the beginning and after 24 hours, samples were taken
and analyzed for enniatins A, A1, B, B1, beauvericin and apicidin
by means of LC-MS/MS as described above. No reduction of the toxin
concentration could be measured in any of the tested extract
concentrations (3 g/l or 3 mg/l). It is, therefore, excluded that
the positive, protective effect of Glycyrrhiza plant preparations
is brought about by an adsorption or absorption of the toxins on
components or by dissolved substances of the Glycyrrhiza plant
preparation.
Example 3: Feeding Test with Broilers
[0075] For assessing the effect of Glycyrrhiza plant preparations
against polypeptide fungitoxins in poultry, feeding tests with
broilers were performed using a Glycyrrhiza antidote as feed
additive. The Glycyrrhiza antidote comprised a 100% aqueous
Glycyrrhiza dry extract from the roots of the Glycyrrhiza glabra
plant with a glycyrrhizic acid concentration of 7% (w/w). The
aqueous Glycyrrhiza dry extract was prepared as described in
Example 1.
[0076] To this end, 800 Ross broilers having a starting weight of
40 g were assigned to four test groups, each in 10 bays with 20
chicks each. The feed was administered ad lib.
[0077] The positive control group received regular chicken feed
(phase 1, days 0-14: corn 58%, soy HP 31.25%, premix BR 5%,
universal 6.25%, megafat 1.25%, soy oil 2.50%, amino acids 0.50%,
monocalcium phosphate 0.25%. Phase 2, days 15-35: corn 6%, soy HP
29.35%, premix BR 5% universal 6.0%, megafat 2.50%, soy oil 2.00%,
amino acids 0.15%).
[0078] The negative control group received chicken feed of the same
formulation as the positive control group, yet contaminated with
polypeptide fungitoxins. The overall contamination with polypeptide
fungitoxins was 4986.34 ppb, the final chicken feed comprising
beauvericin at 1197 ppb, enniatins at 2763.34 ppb (ENN A 34 ppb,
ENN A1 175 ppb, ENN B 1700 ppb, ENN B1 803 ppb, ENN B2 51 ppb, ENN
B3 0.34) and apicidin at 1026 ppb. The natural contamination by
aflatoxin and deoxynivalenol was each <1 ppb and thus
negligible.
[0079] The two test groups received the same feed contaminated with
polypeptide fungitoxins as the negative control group and, in
addition, the Glycyrrhiza antidote at an admixture rate of 7 g
(test group 1) and 50 g (test group 2) per ton of feed. The test
period was 35 days, the animals having been weighed on days 0 and
35.
[0080] The performance parameters, live weight and feed conversion
ratio, are represented in Tables 4 and 5. The animals of the
negative control group suffered from polypeptide fungitoxin
toxicosis, which caused liquid feces and a significant reduction of
the live weight (deterioration by 11.4%) and a deterioration of the
feed conversion ratio (deterioration by 4.9%) as compared to the
positive control group. The administration of the Glycyrrhiza
antidote in the two test groups caused a reduction of the toxic
effect of the polypeptide fungitoxins, and hence a far less
pronounced or no longer present polypeptide fungitoxin toxicosis,
thus markedly improving, at both admixture rates, the live weight
of the broilers and the feed conversion ratio relative to the
negative control group.
[0081] The Glycyrrhiza antidote or Glycyrrhiza plant preparation
according to the invention can thus be used for reducing the toxic
effect of at least one polypeptide fungitoxin in agrarian products
and for increasing the performance parameters, live weight and feed
conversion ratio, of feed contaminated with polypeptide fungitoxins
for farm animals, in particular broilers, and also for treating and
preventing polypeptide fungitoxin toxicoses.
TABLE-US-00003 TABLE 4 Effect of the Glycyrrhiza antidote or
Glycyrrhiza plant preparation on the live weight Positive Negative
Test Test control group control group group 1 group 2 Day [g] [g]
[g] [g] 35 1963 1739 1870 1897
TABLE-US-00004 TABLE 5 Effect of the Glycyrrhiza antidote or
Glycyrrhiza plant preparation on the feed conversion ratio in an
observation period of 35 days Positive Negative Test Test control
group control group group 1 group 2 Day [g/g] [g] [g/g] [g/g] 1-35
1.74 1.83 1.76 1.74
Example 4: Feeding Test with Laying Hens
[0082] For assessing the effect of Glycyrrhiza plant preparations
against polypeptide fungitoxins in poultry, feeding tests with
laying hens were performed using a Glycyrrhiza antidote as feed
additive. The Glycyrrhiza antidote comprised a 100% aqueous
Glycyrrhiza dry extract from the roots of the Glycyrrhiza glabra
plant with a glycyrrhizic acid concentration of 7% (w/w). The
aqueous Glycyrrhiza dry extract was prepared as described in
Example 1.
[0083] To this end, 160 Lohmann Brown laying hens were assigned to
four test groups, each in 10 bays with 4 hens each. The feed was
administered ad lib. The test started at the age of 22 weeks.
[0084] The positive control group received regular laying hen feed
(wheat 32.1%, corn 30.00%, soy HP 25.00%, calcium carbonate 8.60%,
laying hen premix 2.00%, rapeseed oil 1.90%, Biotronic SE forte
0.40%) (Biotronic is a trademark of Erber Aktiengesellschaft).
[0085] The negative control group received laying hen feed of the
same formulation as the positive control group, yet contaminated
with polypeptide fungitoxins. The overall contamination with
polypeptide fungitoxins was 1985 ppb, the final laying hen feed
comprising beauvericin at 835 ppb, enniatins at 1028 ppb (ENN A 35
ppb, ENN A1 76 ppb, ENN B 510 ppb, ENN B1 392 ppb, ENN B2 15 ppb)
and apicidin at 122 ppb. The natural contamination by aflatoxin and
deoxynivalenol was each <1 ppb and thus negligible.
[0086] The two test groups received the same feed contaminated with
polypeptide fungitoxins as the negative control group and, in
addition, the Glycyrrhiza antidote at an admixture rate of 5 g
(test group 1) and 40 g (test group 2) per ton of feed. The test
period was 14 days.
[0087] The performance parameters, egg-laying rate (percentage of
hens laying an egg per day), average egg weight and feed conversion
ratio, were determined during the test period and are represented
in Table 6. The animals of the negative control group suffered from
polypeptide fungitoxin toxicosis, which caused liquid feces and, in
particular, a significant deterioration of the performance
parameters (egg-laying rate: deterioration by 6.1%; egg weight:
deterioration by 3.6%; feed conversion ratio: deterioration by
7.1%). The administration of the Glycyrrhiza antidote in the two
test groups caused a reduction of the toxic effect of the
polypeptide fungitoxins, and hence a far less pronounced or no
longer present polypeptide fungitoxin toxicosis, thus markedly
improving, at both admixture rates, the performance parameters
relative to the negative control group.
[0088] The Glycyrrhiza antidote or Glycyrrhiza plant preparation
according to the invention can thus be used for reducing the toxic
effect of at least one polypeptide fungitoxin in agrarian products
and also for increasing the performance parameters, egg-laying
rate, average egg weight and feed conversion ratio, of feed
contaminated with polypeptide fungitoxins for farm animals, in
particular laying hens, and also for treating and preventing
polypeptide fungitoxin toxicoses.
TABLE-US-00005 TABLE 6 Performance parameters Positive Negative
Test Test control group control group group 1 group 2 Egg-laying
rate [%] 99 93 96 98 Average egg weight 56 54 55 56 [g] Feed
conversion 1.83 1.97 1.89 1.84 ratio [g/g]
Example 5: Feeding Test with Breeding Piglets
[0089] For assessing the effect of Glycyrrhiza plant preparations
against polypeptide fungitoxins in pigs, a feeding test with
breeding piglets was performed using a Glycyrrhiza antidote as feed
additive. The Glycyrrhiza antidote comprised a 100% aqueous
Glycyrrhiza dry extract from the roots of the Glycyrrhiza glabra
plant with a glycyrrhizic acid concentration of 7% (w/w). The
aqueous Glycyrrhiza dry extract was prepared as described in
Example 1.
[0090] To this end, 120 breeding piglets were assigned to four test
groups, each in 10 pens with 3 piglets each. The feed was
administered ad lib. The test started with 4-week-old piglets
weighing 7.7 kg.
[0091] The positive control group received regular breeding piglet
feed (phase 1, days 1-14: corn 32.00%, barley 34.90%, protein
premix 23%, sunflower oil 1.00%, dextrose 4.00%, lactose 3.00%,
piglet premix 2.1%. Phase 2, days 15-56: corn 41.00%, barley
35.00%, soy HP 20.00%, sunflower oil 0.50%, piglet premix
3.5%).
[0092] The negative control group received breeding piglet feed of
the same formulation as the positive control group, yet
contaminated with polypeptide fungitoxins. The overall
contamination with polypeptide fungitoxins was 7183.6 ppb, the
final breeding piglet feed comprising beauvericin at 717 ppb,
enniatins at 4733.6 ppb (ENN A 86 ppb, ENN A1 40 ppb, ENN B 1492
ppb, ENN B1 3111 ppb, ENN B2 4 ppb, ENN B3 0.6 ppb) and apicidin at
1733 ppb. The natural contamination by aflatoxin and deoxynivalenol
was each <1 ppb and thus negligible.
[0093] The two test groups received the same feed contaminated with
polypeptide fungitoxins as the negative control group and, in
addition, the Glycyrrhiza antidote at an admixture rate of 5 g
(test group 1) and 30 g (test group 2) per ton of feed. The test
period was 56 days.
[0094] The performance parameters, live weight and feed conversion
ratio, were determined at the end of the test period and are
represented in Table 7. The animals of the negative control groups
suffered from polypeptide fungitoxin toxicosis, which caused lack
of appetite, diarrhea and, in particular, a significant
deterioration of the performance parameters (live weight:
deterioration by 17.1%; feed conversion ratio: deterioration by
6.4%). The administration of the Glycyrrhiza antidote in the two
test groups caused a reduction of the toxic effect of polypeptide
fungitoxins, and hence a far less pronounced or no longer present
polypeptide fungitoxin toxicosis, thus markedly improving, at both
admixture rates, the performance parameters relative to the
negative control group.
[0095] The Glycyrrhiza antidote or Glycyrrhiza plant preparation
according to the invention can thus be used both for reducing the
toxic effect of at least one polypeptide fungitoxin in agrarian
products and for increasing the performance parameters, live weight
and feed conversion ratio, of feed contaminated with polypeptide
fungitoxins for farm animals, in particular swine, and also for
treating and preventing polypeptide fungitoxin toxicoses.
TABLE-US-00006 TABLE 7 Performance parameters Positive Negative
Test Test control group control group group 1 group 2 [kg] [kg]
[kg] [kg] Average live weight 35 29 32 33 day 56 Feed conversion
1.59 1.70 1.65 1.62 ratio [kg/kg]
* * * * *