U.S. patent application number 13/763262 was filed with the patent office on 2013-08-22 for antimicrobial, antibacterial and spore germination inhibiting activity from an avocado extract enriched in bioactive compounds.
This patent application is currently assigned to AVOMEX, INC. The applicant listed for this patent is AVOMEX, INC., INSTITUTO TECNOLOGICO Y DE ESTUDIOS SUPERIORES DE MONTERREY. Invention is credited to Jorge Alejandro Benavides-Lozano, Maria Isabel Garcia-Cruz, Janet Alejandra Gutierrez-Uribe, Carmen Hernandez-Brenes, Dariana Graciela Rodriguez-Sanchez.
Application Number | 20130216488 13/763262 |
Document ID | / |
Family ID | 45893587 |
Filed Date | 2013-08-22 |
United States Patent
Application |
20130216488 |
Kind Code |
A1 |
Hernandez-Brenes; Carmen ;
et al. |
August 22, 2013 |
ANTIMICROBIAL, ANTIBACTERIAL AND SPORE GERMINATION INHIBITING
ACTIVITY FROM AN AVOCADO EXTRACT ENRICHED IN BIOACTIVE
COMPOUNDS
Abstract
The present disclosure relates to extracts from Persea sp.
(avocado) enriched in bioactive compounds which can be used as
antimicrobial, antibacterial or spore germination inhibiting
agents, the process for obtaining the extracts, acetogenins and
isolated molecules and methods for using the extracts enriched in
bioactive compounds for providing antimicrobial, antibacterial or
spore germination inhibiting effect.
Inventors: |
Hernandez-Brenes; Carmen;
(Monterrey, MX) ; Garcia-Cruz; Maria Isabel;
(Monterrey, MX) ; Gutierrez-Uribe; Janet Alejandra;
(Monterrey, MX) ; Benavides-Lozano; Jorge Alejandro;
(Monterrey, MX) ; Rodriguez-Sanchez; Dariana
Graciela; (Monterrey, MX) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
ESTUDIOS SUPERIORES DE MONTERREY; INSTITUTO TECNOLOGICO Y DE
AVOMEX, INC.; |
|
|
US
US |
|
|
Assignee: |
AVOMEX, INC
Fort Worth
TX
INSTITUTO TECNOLOGICO Y DE ESTUDIOS SUPERIORES DE
MONTERREY
Monterrey
|
Family ID: |
45893587 |
Appl. No.: |
13/763262 |
Filed: |
February 8, 2013 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
PCT/IB2011/053535 |
Aug 8, 2011 |
|
|
|
13763262 |
|
|
|
|
61371984 |
Aug 9, 2010 |
|
|
|
Current U.S.
Class: |
424/59 ; 424/64;
514/546 |
Current CPC
Class: |
A01N 65/00 20130101;
A61P 31/00 20180101; A61K 31/12 20130101; A61K 31/22 20130101; A61Q
17/005 20130101; A61K 8/9789 20170801; A61K 2236/33 20130101; C07C
69/145 20130101; C11D 3/2093 20130101; A01N 37/12 20130101; A61K
45/06 20130101; A23L 3/3499 20130101; A23V 2002/00 20130101; A61K
2800/524 20130101; A61K 8/375 20130101; A61Q 19/00 20130101; A61K
2236/39 20130101; A61K 31/23 20130101; A61P 31/04 20180101; A61K
36/54 20130101; A61K 8/37 20130101; A61K 2236/35 20130101; A01N
65/24 20130101; C07C 69/16 20130101; A61K 8/97 20130101; A61K 31/23
20130101; A61K 2300/00 20130101; A61K 31/12 20130101; A61K 2300/00
20130101; A01N 65/00 20130101; A01N 65/24 20130101; A01N 2300/00
20130101 |
Class at
Publication: |
424/59 ; 514/546;
424/64 |
International
Class: |
A01N 37/12 20060101
A01N037/12 |
Claims
1. A process to obtain an extract enriched in naturally occurring
acetogenins from Persea sp. having a bacterial spore germination
inhibiting effect, which has a starting point a raw extract of the
dried or fresh seeds, and/or other Persea spp. tissue such as
mesocarp, peel, leafstalks, branches or leaves, and which comprises
the following steps: d) Partitioning of the raw extract serving as
an starting point, into a two-phase solvent system to obtain a
phase with a high content of acetogenins, further evaporated or
concentrated to obtain an extract with a high content of
acetogenins; e) Fractionating the extract with a high content of
acetogenins obtained in step a) by Fast or High Performance
Centrifugal Partition Chromatography (FCPC or HPCPC) or
Countercurrent chromatography (CCC) based on their corresponding
partition coefficient, to obtain fractions with higher
concentration of acetogenins presenting bacterial spore germination
inhibiting effect and separate them from fractions comprising
contaminants; f) Recovering and mixing of the fractions comprising
acetogenins with bacterial spore germination inhibiting effect
obtained in step b), and concentrating them to finally obtain an
extract enriched in naturally occurring acetogenins from Persea sp.
having bacterial spore germination inhibiting effect.
2. The process of claim 1; wherein the two-phase solvent system
said in step a) comprises: at least one polar solvents selected
from the group: water, C.sub.1-C.sub.4 alcohol (e.g. ethanol,
isopropanol, methanol), dimethyl sulfoxide, tetrahydrofuran,
acetone, acetonitrile; and at least one non-polar solvents selected
from the group: hexanes, heptanes, ethyl ether, ethyl acetate,
petroleum ether, butyl alcohol, chloroform, toluene, methyl
tert-butyl ether, methyl isobutyl ketone and mixtures therein.
3. The process of claim 1; wherein the fractionation by FCPC, HPCPC
or CCC said in step b) is carried out by use of a two-phase solvent
system comprising at least one of the following combinations, all
of them at different ratios, alone or in parallel:
methanol:heptanes, water:hexane, water:butanol, methyl tert-butyl
ether: acetonitrile:water, heptanes:ethyl acetate: acetonitril,
heptanes:ethyl acetate:methanol:water.
4. The process of claim 1; wherein the recovered fractions
comprising acetogenins with bacterial spore germination inhibiting
effect said in step c) have a partition coefficient value lower
than 0.5, and preferably in the in the range of between 0.19 to
0.35, when fresh seeds are used and FCPC, HPCPC or CCC is carried
out with a heptane:methanol two-phase solvent system and heptane as
initial stationary phase.
5. An extract enriched in acetogenins from Persea spp; wherein the
extract is characterized by having bacterial spore germination
inhibiting effect.
6. The extract of claim 5; wherein the spore germination inhibiting
effect of said extract can be applied to bacterial spores from the
genera which includes, but is not limited to Clostridium, Bacillus,
Alicyclobacillus.
7. The extract of claims 5 and 6; wherein the extract inhibits
bacterial spores from at least one bacteria selected from
Clostridium botulinum, Clostridium perfringens, Clostridium
difficile, Bacillus anthracis, Bacillus cereus, Bacillus subtilis,
Bacillus lichniformis, Alicyclobacillus acidoterrestris,
Alicyclobacillus acidiphilus.
8. The extract of claim 5; wherein the extract comprises at least
one compound characterized by the formula (I) ##STR00012## wherein:
R.sup.1 is an acetyl group; R.sup.2 is hydrogen or a hydroxy
protecting group; and R.sup.3 is an is an alkenyl group with at
least one carbon-carbon double bond; and/or compounds of formula
(II) ##STR00013## wherein: R.sup.1 is an acetyl group; R.sup.2 and
R.sup.4 hydrogen or a hydroxy protecting group; and R.sup.3 is an
alkenyl group with at least one carbon-carbon double bond.
9. The extract of claim 8; wherein all the stereoisomeric forms (R)
and (S), and the double bonds in cis (Z) or trans (E) configuration
are included.
10. The extract of claims 8 and 9; wherein the extract comprises at
least one compound preferably characterized by the formula (I), and
wherein there is at least one double bond with trans configuration
at the C-5 and C-6 position of the compound.
11. The extract of claims 8 and 9; wherein the extract comprises at
least one compound preferably characterized by the formula (I), and
wherein there is at least one double bond with trans configuration
at the C-16 and C-17 position of the compound.
12. The extract of claims 8 and 9; wherein the extract comprises at
least one compound preferably characterized by the formula
##STR00014##
13. Use of the extract of claims 5 to 12 to make a composition or
product for providing an spore germination inhibiting effect;
wherein the composition or product is selected from the group
consisting of: a pharmaceutical composition, comprising the extract
and a pharmaceutically acceptable carrier; wherein the
pharmaceutical composition is suitable for one or more of the
following administration vias: oral, dermal, parenteral, nasal,
ophthalmical, optical, sublingual, rectal, gastrical or vaginal; a
food additive composition comprising the extract and a food grade
acceptable carrier, suitable for inclusion into food products;
wherein the food product is selected from one of more of the
following: fish, crustaceans, fish substitutes, crustacean
substitutes, meat, meat substitutes, poultry products, vegetables,
greens, sauces, emulsions, beverages, juices, wines, beers, dairy
products, egg-based products, jams, jellies, grain-based products,
baked goods and confectionary products; a personal care products;
wherein the personal care composition is selected from one or more
of the following: creams, gels, powders, lotions, sunscreens,
lipstick, body wash, herbal extracts, and formulations that support
the growth of bacteria; and a cleaning composition; wherein the
cleaning composition is suitable for application to one of the
following: counter tops, doors, windows, handles, surgical
equipment, medical tools, and contact surfaces that can contaminate
humans or animals.
14. Composition or product comprising at least one of the extracts
of claims 5 to 12 characterized by having an inhibitory effect in
spore germination.
15. Composition or product comprising at least one of the extracts
of claim 10 characterized by having an inhibitory effect over the
genera Listeria at storage temperatures in the range of 0 to
10.degree. C.
16. Composition or product comprising at least one of the extracts
of claims 11 and 12 characterized by having an antibacterial,
antifungical, antiviral, anti-yeast, and in spore germination
inhibitory effect.
17. Use of the compositions or products of claims 14 to 16, wherein
the composition or product is selected from the group consisting
of: a pharmaceutical composition, comprising the extract and a
pharmaceutically acceptable carrier; wherein the pharmaceutical
composition is suitable for one or more of the following
administration vias: oral, dermal, parenteral, nasal, ophthalmical,
optical, sublingual, rectal, gastrical or vaginal; a food additive
composition comprising the extract and a food grade acceptable
carrier, suitable for inclusion into food products; wherein the
food product is selected from one of more of the following: fish,
crustaceans, fish substitutes, crustacean substitutes, meat, meat
substitutes, poultry products, vegetables, greens, sauces,
emulsions, beverages, juices, wines, beers, dairy products,
egg-based products, jams, jellies, grain-based products, baked
goods and confectionary products; a personal care products; wherein
the personal care composition is selected from one or more of the
following: creams, gels, powders, lotions, sunscreens, lipstick,
body wash, herbal extracts, and formulations that support the
growth of bacteria; and a cleaning composition; wherein the
cleaning composition is suitable for application to one of the
following: counter tops, doors, windows, handles, surgical
equipment, medical tools, and contact surfaces that can contaminate
humans or animals.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of International PCT
Application No. PCT/IB2011/053535 filed Aug. 8, 2011. This
application also claims priority to U.S. Provisional Application
No. 61/371,984 filed Aug. 9, 2010. The contents of all of the above
are hereby incorporated in their entirety by reference.
[0002] Any foregoing applications and all documents cited therein
or during their prosecution ("application cited documents") and all
documents cited or referenced in the application cited documents,
and all documents cited or referenced herein ("herein cited
documents"), and all documents cited or referenced in herein cited
documents, together with any manufacturer's instructions,
descriptions, product specifications, and product sheets for any
products mentioned herein or in any document incorporated by
reference herein, are hereby incorporated herein by reference, and
may be employed in the practice of the disclosure.
BACKGROUND
[0003] 1. Technical Field
[0004] Some technical definitions relevant to the disclosure
include "non-spore forming bacteria" which is a known term used for
pathogenic and spoilage bacteria that cannot form bacterial spores
and can be destroyed or controlled by a heat treatment,
refrigerated anaerobic storage, antibacterial substances and other
methods known in the art used alone or in combination. Another
relevant term is "spore forming bacteria", which includes
pathogenic and spoilage bacterial capable of forming very resistant
structures called bacterial spores (also termed endospores) that
are not necessarily destroyed or controlled by the common methods
known in the art for the control of non-spore forming bacteria and
require specific treatments for their inhibition and/or
inactivation. Additionally, both types of bacteria can exist in
nature in a "vegetative state" also termed viable cells; however
spore-forming bacteria can also exist in a "spore-state" which is
more resistant to chemical and physical treatments for their
inactivation. In the field of food technologies there are
additional bacterial states for spore forming bacteria that are
artificially created by the application of heat termed
"heat-shocked spores" and/or pressure "pressure-shocked spores".
The artificial states generated in the food industry result in an
even higher resistance of the spores to their inactivation by
chemical and physical means and in some food systems need to be
controlled in order to inhibit their germination into the
vegetative form of the spore forming bacteria and subsequent
spoilage of the food and/or toxin production.
[0005] Some additional technical definitions relevant to the
disclosure include "antimicrobial" which is a term used to describe
an agent able of inhibiting the growth of a wide class of
microorganisms including bacterias, fungus, molds, viruses or
yeast. Whereas "antibacterial" is a term used to describe an agent
able of inhibiting the growth of spore forming or non-spore forming
bacterias in a vegetative state. And the term "spore germination
inhibiting activity" or "spore germination inhibiting effect"
refers to spores from spore forming bacteria, except for where
otherwise indicated. Additionally "raw extract" is a term used to
define an extract obtained by mixing Persea spp. (avocado) tissue
with a non-polar or polar solvent and that contains a broad
spectrum of chemical compounds other than acetogenins with
antimicrobial, antibacterial and spore germination inhibiting
effect. Whereas "extract enriched in acetogenins" is the term used
to define an extract obtained after the removal of compounds
different from acetogenins with antimicrobial, antibacterial and
spore germination inhibiting effect.
[0006] This disclosure relates to the food and pharmaceutical arts.
In particular it relates to a method of inhibiting vegetative
cells, spore germination and growth of gram positive bacteria by
the use of chemical compounds naturally present in Persea spp.
[0007] The disclosure also relates to the medical arts. In
particular it relates to a method of inhibiting the growth of
pathogenic spore forming bacteria in the body including the
gastrointestinal tract of a human or non-human vertebrate by the
use of an antimicrobial extract with specificity for this type of
bacteria.
[0008] It is known in the discipline of food processing that food
products with pH values>4.6 (commonly known in the food industry
as low-acid foods) can experience the germination and growth of
spore forming bacteria. Of particular interest for the food
industry is the use of food additives capable of inhibiting spore
germination and vegetative cell growth from pathogenic spore
forming microorganisms such as Clostridium botulinum, Clostridium
perfringens and Bacillus cereus, among others. Under the proper
food environments such as enclosed containers or anaerobic
conditions generated within the food matrix the spores from these
pathogenic microorganisms can germinate and grow to harmful numbers
of bacterial cells and in some cases can produce toxins
jeopardizing human health. Particularly, the proteolytic and
non-proteolytic strains of Clostridium botulinum are a major
concern for the food industry because of the potential germination
of their bacterial spores in foods and the production of potent
neurotoxins. Nitrites are the most commonly used food additives in
the food industry to retard/inhibit the growth of spore forming
pathogenic bacteria in refrigerated low-acid foods.
[0009] However, there is a consumer and industrial long standing
interest to reduce the utilization of synthetic food additives,
particularly nitrite compounds. Other food additives that have been
used for the same purposes include nisin (Rayman, 1981),
recombinant peptides (Tang et al., 2008), 5-aminosalicylates (Lin
and Pimentel, 2001) and ethyl lauroyl arginate (Beltran et al.,
2011). Additionally, there have been prior patents and articles
related to antimicrobial compounds from natural origin that act
against bacterial vegetative cells. Many natural sources have been
reported to contain antimicrobial compounds mainly lipophilic,
although some hydrophilic compounds have also shown activity.
Reports of antimicrobial compounds of this nature are available in
literature.
[0010] The disclosure also relates to an important public health
concern that is the ability of pathogenic species, especially the
gram positive Listeria monocytogenes, to grow at commercial
refrigeration temperatures at which processed foods are normally
stored before final consumption. Listeria monocytogenes is a
non-spore forming pathogenic bacteria of special concern for
ready-to-eat meats and dairy products; as such foods are frequently
not heated by the user prior to consumption. Consumption of foods
contaminated with Listeria monocytogenes are known in the art to
increase the risk of infection, especially among infants, the
elderly, pregnant women, and any immune compromised
individuals.
[0011] For the purposes of this disclosure a sporocidal agent is a
substance with the ability to kill at least some types of bacterial
spores whereas a sporostatic agent is a substance that has the
ability to inhibit the growth and reproduction of at least some
types of bacterial spores. Spore germination inhibitors include
both sporicidal and sporostatic agents.
[0012] In addition, except for where otherwise indicated,
depictions of the compounds described below are intended to
encompass all stereoisomeric forms thereof which includes (R) and
(S) forms and cis (Z) and trans (E) forms of the compounds. For the
purposes of this disclosure, the trans (E) form can include a
terminal alkene which has the formula --CH.dbd.CH.sub.2 (see e.g.
(2R,16E)-1-acetoxy-2-hydroxy-4-oxo-nonadeca-16,18-diene below).
[0013] 2. Description of the Related Art
[0014] Jensen in 1951 (U.S. Pat. No. 2,550,254) obtained an acetone
extract from avocado (Persea gratissima) seed having antibacterial
activity against vegetative cells from Staphylococcus aureus,
Bacillus subtilis, Aspergillus glaucus, Penicillium notatum, and
Achromobacter perolens. This extract was found to be inactive
against Esherichia coli, Pseudomonas fluorescens and Penicilliun
camemberti. The same author in 1953 (Canada Patent 494,110) refers
to avocado (Persea americana) seed as another natural source that
might be used to obtain an extract with antimicrobial activity.
Valeri and Gimeno (1954) extracted avocado seeds with petroleum
ether and reported that the resulting crude wax inhibited growth of
Micrococcus pyogenes and Sarcina lutea, but not growth of B.
subtilis or of E. coli. The prior art indicates that avocado seeds
contain antimicrobial compounds but the specific bioactivity of the
extract against particular microorganisms clearly depends on the
method of extraction, which in the end impacts the chemical
composition of the extract.
[0015] In the related art, some compounds have been isolated from
avocado seed extracts and tested to inhibit the growth of certain
microorganisms (bacteria, yeasts and fungi). Kashman et al. (1969)
isolated and elucidated the structure of eight compounds from a
hexane extract of avocado fruit and seeds and a number of derivates
thereof were prepared, obtaining higher yields from the seeds than
the fruit. All compounds showed by Kashman (1969) belong to the
same group of long chain aliphatic compounds, with one end being
unsaturated and the other end highly oxygenated. Interestingly the
compounds were divided by the authors in pairs differing only by
having a double or triple bond at the end of the chain. The
isolation of these compounds was with the aim of performing a
chemical characterization and not for obtaining bioactive
components (not bioactivity-guided isolation). Additional studies
were then performed to evaluate their antimicrobial activity
against Bacillus subtilis, Bacillus cereus, Salmonella typhi,
Shigella dysenteriae, Staphylococcus aureus, Candida albicans,
Saccharomyces cerevisiae (ATCC 7752 and S 288C) (Neeman et al.
1970). Only six of twelve long-chain aliphatic compounds tested
demonstrated inhibitory effects against some of the microorganisms
but only 1, 2, 4-trihydroxy-n-hepadeca-16-en was capable of
inhibiting the growth of all the microorganisms included in their
study in a disc inhibition antimicrobial test that used 0.05 mg of
the compound. The authors concluded that when the hydroxyl groups
on the oxidized part of the compound were totally, or partially,
acetylated, the antibacterial activity was greatly weakened (Neeman
et al. 1970). Therefore acetogenins, which are the acetylated form
of the above mentioned long chain aliphatic compounds, did not
inhibit the growth of the previously mentioned microorganisms.
Baratta et al. (1998) more recently conducted a study to evaluate
the antimicrobial and antioxidant properties of an extract of
essential oils from plants including laurel (Laurus nobilis) form
the Lauraceae family but did not include the genus Persea.
[0016] Recently, Ugbogu and Akukwe (2009) reported on the
antimicrobial effects of seed oils from Persea gratissima Gaerth F,
among other plant seed oils, against clinical isolates of non-spore
forming bacteria that included Escherichia coli, Proteus mirabilis,
Pseudomonas aeruginosa, Staphylococcus aureus and Staphylococcus
epidermis. The authors reported potential use of Persea seed oils
in the treatment of wounds. Chia and Dykes (2010) also prepared
ethanolic extracts from the epicarp and seed of Persea Americana
Mill. vars. Hass, Shepard and Fuerte. They reported that at
concentrations between 104.2-416.7 .mu.g/ml, the extract showed
antimicrobial activities against the growth of vegetative cells of
both gram positive and gram negative bacteria; the authors also
prepared a water extract that only inhibited the growth of Listeria
monocytogenes (93.8-375 .mu.g/ml) and Staphylococcus epidermis
(354.2 .mu.g/ml). Activity against Clostridium or Bacillus genus
was not evaluated for the ethanolic or aqueous extract.
Rodriguez-Carpena et al. (2011), in an attempt to isolate molecules
with antibacterial activities, prepared extracts from the peel,
pulp, and seed of two avocado cultivars (Persea Americana Mill.)
using three different solvents that included ethyl acetate, acetone
(70%) and methanol (70%). The authors tested the antibacterial
properties of the extracts against a panel of vegetative cells from
non-spore forming and spore forming bacteria, concluding that their
antibacterial activity was moderate and it was attributed to the
presence of phenolic compounds in their extracts. Therefore the
prior cited studies did not successfully performed the isolation or
chemical identification of the components potentially responsible
for the observed bioactivities or tested bacterial spores,
heat-shocked spores or pressure-shocked spores.
[0017] Similarly, other authors have tested the antimicrobial
properties of the avocado plant, against microorganisms other than
bacteria. Prusky et al. (1982) described the presence of
1-acetoxy-2-hydroxy-4-oxo-heneicosa-12,15-diene (Persin) in the
peel of unripe avocado fruits and attributed to the molecule the
antimicrobial activity against Colletotrichum gloeosporioides, a
fungus that causes anthracnose, a known problem encountered during
storage of avocado fruits.
[0018] The compound was isolated by Thin Layer Chromatography from
an ethanolic extract partitioned with dichloromethane. This
compound was later termed "persin" (Oelrichs et al., 1995), and was
confirmed by other authors as the constituent of avocado with the
highest inhibitory activity against the vegetative growth of the
fungi Colletotrichum gloeosporioides tested in vitro (Sivanathan
and Adikaram, 1989; Domergue et al., 2000), and with the capability
to inhibit its fungi spore germination and germ tube elongation
(Prusky et al., 1991a). Persin inhibited fungi spore germination
completely at 790 .mu.g/ml and the concentration of this compound
in the peels was greatly reduced during ripening (Prusky et al.,
1982). A monoene with similar structure,
1-acetoxy-2,4-dihydroxy-n-heptadeca-16-ene, also demonstrated
bioactivity against Colletotrichum gloeosporioides but it was 3
fold lower than that of persin. Interestingly, a 1:1 mixture of
both antifungal compounds showed synergistic activity and increased
the percent of inhibited germ tube elongation of germinated conidia
(Prusky et al., 1991b). Other compounds such as
1-acetoxy-2-hydroxy-4-oxo-heneicosa-5,12,15-triene (Domergue et
al., 2000) have also been proven to have antifungal bioactivity.
This last compound has been termed "Persenone A" (Kim et al.,
2000a), however none of the isolations has been performed based on
its bioactivity or with the aim of discovering novel compounds or
mixtures with increased bioactivity. Most of the prior art
publications have focused on finding molecules to prevent
postharvest damage.
[0019] Additional bioactivities that have been reported for
acetogenins included insecticidal, antitumoral, and antihelmintic
properties. Persin has shown to have insecticidal activity,
inhibiting the larval feeding of silkworm larvae Bombyx mori L., at
a concentration in the artificial diet of 200 .mu.g/g or higher
(Chang et al., 1975; Murakoshi et al., 1976). More recently,
Rodriguez-Saona et al. (1997) demonstrated the effects of persin on
Spodoptera exigua, a generalist feeder insect, that does not feed
on avocados, but is one of the major pests of many vegetables.
Inhibitory effects were observed for both larval growth and feeding
at concentrations of 200 .mu.g/g and 400 .mu.g/g of diet,
respectively.
[0020] Persin was also identified as the active principle present
in avocado leaves that induces lactating mammary gland necrosis of
mice at a dose rate of 60-100 mg/kg, at doses above 100 mg/kg
necrosis of mice myocardial fibers may occur, and hydrothorax may
be present in severely affected animals (Oelrichs et al., 1995).
Derived from this effect, this compound and others obtained from
avocado leaves were patented as treatment for ovarian and breast
cancer in mammals (Seawright et al., 2000). The compounds were
administered orally up to 100 mg/kg of body weight of mammal being
treated, but preferably on a number of consecutive days at a
concentration of 20-40 mg/kg of body weight to avoid the previously
reported toxic effects. As it was previously noted, the
concentration of these compounds in the avocado pulp is greatly
reduced during ripening to values lower than 1500 .mu.g/g (Kobiler
et al, 1993); therefore more than 0.8 kg of avocado pulp should be
consumed daily by a 60 kg human to reach the anticancer effect and
even a higher concentration to reach the cytotoxic effects. The
annual therapeutic dose proposed for cancer treatment is 160-fold
higher than the actual annual per capita consumption of avocado in
the United States (1.8 kg or 4.1 pounds) reported by Pollack et al
(2010).
[0021] Persenone A, and its analog
1-acetoxy-2-hydroxy-5-nonadecen-4-one (Persenone B), along with
Persin were found to inhibit superoxide (O2.sup.-) and nitric oxide
(NO) generation in cell culture, activities that were associated by
the authors to therapeutic uses as cancer chemopreventive agents in
inflammation-related organs (Kim et al., 2000a, 2000b and 2000c).
In vitro results demonstrated that they have equal or better
activity than DHA (docosahexaenoic acid), a natural NO generation
inhibitor. The IC50 values were in the range of 1.2-3.5 .mu.M for
acetogenins and 4.5 .mu.M for DHA (Kim et al., 2000a).
Additionally, 1-acetoxy-2,4-dihydroxy-n-heptadeca-16-ene, persin
and persenone A showed inhibition of acetyl CoA carboxylase (ACC)
activity, in the IC50 value range 4.0-9.4 .mu.M (Hashimura, 2001).
Authors concluded that since ACC is involved in fatty acids
biosynthesis, those compounds have a potential use as fat
accumulation suppressors to avoid obesity.
[0022] Most of the extraction methods for long-chain fatty acid
derivatives require a previous step to recover the oil or the use
of organic solvents such as hexane. The method of extraction for
the identified antimicrobial compounds used by Kashman, Neeman and
Lifshitz, (1969) used hexane at boiling temperatures. Broutin et
al. in 2003 (U.S. Pat. No. 6,582,688 B1) developed a method for
obtaining an extract from avocado fruit oil enriched in certain
class of long chain aliphatic compounds, such as furan lipid
compounds and polyhydroxylated fatty alcohols. The authors claimed
that different compositions of those non polar compounds may be
used in different therapeutic, cosmetic and food applications.
However the chemical composition of the extract obtained by their
process or the content of the active molecule(s) was not specified
for its use as an antimicrobial agent. Considering the toxicity of
some of the compounds that might be present in a raw extract, it is
extremely important to define the minimal concentration required to
attain the desired effects (see U.S. Patent Application
Publications 2006-0099323 and 2009-0163590).
[0023] Even if some acetogenins have been proven to have
antimicrobial activity against vegetative cells of bacteria, the
preliminary art does not show any reports on the bio-assay guided
isolation of the antimicrobial compounds from avocado (Persea
americana) against microorganisms, particularly sporulated forms.
The present disclosure provides a series of steps for a process to
obtain isolated compounds and/or a composition that concentrates
the naturally occurring antibacterial compounds in Persea americana
that inhibit the growth of vegetative and sporulated states of
spore forming bacteria. The isolation of compounds based on
inhibition of sporulated microorganisms do not form part of the
teaching of the prior art. More importantly, the synergistic effect
of the specific compounds in partially purified mixtures is also
part of the present disclosure. The inventors found intriguing that
the partially purified extracts and/or mixtures of isolated
compounds possess spore germination inhibiting properties, such as
sporostatic and/or sporocidal properties, and in some instances
even better effects than the isolated compounds alone. The chemical
identity and specificity of the active compounds against spore
forming microorganisms has never been previously reported nor the
heat or pressure stabilities of the bioactive compounds under
commercially applicable processing conditions.
[0024] Maseko (2006) proposed a simple method to produce a non
acetylated fatty acid derivative called
(2R,4R)-1,2,4-trihydroxyheptadeca-16-ene by using (S)-malic acid as
a cheap source of the triol fragment and the Grignard reaction to
achieve the elongation of the aliphatic chain. This precursor could
be used for the synthesis of most acetogenins in avocado oil. This
molecule was produced as an analytical standard in Masenko (2006)
and in prior art Neeman et al. (1970) had shown the potential of
the compound as an antimicrobial agent against Staphylococcus spp.,
a non-spore forming bacteria. None of the cited authors tested any
specific antimicrobial properties against spore forming bacteria
nor a method to produce acetogenins with this particular
effect.
[0025] In reference to the prior art on antimicrobial substances to
be used for the specific control of vegetative cells of Listeria
monocytogenes in refrigerated foods, U.S. Pat. No. 5,217,950
suggested the use of nisin compositions as bactericides for gram
positive bacteria. U.S. Pat. Nos. 5,573,797, 5,593,800 and
5,573,801 disclose antibacterial compositions which include a
combination of a Streptococcus or Pediococcus derived bacteriocin
or synthetic equivalent antibacterial agent in combination with a
chelating agent. U.S. Pat. No. 5,458,876 suggests the combination
of an antibiotic (such as nisin) with lysozyme as an antibacterial.
In this case, lysozyme breaks down the cell wall and weakens the
structural integrity of the target cell so that the antibacterial
agent becomes more effective in damaging or killing the bacterial
cell. In particular, this combination proves to be effective in
improving the antibacterial-efficacy of nisin against Listeria
monocytogenes, yielding a significant reduction, though not a
complete elimination, of Listeria at safe and suitable levels of
use. U.S. Pat. No. 6,620,446B2, describes an antibacterial
composition for control of gram positive bacteria in food
applications that may be used as an ingredient or applied to a food
surface. This composition includes nisin, and/or lysozyme and beta
hops acids in order to reduce or eliminate gram positive spoilage
or pathogenic bacteria, and, most especially, all strains of the
harmful pathogen Listeria monocytogenes. Perumalla and
Hettiarachchy (2011) reported that green tea extract and grape seed
extract (polyphenolic and proanthocyanidin rich compounds) had
antimicrobial activities against major food borne pathogens like
Listeria monocytogenes, Salmonella typhimurium, Escherichia coli
O157:H7, and Campylobacter jejuni. Furthermore, they have
demonstrated synergism in antimicrobial activity when used in
combination with organic acids (malic, tartaric acid, benzoic acids
etc.), bacteriocins like nisin or chelating agents like EDTA in
various model systems including fresh products (fruits and
vegetables), raw and ready-to-eat meat and poultry products.
[0026] Given the difficulties associated with obtaining extracts
with adequate antibacterial, antimicrobial or spore germination
inhibiting activities, the development of resistance by bacteria,
microbes and spores to known antibacterial, antimicrobial, spore
germination inhibiting compounds and compositions, and the desire
for food products and medicaments of natural origin, there still
exists a need in the art for additional antibacterial,
antimicrobial or sporicidal compounds and compositions preferably
obtained from economically feasible sources such as plant
processing by-products and waste.
BRIEF SUMMARY
[0027] This disclosure is directed to an extract enriched in
naturally occurring acetogenins from Persea spp. characterized by
having antimicrobial, antibacterial or spore germination inhibiting
effect and the process to obtain the said extract. The disclosure
is also directed to the use of the acetogenin enriched extract that
presents spore germination inhibiting activity, as a sporicidal
and/or sporostatic agent against native bacterial spores from
Clostridium spp., Bacillus spp. and Alicyclobacillus spp., among
other pathogenic and non-pathogenic bacteria. The disclosure is
also directed to pharmaceutical, foods, personal care and cleaning
compositions or products comprising the said extract and thus
having antimicrobial, antibacterial or spore germination inhibiting
effect. We also discovered that the enriched extract is effective
as an antimicrobial agent to inhibit the growth of viable cells of
other non-spore forming gram positive bacteria such as Listeria
monocytogenes, in combination with refrigerated conditions.
Additionally, we also discovered that the enriched extract contains
two natural occurring acetogenins not previously characterized,
which have antimicrobial and spore germination inhibiting effect.
It is also part of this disclosure to protect the use of the
acetogenin enriched extract in formulations that are heat treated,
pressure treated or stabilized by other thermal or non-thermal
conservation technologies.
BRIEF DESCRIPTION OF THE DRAWINGS
[0028] FIG. 1. Primary extraction diagram for the compounds present
in avocado seed used to evaluate their antimicrobial activities
against vegetative cells, native spores and heat shocked spores of
gram positive bacteria.
[0029] FIG. 2. Effect of the type of extraction solvent on the
antimicrobial activities of crude avocado pit extracts against the
growth of vegetative cells, native spores and heat shocked spores
of Clostridium sporogenes (ATCC 7955). The extracts were tested at
final concentration of 12.5 .mu.g of solids. Data represents the
average of three replications.+-.the standard error of the
mean.
[0030] FIG. 3. Effect of shaking on the extraction of antimicrobial
compounds from avocado pit extracts using hexane and evaluation of
their antimicrobial activities against the growth of vegetative
cells, native spores and heat shocked spores of Clostridium
sporogenes (ATCC 7955). The extracts were tested at final
concentration of 12.5 .mu.g of solids. Data represents the average
of three replications.+-.the standard error of the mean. Values
with the same letter are not significantly different (LSD test,
p<0.05).
[0031] FIG. 4. Comparisons of the effect of extraction time using
acetone or ethanol instead of hexane to obtain bioactive compounds
from avocado pit that inhibit the growth of vegetative cells of C.
sporogenes (ATCC 7955). The extracts were tested at final
concentration of 12.5 .mu.g of solids. Data represents the average
of three replications.+-.the standard error of the mean. Values
with the same letter are not significantly different (LSD test,
p<0.05).
[0032] FIG. 5. Comparisons of the effect of extraction time using
acetone or ethanol instead of hexane to obtain bioactive compounds
from avocado pit that inhibit the growth of native spores of C.
sporogenes (ATCC 7955). The extracts were tested at final
concentration of 12.5 .mu.g of solids. Data represents the average
of three replications.+-.the standard error of the mean. Values
with the same letter are not significantly different (LSD test,
p<0.05).
[0033] FIG. 6. Comparisons of the effect of extraction time using
acetone or ethanol instead of hexane to obtain bioactive compounds
from avocado pit that inhibit the growth of heat-shocked spores of
C. sporogenes (ATCC 7955). The extracts were tested at final
concentration of 12.5 .mu.g of solids. Data represents the average
of three replications.+-.the standard error of the mean. Values
with the same letter are not significantly different (LSD test,
p<0.05).
[0034] FIG. 7. A) Primary extraction diagram for the compounds
present in avocado seeds using acetone and their subsequent
partition in a heptane:methanol system to obtain fractions F001 and
F002, in each phase respectively, later used to evaluate their
antimicrobial activities against vegetative cells, native spores
and heat shocked spores of gram positive bacteria. B) Simultaneous
extraction and partition diagram for the compounds present in
avocado seeds using a heptane:methanol system to obtain fractions
F003 and F004, respectively, later used to evaluate their
antimicrobial activities against vegetative cells, native spores
and heat shocked spores of gram positive bacteria.
[0035] FIG. 8. Evaluation of the antimicrobial activities against
the growth of vegetative cells, native spores and heat shocked
spores of Clostridium sporogenes (ATCC 7955) of extracts F001-F004
obtained as described in FIG. 7. The extracts were tested at final
concentration of 12.5 .mu.g of solids. Data represents the average
of three replications.+-.the standard error of the mean. Values
with the same letter are not significantly different (LSD test,
p<0.05).
[0036] FIG. 9. Evaluation of the antimicrobial activities against
the growth of vegetative cells, native spores and heat shocked
spores of Clostridium sporogenes (ATCC 7955), of the upper and
lower phases of a two phase system (ethyl acetate:water) used as a
second partition of lower phase F002 (methanol) obtained as
described in FIG. 7A. The extracts were tested at final
concentration of 12.5 .mu.g of solids. Data represents the average
of three replications.+-.the standard error of the mean. Values
with the same letter are not significantly different (LSD test,
p<0.05). (The letter c indicates a zero cm value for the disc
inhibition zone)
[0037] FIG. 10. Evaluation of the antimicrobial activities against
the growth of vegetative cells, native spores and heat shocked
spores of Clostridium sporogenes (ATCC 7955), of the upper and
lower phases of a two phases system (hexane:methanol) used for
partiton of the acetonic crude extract obtained as described in
Example 1. The extracts were tested at final concentration of 12.5
.mu.g of solids. Data represents the average of three
replications.+-.the standard error of the mean. Values with the
same letter are not significantly different (LSD test,
p<0.05).
[0038] FIG. 11. Evaluation of the antimicrobial activities against
the growth of vegetative cells, native spores and heat shocked
spores of Clostridium sporogenes (ATCC 7955), of the
unsaponifiables compounds from the acetone raw extract obtained as
described in Example 1, and the unsaponifiables compounds from the
upper phase of the two phases system (hexane:methanol) used for
partiton of the acetonic raw extract as described in Example 5. The
extracts were tested at final concentration of 12.5 .mu.g of
solids. Data represents the average of three replications.+-.the
standard error of the mean. Values with the same letter are not
significantly different (LSD test, p<0.05). (The letter d
indicates a zero cm value for the disc inhibition zone)
[0039] FIG. 12. Evaluation of the antimicrobial activities against
the growth of vegetative cells and heat shocked spores of
Clostridium sporogenes (ATCC 7955), of the fractions obtained by
reverse phase Fast Centrifugal Partition Chromatography (RP-FCPC)
of the upper phase (heptane) of the two phases system
(heptane:methanol) used for partiton of the acetonic raw extract as
described in Example 5. The solvent system used to achieve the
RP-FCPC was heptane:methanol (1:1) and methanol was used as mobile
phase. The fractions were tested at final concentration of 12.5
.mu.g of solids.
[0040] FIG. 13. Evaluation of the antimicrobial activities against
the growth of vegetative cells, native spores and heat shocked
spores of Clostridium sporogenes (ATCC 7955), of the fractions
obtained by Normal phase Fast Centrifugal Partition Chromatography
(NP-FCPC) of the upper phase (heptane) of the two phases system
(heptane:methanol) used for partiton of the acetonic raw extract as
described in Example 5. The solvent system used to achieve the
NP-FCPC was heptane:methanol (1:1) and heptane was used as mobile
phase. The fractions were tested at final concentration of 12.5
.mu.g of solids.
[0041] FIG. 14. Evaluation of the antimicrobial activities against
the growth of vegetative cells of S. aureus and B. subtilis, of the
fractions obtained by Normal phase Fast Centrifugal Partition
Chromatography (NP-FCPC) of the upper phase (heptane) of the two
phases system (heptane:methanol) used for partiton of the acetonic
raw extract as described in Example 5. The solvent system used to
achieve the NP-CPC was heptane:methanol (1:1) and heptane was used
as mobile phase. The fractions were tested at final concentration
of 12.5 .mu.g of solids.
[0042] FIG. 15. Effect of temperature (25-100.degree. C./60 min)
treatments of hexane and ethyl acetate upper phases obtained as
described in Example 5, on the inhibitory activity of the growth of
vegetative cells of Clostridium sporogenes (ATCC 7955).
[0043] FIG. 16. Effect of temperature (25-100.degree. C./60 min)
treatments of hexane and ethyl acetate upper phases obtained as
described in Example 5, on the inhibitory activity of the growth of
native spores cells of Clostridium sporogenes (ATCC 7955).
[0044] FIG. 17. Progressive change in the chromatographic profiles
of the fractions present in the active pool, obtained as described
in Example 10, as their partition coefficient (Kd) increases. The
fractions were analyzed by means of high performance liquid
chromatography and diode array detector set at 220 nm. The numbers
represent the common peaks present in different fractions.
[0045] FIG. 18. Concentration of the active compounds present in
the pool of active fractions described in Example 10.
DETAILED DESCRIPTION
[0046] The present disclosure provides a series of steps to obtain
an extract enriched in naturally occurring antimicrobial,
antibacterial or bacterial spore germination inhibiting compounds,
termed acetogenins, from Persea spp. (avocado) for providing
antimicrobial, antibacterial or bacterial spore germination
inhibiting effect.
[0047] In one aspect of the disclosure is the a process to obtain
an extract enriched in naturally occurring acetogenins with
antimicrobial, antibacterial or bacterial spore germination
inhibiting effect, from Persea sp., which includes, but is not
limited to Persea americana and gratissima (avocado) for providing
antimicrobial, antibacterial or bacterial spore germination
inhibiting effect, which includes but is not limited to the growth
of vegetative cells and spores of gram positive bacteria.
[0048] In other aspect of the disclosure the process to obtain the
said enriched extract has as an starting point a raw extract of the
dried or fresh seeds, and/or other Persea sp. tissue such as
mesocarp, peel, leafstalks, branches or leaves, which
comprises:
[0049] a) Partitioning of the raw extract serving as an starting
point, into a two-phase solvent system to obtain a phase with a
high content of acetogenins, further evaporated or concentrated to
obtain an extract with a high content of acetogenins;
[0050] b) Fractionating the extract with a high content of
acetogenins obtained in step a) by Fast or High Performance
Centrifugal Partition Chromatography (FCPC or HPCPC) or
Countercurrent chromatography (CCC) based on their corresponding
partition coefficient, to obtain fractions with higher
concentration of acetogenins presenting bacterial spore germination
inhibiting effect and separate them from other fractions comprising
contaminants;
[0051] c) Recovering and mixing of the fractions comprising
acetogenins with bacterial spore germination inhibiting effect
obtained in step b), and concentration them to finally obtain an
extract enriched in naturally occurring acetogenins from Persea sp.
having bacterial spore germination inhibiting effect.
[0052] In one embodiment of this aspect of the disclosure, the
two-phase solvent system said in step a) comprises:
[0053] at least one polar solvents selected from the group
including, but is not limited to water, C.sub.1-C.sub.4 alcohol
(e.g. ethanol, isopropanol, methanol), dimethyl sulfoxide,
tetrahydrofuran, acetone, acetonitrile; and
[0054] at least one non-polar solvents selected from the group
including, but is not limited to hexanes, heptanes, ethyl ether,
ethyl acetate, petroleum ether, butyl alcohol, chloroform, toluene,
methyl tert-butyl ether, methyl isobutyl ketone and mixtures
therein.
[0055] In another embodiment of this aspect of the disclosure, the
fractionation by FCPC, HPCPC or CCC said in step b) is carried out
to separate the compounds based on their corresponding partition
coefficient with the aim of reducing and/or eliminating
contaminants obtained during the extraction. See e.g. Alain P.
Foucalt. Centrifugal Partition Chromatography, Chromatographic
Sciences Series, vol. 68, Marcel-Dekker (1995). Additionally,
fractionation by FCPC, HPCPC or CCC can increase the concentration
of naturally occurring antimicrobial compounds from avocados (more
than 4-fold), that inhibit the growth of vegetative cells and
spores of gram positive bacteria, to provide at least 1.2 to 2
times or greater antibacterial properties when compared to an
acetone crude extract from avocado seed evaluated at the same
concentration of solids (2.5 mg/mL).
[0056] In another embodiment of this aspect of the disclosure, the
process to obtain the said enriched extract wherein the
fractionation by FCPC, HPCPC or CCC said in step b) is carried out
by use of a two-phases solvent system which include, but is not
limited to:
[0057] methanol:heptane and/or water:hexane and/or water:butanol
and/or methyl tert-butyl ether:acetonitrile:water, and/or
heptanes:ethyl acetate:acetonitril, heptanes:ethyl
acetate:methanol:water (at different ratios) of alone or in
parallel. See e.g. Alain P. Foucault, L. Chevolot. Counter-current
chromatography: instrumentation, solvent selection and some recent
applications to natural product purification. J. Chromatogr. A 808
(1998) 3-22.
[0058] In another embodiment of this aspect of the disclosure,
recovered fractions comprising acetogenins with bacterial spore
germination inhibiting effect said in step c) have a partition
coefficient value lower than 0.5, and preferably in the in the
range of between 0.19 to 0.35, when fresh seeds are used and FCPC,
HPCPC or CCC is carried out with a heptane:methanol two-phase
solvent system and heptane as initial stationary phase.
[0059] In another aspect of the disclosure, the extraction and
purification process to obtain the enriched extract, optionally
does not result in saponification of the enriched or isolated
compounds. In another embodiment of this aspect of the disclosure,
the extraction and purification process optionally does not result
in saponification of the enriched or isolated compounds.
[0060] In another aspect of the disclosure, is the extract enriched
in naturally occurring acetogenins, with antimicrobial,
antibacterial or bacterial spore germination inhibiting compounds,
comprised of at least one acetogenins with m/z in the range of 329
to 381, including, but is not limited to: Persenone A, Persenone B,
persin or the newly discovered
(2R,5E,16E)-1-acetoxy-2-hydroxy-4-oxo-nonadeca-5,16-diene or the
also newly discovered
(2R,16E)-1-acetoxy-2-hydroxy-4-oxo-nonadeca-16,18-diene that can be
purified from Persea spp., or chemically synthesized to enrich the
bioactivity.
[0061] In another aspect, the extract of the disclosure, enriched
in naturally occurring acetogenins with antimicrobial,
antibacterial or bacterial spore germination inhibiting effect is
comprised of at least one compound characterized by the formula
(I)
##STR00001##
wherein:
[0062] R.sup.1 is an acetyl group;
[0063] R.sup.2 is hydrogen or a hydroxy protecting group; and
[0064] R.sup.3 is an alkenyl group with at least one carbon-carbon
double bonds; and/or compounds of formula (II)
##STR00002##
wherein:
[0065] R.sup.1 is an acetyl group;
[0066] R.sup.2 and R.sup.4 hydrogen or a hydroxy protecting group;
and
[0067] R.sup.3 is an alkenyl group with at least one carbon-carbon
double bond.
[0068] In other embodiment of this aspect of the disclosure, the
hydroxy protecting group can be any known hydroxy protecting group,
e.g. those described in Greene and Wuts, Protective Groups in
Organic Synthesis (Third Edition), Wiley-Interscience (1999). As
noted above, the compounds of formula (I) and (II) include all
stereoisomeric forms which includes (R) and (S) forms and cis (Z)
and trans (E) forms of the compounds. For the purposes of this
disclosure, the trans (E) form can include a terminal alkene which
has the formula --CH.dbd.CH.sub.2 (see e.g.
(2R,16E)-1-acetoxy-2-hydroxy-4-oxo-nonadeca-16,18-diene below).
[0069] The compounds of formula (I) can be synthesized by reacting
dimethyl-1,3-dioxolane-ethylmagnesium halide (e.g. chloride or
bromide) with a reagent of the formula R.sup.3COX wherein R.sup.3
is as defined above and X is a halide and subsequently forming a
diol from the dioxolane ring using the procedures described in Bull
et al. (1994).
[0070] Alternatively, the compounds of formula (I) can be
synthesized by obtaining an unsaturated fatty acid and converting
it to its corresponding methyl ketone and then reacting the
corresponding methyl ketone with 2-acetoxyacetaldehye using the
procedures described in MacLeod et al. (1995).
[0071] The compounds of formula (II) can be synthesized via
reduction of ketone from the compounds of Formula (I) or
synthesized by reacting dimethyl-1,3-dioxolane-4-ethanal with a
compound of R.sup.3MgX wherein R.sup.3 is as defined above and X is
a halide using procedures disclosed by Sugiyama et al. (1982).
[0072] The methods of forming the compounds of formula (I) and
formula (II) are intended to be illustrative in nature and is not
intended to encompass all possible means of making the
compounds.
[0073] In another aspect, the extract of the disclosure is
comprised of at least one compounds preferably characterized by the
formula (I), and wherein there is at least one carbon-carbon double
bond at the C-5 and C-6 position of the compound.
[0074] In one embodiment of this aspect of the disclosure, the said
extract, comprised of at least one compounds preferably
characterized by the formula (I) and wherein there is at least one
carbon-carbon double bond at the C-5 and C-6 position of the
compound, is characterized by having an inhibitory effect over
bacterial spores from the genera which includes, but is not limited
to Clostridium, Bacillus, Alicyclobacillus and can be used as a
bacterial spore germination inhibiting agent.
[0075] In other embodiment of this aspect of the disclosure, the
said extract, comprised of at least one compounds preferably
characterized by the formula (I) and wherein there is at least one
carbon-carbon double bond at the C-5 and C-6 position of the
compound, is characterized by having an inhibitory effect over
bacterial spores from the group which includes, but is not limited
to Clostridium botulinum, Clostridium perfringens, Clostridium
difficile, Bacillus anthracis, Bacillus cereus, Bacillus subtilis,
Bacillus lichniformis, Alicyclobacillus acidoterrestris,
Alicyclobacillus acidiphilus and can be used as an bacterial spore
germination inhibiting agent.
[0076] In other embodiment of this aspect of the disclosure, the
said extract is characterized by having an inhibitory effect over
the genera Listeria at storage temperatures in the range of 0 to
10.degree. C. and can be used as an anti-Listeria agent.
[0077] In another aspect, the extract of the disclosure is
comprised of at least one compound preferably characterized by the
formula (I), wherein there is a double bond with trans
configuration at the C-16 and C-17 position of the compound.
[0078] In one embodiment of this aspect of the disclosure, the
extract of the disclosure is comprised of at least one compound
characterized by the formula:
##STR00003##
[0079] In other embodiment of this aspect of the disclosure, the
said extract is characterized by having an antibacterial,
antifungical, antiviral, anti-yeast, and in spore germination
inhibitory effect and can be used as an anti-microbial or spore
germination inhibiting agent.
[0080] In another aspect, the extract of the disclosure can be used
in compositions or products that inhibit the growth of bacterial
spores, alone or in combination with other antimicrobial substances
commonly known in the art which include but are not limited to
nitrite compounds, nisin, bacteriocins, ethyl lauroyl arginate,
essential oils, enthylenediaminetetraacetic acid (EDTA) and
ascorbic acid derivatives, benzoic acid derivatives, among others
in order to improve the antimicrobial activities against the growth
of vegetative and sporulated states of bacteria.
[0081] In another aspect, the extract of the disclosure or
compounds there in contained, or extracts derived therefrom can be
used in compositions or products providing an antimicrobial,
antibacterial or bacterial spore germination inhibiting effect and
can be formulated in solid or oily form, with antioxidants,
emulsifying agents, carriers, excipients, encapsulating agents and
other formulation components to improve the application and
stability of the bioactive components.
[0082] In another aspect, is the use of the extract of the
disclosure to make a composition or product for providing
antimicrobial, antibacterial and bacterial spore germination
inhibiting effect, wherein the composition or product is selected
from the group consisting of:
[0083] a pharmaceutical composition, comprising the extract and a
pharmaceutically acceptable carrier;
[0084] wherein the pharmaceutical composition is suitable for one
or more of the following administration vias: oral, dermal,
parenteral, nasal, ophthalmical, optical, sublingual, rectal,
gastrical or vaginal; Dermal administration includes topical
application or transdermal administration. Parenteral
administration includes intravenous, intraarticular, intramuscular,
and subcutaneous injections, as well as use of infusion techniques.
The extracts, compounds and compositions or products of the
disclosure may be present in association with one or more non-toxic
pharmaceutically acceptable ingredients to form the composition.
These compositions can be prepared by applying known techniques in
the art such as those taught in Remington--The Science and Practice
of Pharmacy, 21st Edition (2005), Goodman & Gilman's The
Pharmacological Basis of Therapeutics, 11th Edition (2005) and
Ansel's Parmaceutical Dosage Forms and Drug Delivery Systems (8th
Edition), edited by Allen et al., Lippincott Williams &
Wilkins, (2005).
[0085] a food additive composition comprising the extract and a
food grade acceptable carrier, suitable for inclusion into food
products; wherein the food product is selected from one of more of
the following: fish, crustaceans, fish substitutes, crustacean
substitutes, meat, meat substitutes, poultry products, vegetables,
greens, sauces, emulsions, beverages, juices, wines, beers, dairy
products, egg-based products, jams, jellies, grain-based products,
baked goods and confectionary products;
[0086] a personal care products; wherein the personal care
composition is selected from one or more of the following: creams,
gels, powders, lotions, sunscreens, lipstick, body wash, herbal
extracts, and formulations that support the growth of bacteria;
and
[0087] a cleaning composition; wherein the cleaning composition is
suitable for application to one of the following: counter tops,
doors, windows, handles, surgical equipment, medical tools, and
contact surfaces that can contaminate humans or animals.
[0088] Another aspect of the disclosure is the use of the extracts
or isolated compounds of the disclosure or compositions comprising
the same, to provide an antibacterial, antimicrobial or sporicidal
effect to a patient in need thereof.
[0089] Another aspect of the disclosure is the use of compositions
comprising the extract of the disclosure to provide an
antibacterial, antimicrobial or sporicidal effect to a
pharmaceutical, food, personal care, or cleaning composition or
cleaning products.
[0090] Another aspect of the disclosure is the use of the extracts
or isolated compounds of the disclosure or compositions comprising
the same to provide an antibacterial, antimicrobial or sporicidal
effect to a surface. The effect may be produced by exposing the
surface with the extracts or isolated compounds of the disclosure
or by laminating or embedding the extracts or isolated compounds of
the disclosure onto the surface itself.
[0091] The novel compounds from the extract and purification of the
disclosure were depicted above in Formula (I). For the purposes of
providing an antimicrobial, antibacterial and/or sporicidal effect,
the compounds of Formula (I) can have as few as one carbon-carbon
double bond for R.sup.3 and this double bond can either be in the
cis (Z) or trans (E) configuration. One embodiment of this scope of
the compounds of Formula (I) is that the carbon-carbon double bond
are at C-5/C-6, C-12/C-13, C-15/C-16, C-16/C-17 or any combination
thereof, with the bonds being trans or cis bonds. Another
embodiment of the scope of the compounds, include where the
carbon-carbon double bond is at C-5 and C-6 alone, and/or C-16 and
C-17, and/or C-12 and C-13, and/or C-15 and C-16 positions, either
being trans or cis bonds.
[0092] Examples of this enhanced scope of the compounds of formula
(I) include, but are not limited to:
##STR00004##
[0093] Moreover, for the purposes of providing an antimicrobial,
antibacterial and/or sporicidal effect, the compound of Formula (I)
can be used alone or in combination with the compounds for formula
(II).
[0094] Another embodiment of this aspect of the disclosure is use
of the compound of formula (II) depicted below:
##STR00005##
[0095] In another aspect of the disclosure, the antibacterial,
antimicrobial or spostatic/sporicidal effects are at least as
effective as other known antibacterial, antimicrobial or
spostatic/sporicidal agents such as LAE (ethyl ester of lauramide
of arginine monohydrochloride), nitrites or nisin (a polycyclic
peptide with 34 amino acids). Use of the extracts or isolated
compounds of the disclosure being a natural product or easily
derived therefrom is advantageous over other known agents which are
either not natural products or are not easily obtained. The use of
non-natural products especially has ramifications when making food
or cosmetic products which may require regulatory approval for
their use.
[0096] The disclosure is further described by the following
non-limiting examples which further illustrate the disclosure, and
are not intended, nor should they be interpreted to, limit the
scope of the disclosure.
EXAMPLES
Example 1
Antimicrobial and Sporicidal Activity of Acetone and Hexane Avocado
Seed Extracts
[0097] Avocado seeds were ground using a colloidal mill to obtain
particles with an average radius of 0.5-2 mm. Ground avocado seeds
(50 g) were mixed with either acetone or hexane at a
material-to-solvent ratio of 1:2 (w/v). Mixtures were stored for 24
hr at 25.degree. C. in order to obtain an avocado seed raw extract.
The seed was separated from the extract by means of vacuum
filtration. The raw extracts were evaporated under vacuum to
dryness using a rotary evaporator (35.degree. C., 22 in Hg) and the
obtained dry matter was weighed and redissolved in acetone to a
final concentration of 2.5 mg/ml. Adjusted samples were used for
antimicrobial and sporicidal tests (see FIG. 1).
[0098] For the antibacterial evaluations, adjusted solutions (5
.mu.g) were transferred to sterile 6-mm diameter discs made from
Whatman no. 1 filter paper, so that after solvent evaporation each
disc contained 12.5 .mu.g of solids from the enriched avocado seed
extract. Experimental controls were treated under the same
conditions that the extracts and included negative control discs
that contained 5 .mu.L of acetone, and for positive control discs 5
.mu.L of a nisin solution (30 mg/ml in sterile water) were added to
provide a residual concentration of 150 .mu.g of nisin in each
disc. All test discs were left for about 1-2 hr in a Biological
Safety Cabinet to evaporate the solvent. Suspensions of about 0.1
optical density (at 600 nm) containing approximately 1 to
2.times.10.sup.8CFU/ml of Clostridium sporogenes (ATCC 7955)
vegetative cells, isolated native spores or isolated heat shocked
spores were prepared as described in official protocols of Health
Canada (Food Directorate, 2010). Aliquots of the suspensions (100
.mu.L) were transferred to Petri dishes containing 15 ml of solid
medium (TPGY medium) and spread evenly with a sterile plastic rod.
Four discs, each containing 12.5 .mu.g of the test extract, and two
more discs (one solvent blank and one nisin positive control) were
placed each dish and incubated at 37.degree. C. under anaerobic
conditions. The diameter of the inhibition zones (cm) around the
discs were measured after 36 hrs.
[0099] Acetone and hexane avocado seed extracts showed significant
antimicrobial activity against vegetative bacterial cells, as well
as native and heat-shocked spores of the spore forming bacteria
Clostridium sporogenes (see FIG. 2). Non-significant differences
between the activity of acetone and hexane extracts was observed,
except for heat shocked spores were the hexane extract showed
around 20% higher sporicidal activity than the acetone extract.
Both acetone and hexane avocado seed extracts presented higher
antibacterial activities than the positive control (nisin, 150
.mu.g). Positive control treatments (nisin) gave inhibition zones
of 1.3, 1.0 and 0.9 cm for vegetative bacterial cells, spores and
heat shocked spores, respectively.
[0100] Avocado seeds used to obtain the crude extracts, once
ground, can be stored at temperatures below 25.degree. C. in
presence or absence of oxygen for at least 14 days without
affecting the antibacterial activity against spore forming
bacteria. Therefore avocado seeds can be stored as a whole or as a
meal prior to the preparation of the extracts enriched in bioactive
compounds.
Example 2
Specific Activity of Avocado Seed Extracts Against Vegetative Cells
and Heat-Shocked Bacterial Spores of Spore-Forming Bacteria as
Compared to Other Plant Sources
[0101] The efficacy of the present disclosure can be observed by
the preparation of crude antibacterial extracts from mango seed
kernel, which has been reported in the prior art to exhibit
antibacterial activity against vegetative cells of spore-forming
bacteria (Kabuki et al., 2000).
[0102] Crude extracts from avocado (Persea americana) and mango
kernel (Mangifera indica) were prepared as described in Example 1
and their antibacterial activities tested against the growth of
vegetative cells and heat-shocked spores of C. sporogenes (See
Table 1).
TABLE-US-00001 TABLE 1 Antibacterial activities of avocado seed and
mango kernel extracts against vegetative cells and heat shocked
spores of Clostridium sporogenes (ATCC 7955). Antibacterial
Activity against Clostridium sporogenes Extract (Disc inhibition
zone (cm)) Concentration Vegetative Heat-shocked Plant Source
(mg/mL) cells spores Avocado Seed 2.5 (acetone 2.0 1.0 (Persea
americana) extract) 1.25 (acetone 1.4 1.0 extract) Mango Seed
Kernel 100 (hexane 0.7 0.0 (Mangifera indica) extract) 250 (hexane
1.0 0.0 extract) 100 (acetone 0.0 0.0 extract) 250 (acetone 0.0 0.0
extract) Nisin 40 1.0 0.9 (positive control) 2.5 0.0 0.0 Methanol
(negative 0.0 0.0 0.0 control)
[0103] Contrary to the expected only the avocado seed extracts
presented activity against the two bacterial physiological stages
tested herein, vegetative cells and heat shocked spores. Mango
kernel extracts presented antibacterial activity against vegetative
cells of spore forming bacteria but not against the growth of
bacterial spores or heat-shocked spores.
[0104] The present example therefore demonstrates that the chemical
nature of avocado phytochemicals is particularly useful for the
inhibition of the growth of vegetative cells, spores and
heat-shocked pores of spore-forming bacteria.
Example 3
Effect of Shaking on the Antimicrobial Activities of Crude Acetone
and Hexane Avocado Seed Extracts
[0105] Similarly to Example 1, avocado seeds were ground using a
colloidal mill obtaining particles with an average diameter of
0.5-2 mm. Ground avocado seeds (50 g) were mixed with hexane at a
material-to-solvent ratio of 1:2 (m/v). Mixtures were shaken or
soaked at 200 rpm for 24 hr at 25.degree. C. in order to obtain an
avocado seed raw extract. The raw extracts were evaporated to
dryness using a Rotary evaporator (35.degree. C., 22 in Hg) and the
obtained dry matter was weighed.
[0106] As in Example 1, dry matter was re-dissolved in acetone to a
final concentration of 2.5 mg/ml for the antibacterial evaluations.
Clostridium sporogenes (ATCC 7955) was used as test microorganism
since it is a known surrogate microorganism for Clostridium
botulinum. Antimicrobial activities against vegetative bacterial
cells, as well as native and heat-shocked spores were conducted as
described in Example 1.
[0107] A significant effect was observed for the shaking treatment
on the antimicrobial properties of the avocado seed hexane extract
against vegetative bacterial cells, native spores and heat shocked
spores (FIG. 3). Extracts obtained without shaking presented a
higher antibacterial activity when compared with those obtained
with shaking, even though the yields of extracted dry mass are
higher when shaking. Through the example we can observe that
shaking enhances the extraction of other non-antimicrobial
compounds present in the avocado seed, therefore diluting the
concentration of compounds with antibacterial activity. Therefore,
the antibacterial avocado seed extract must be obtained by
maceration, preferently without shaking.
[0108] Due to the dilution of compounds, the extract obtained with
shaking gave similar or lower inhibition zones than the positive
control (nisin, 150 .mu.g) which showed 1.3, 1 and 0.9 cm for
vegetative cells, spores and heat shocked spores, respectively.
Example 4
Effect of Extraction Time and Extraction Solvent Type (Acetone,
Ethanol and Hexane) on the Antimicrobial Properties of Crude
Avocado Seed Extracts
[0109] Avocado seeds were ground using a colloidal mill obtaining
particles with average radio of 0.5-2 mm. Ground avocado pits (50
g) were mixed with either acetone or ethanol or hexane at a
material-to-solvent ratio of 1:2 (m/v). Mixtures were shaked at 200
rpm 24 hr at 35.degree. C. in order to obtain an avocado seed crude
extracts. Aliquots from each crude extract were sampled at times
0.5, 5 and 24 hr during extraction. Crude extracts obtained at
different extraction times were evaporated to dryness using a
Rotary evaporator (35.degree. C., 22 in Hg) and the obtained dry
matter was weighed.
[0110] Dry matter was re-dissolved in acetone to a final
concentration of 2.5 mg/ml. Clostridium sporogenes (ATCC 7955) was
used as test microorganism in the antimicrobial assays.
Antibacterial activities against vegetative cells, native spores
and heat shocked spores (using the disc inhibition zone
determination) were conducted as described in Example 1.
[0111] Antimicrobial activities of hexane extracts against
vegetative bacterial cells, spores and heat-shocked spores were
considered as a 100% inhibition for comparison purposes with the
other solvents (acetone and ethanol) at the same time interval.
Results of the antibacterial activity against vegetative cells are
shown in FIG. 4 and indicated that an ethanol extract obtained
after an extraction time of 30 minutes had exactly the same
activity as the one obtained with hexane under the same conditions.
In contrast at an extraction time of 30 min with acetone the
extract presented only 70% of the antimicrobial activity observed
for the hexane extract, value that reached a maximum of
antimicrobial activity of 80% of the activity observed in hexane
extract after an extraction time of 5 hrs. Therefore this example
demonstrates that since acetone and ethanol are polar solvents,
increasing the extraction time at the conditions tested diluted the
concentration of bioactive compounds and/or saturated the solution.
Additionally and contrary to the expected, the nature of
antibacterial compounds against vegetative cells of spore forming
bacteria allows a better recovery using ethanol than acetone (FIG.
4).
[0112] The results for antimicrobial properties of the extracts
against native spores are presented in FIG. 5; and indicated that
increases in extraction times (0.5-24 hr) did not present any
differences using either solvent acetone or ethanol as the
extraction solvents. Ethanol also was more selective for the
extraction of the compounds with antibacterial properties against
native spores.
[0113] Results for the antimicrobial activities of the different
extracts against the growth of heat-shocked spores are presented in
FIG. 6, and indicated a different trend, at 30 min of extractions
both solvents (acetone and ethanol) were equally efficient for the
extraction of the antibacterial molecules. However, when acetone
was used as solvent over time a significant decrement on the
concentrations of antibacterial molecules in the extracts was
observed that varied from 100% to less than 80% bacterial
inhibition for extraction times of 0.5 to 5 hr, respectively, and
then the activity remained constant. Ethanol did not get as easily
saturated over the extraction time with the compounds of interest
as the acetone extract did and therefore, for this solvent, no
differences were observed for the extraction times between 0.5 and
5 hours. Therefore the present example demonstrates that ethanol
was as effective as hexane for the extraction of the antimicrobial
compounds with inhibitory activities against the growth vegetative
cells, native spores and heat-shocked spores from spore forming
bacteria.
Example 5
Comparison of the Fractionation of an Acetone Avocado Seed Extract
Versus Ground Avocado Seeds in Heptane: Methanol Two-Phase
Non-Miscible Solvent System
[0114] For the present example an acetone raw extract of avocado
seed was obtained as described in Example 1, and evaporated to
dryness. The dry acetone raw extract obtained from 50 g of ground
avocado seeds was directly added to a separation funnel containing
a two non-miscible solvent system comprised of 100 ml of heptane
(upper phase F002) and 100 ml of methanol (lower phase F001) in
order to allow the partition of polar and non-polar compounds
contained in the extract (FIG. 7A).
[0115] For comparison purposes a second two-phase system was
prepared with 50 g of ground avocado seeds directly added the other
non-miscible solvent system also comprised of 100 ml of heptane
(upper phase) and 100 ml of methanol (lower phase). Mixture was
shaken at 200 rpm 24 hr at 35.degree. C. in order to selectively
extract and partition the compounds present in the seed in one
step. Later, the seed was separated from the extract by means of
vacuum filtration. The upper (F003) and the lower (F004) phases of
this system were allowed to form in a separation funnel and were
collected separately FIG. 7B.
[0116] The different phases previously described (F001-F004) were
evaporated to dryness individually using a rotary evaporator
(35.degree. C., 22 in Hg) and the obtained dry matter was
weighed.
[0117] Dried fractions were re-dissolved in acetone to a final
concentration of 2.5 mg/ml for posterior evaluation of their
antibacterial activities against Clostridium sporogenes (ATCC
7955). Antibacterial activities against vegetative cells, native
spores and heat-shocked spores (disc inhibition zone determination)
were conducted as described in Example 1.
[0118] Results from the disc inhibition zones for heat
shocked-spores indicated that a direct extraction of grounded
avocado seeds with the two-non miscible solvents reduces the amount
of contaminants that may migrate to the upper phase and that would
dilute the effect of active compounds (FIG. 8), therefore
illustrates that is a better option for a one step isolation of
compounds that inhibit spore germination. However based on the
antibacterial results for the inhibition of vegetative cells both
procedures resulted in similar results with no particular benefits
of one over the other one.
[0119] The present example therefore demonstrates that the
antibacterial substances were enriched in the upper phases of the
heptane: methanol two-phase systems in both of the performed
evaluations of direct extraction of the grounded seed and
partitioning of a dried acetone avocado seed extract. However
residual activity was also observed in the lower phases (F002 and
F004), indicating that the upper phases were saturated with active
compounds or that the compounds presented partial solubility in the
lower phases of both systems. Therefore a subsequent extraction was
set up by re-extracting the evaporated solids recovered from the
lower methanol phase F002; the subsequent extraction systems
(second two-non miscible solvent systems) used to recover the
remaining antibacterial compounds were formed by ethyl acetate (100
mL) and water (100 mL). Antibacterial activities of the ethyl
acetate and water phases are shown in FIG. 9. This second two-non
miscible solvent systems were more polar than the first ones used
and no residual antibacterial activity was found in the lower
phases (mainly water).
[0120] To further complete the example other two additional
non-miscible solvents systems were also evaluated as alternatives,
to the heptane:methanol system described above, for partitioning
the dried acetone avocado seed extracts and obtaining formulations
enriched in bioactive molecules. By the use of a two-phase system
of hexane and methanol the antibacterial compounds were also
recovered in the upper hexane phase FIG. 10. However, the
heptane:methanol two-phase system proved to be more effective for
the recovery of compounds in the upper phase since it presented
less migration into the lower phase. Additional tests were
performed by the use of aqueous two-phase systems using water, salt
and ethanol to isolate the antibacterial compounds from ethanol raw
extracts and the desired compounds were recovered in the
upper-phase consisting mainly of ethanol.
Example 6
Effect of Saponification on the Antimicrobial Activities of Acetone
and Hexane Avocado Seed Extracts
[0121] Crude acetone extracts from avocado seeds were partitioned
with hexane and methanol as described in Example 5. The phases were
separated and the hexane rich upper phase, containing less polar
compounds was evaporated to dryness using a Rotary evaporator
(35.degree. C., 22 in Hg). According to Broutin et al (2003),
saponification is a necessary step to obtain a bioactive fraction
that contained aliphatic or terpenic alcohols, sterols,
tocopherols, carotenoids, and xanthophylls that remain in the
unsaponifiable portion and are not soluble in water. However this
example demonstrates that the antibacterial compounds of the
present disclosure could not be obtained in the same way,
indicating a different chemical nature.
[0122] Saponification of the acetone raw extract and the
partitioned hexane upper phase fraction was carried out according
to Broutin et al (2003), with some modifications, in order to
recover the unsaponifiable portion and selectively extract the
furan lipids and polyhydroxylated fatty alcohols present in them.
Separately, 5 g of each extract were mixed with 2.5 ml of 12N
potassium hydroxide and 10 ml of ethanol then allowed to rest for 4
hours. The aqueous-alcoholic mixture was then transferred to a
separations funnel and 17.5 ml of water were added, followed by
addition of 17.5 ml of dichloroethane. The mixture was shaken for
30 s and then allowed to separate into two phases. The organic
phase (lower phase) was recovered. This operation was repeated 6
times, and the organic phases were combined and washed with water.
The dichloroethane was evaporated to dryness using a rotary
evaporator (35.degree. C., 22 in Hg) and the obtained dry matter
was weighed.
[0123] Dry matter was re-dissolved in acetone to a final
concentration of 2.5 mg/ml. Antimicrobial and sporicidal activity
tests (disc inhibition zone determination) were conducted as
described in Example 1, Clostridium sporogenes (ATCC 7955) was used
as test microorganism. As shown in FIG. 11, only the
unsaponifiables extracted from acetone raw extract showed disc
inhibition on spores indicating that partitioning an acetone
extract with hexane and methanol eliminates unsaponifiable
compounds. Interestingly these unsaponifiable portion from the
crude acetone extract had lower activity than the non-alkali
treated crude acetone extract (FIG. 2) particularly in their
inhibitory activities against the bacterial spores.
[0124] Unsaponifiable compounds in the crude acetone extract had a
higher specificity for vegetative cells than for spores.
Partitioning with hexane-methanol reduced the activity of
unsaponifiables against vegetative cells indicating that some of
these compounds could migrate to the alcoholic phase during
partitioning.
[0125] When the antibacterial properties of the upper hexane and
lower methanol phases, in which the unsaponifiable matter from the
crude acetone extract was partitioned, were compared with the
activities for crude acetone an hexane extracts described in
Example 1 they were significantly lower for both phases. Results
therefore indicated that active compounds are sensible to alkaline
treatments or that some desirable chemical features are modified or
removed during the saponification treatment and subsequent
partitioning steps. Therefore, a saponification step with the aim
of isolating or increasing the antimicrobial and sporicidal
activity should not be considered to obtain the active avocado seed
extract.
Example 7
Partitioning Chromatography of an Acetone Avocado Seed Extract
[0126] Acetone raw extract of avocado seed was obtained and
evaporated to dryness as described in Example 1 then partitioned in
a heptane:methanol two-phase system as described in Example 5. The
upper heptane-rich phase (F001), containing less polar compounds
was evaporated to dryness using a Rotary evaporator (35.degree. C.,
22 in Hg) and then injected to a Fast Centrifugal Partition
Chromatographer FCPC.RTM. Bench Scale with a 1000 ml column to
fractionate the chemical compounds using heptane and methanol. The
heptane was pumped into the column and it served as the stationary
phase (740 mL). The methanol (mobile phase) was then pumped into
the column at a flow-rate of 10 mL/min. The rotor was set at 800
rpm. The concentrated extract (65 mL), obtained from the evaporated
upper phase of the heptane:methanol two-phase system in which the
avocado seed acetone extract was partitioned, was injected into the
FCPC after the system had reached the hydrodynamic equilibrium.
Methanol was used to elute fractions during the first 170 min, and
after that time heptane was used as mobile phase for 100 min. The
effluent from the outlet of the column was collected in test tubes
using a fraction collector set at 10 ml for each tube. An aliquot
of 1 ml of each fraction was collected for antibacterial and
sporostatic/sporicidal activity tests. Aliquots were evaporated to
dryness using a Speed Vac concentrator, the weights of the solids
from each fraction were recorded and 70 pools of consecutive
fractions were formed having a final concentration per pool of 2.5
mg/ml. The antibacterial properties of each pool were assessed
against vegetative cells, native spores and heat-shocked spores of
Clostridium sporogenes as described in Example 1. The remaining
volume from each fraction (9 mL), were evaporated to dryness using
a Speed Vac concentrator, stored at 80.degree. C. and further used
for chemical identification evaluations.
[0127] As can be observed in FIG. 12, the antibacterial activity
was present in the fractions with partition coefficients (Kd) lower
than 0.5 (more specifically between Kd values from 0.19 to 0.35)
indicating that the active compounds were at least 2 times more
soluble in heptane than in methanol. Also there was a slight
difference in the activity of those fractions against vegetative
cells compared to spores since inhibitors of vegetative cells
growth were more spread into more polar fractions.
[0128] Partitioning the extract by FCPC increased the desired
antibacterial activities (up to 3 cm diameter inhibition zones) in
comparison with the previous experiments with less pure extracts,
clearly indicating the need to eliminate other phytochemicals that
might be diluting the concentration of the antibacterial compounds
(FIG. 12). The antibacterial activities of some FCPC fractions were
increased at least by 50% when compared to the data observed in
FIG. 2 for the crude hexane and acetone avocado seed extracts.
Results shown in FIG. 12 also demonstrate, as in FIG. 8, that the
active compounds have more affinity for the heptane phase than for
the methanolic phase.
[0129] In order to further characterize the antibacterial
activities of the fractions with the highest activity, it was
important to determine their minimum inhibitory concentration
(MIC), defined as the lowest concentration of an antimicrobial that
will inhibit the visible growth of a microorganism after overnight
incubation. Compared to nisin, the fractions obtained by FCPC with
a Kd of 0.3 and 0.4, showed a lower MIC for vegetative cells than
for the native spores or heat-shocked spores of C. sporogenes
(Table 2). Fraction with Kd of 0.4 was almost 2 times more active
than Nisin for spore growth inhibition but fraction with Kd of 0.3
was about 15 times more active than Nisin. But in the case of
heat-shocked spores, the differences between nisin and the
fractions with Kd of 0.4 were less pronounced, but still presented
the desired inhibitory properties against spore germination.
TABLE-US-00002 TABLE 2 Minimal Inhibitory Concentration (MIC) for
the fractions obtained by reverse phase Fast Centrifugal Partition
Chromatography (RF-FCPC) of the solids recuperated from the upper
phase (heptane) of the two-phase system (heptane:methanol) used to
partiton an acetonic crude avocado extract as described in Example
5. Sample tested and Partition Coefficient (Kd) MIC (.mu.g/ml)
Vegetative Cells Nisin* 5000 Fraction with Kd of <<78 0.4
Fraction with Kd of <<78 0.3 Native Spores Nisin* 5000
Fraction with Kd of >>2500 0.4 Fraction with Kd of 312 0.3
Heat shocked spores Nisin* 5000 Fraction with Kd of 1250 0.4
Fraction with Kd of 312 0.3 *Nisin was tested using initial stock
solutions at 50 mg/ml and for avocado fractions at 2.5 mg/ml.
[0130] As shown in the present example, the same extract portioned
by FCPC under the conditions described above (reverse phase) can
also be partitioned using heptane as a mobile phase (normal phase)
and results from the chromatographic separation followed the same
behavior based on antibacterial activities (FIG. 13). Therefore the
first fractions obtained by FCPC had better activity than the last
ones (more polar) and in FIG. 13 it is shown that antibacterial
activity remained present until partition coefficient reaches 7.2,
indicating that other compounds that are more than 7.2 times more
soluble in heptanes than methanol do not inhibit the growth of
vegetative cells or spores from C. sporogenes.
Example 8
Partitioning Chromatography of Acetone Avocado Seed Extract to
Obtain Fractions with Inhibitory Activities Against Other
Microorganisms Besides C. Sporogenes
[0131] Acetone raw extract of avocado seed was obtained and
evaporated to dryness as described in Example 1 then partitioned in
a heptane:methanol two-phase system as described in Example 5. The
upper heptane-rich phase, containing less polar compounds was
evaporated to dryness using a Rotary evaporator (35.degree. C., 22
in Hg) and then injected into a Fast Centrifugal Partition
Chromatographer FCPC.RTM. using the Normal Phase conditions
described in Example 7.
[0132] The fractions obtained from Normal phase FCPC, were then
used to asses their antimicrobial activities against the growth of
vegetative cells from S. aureus and B. subtilis. As can be observed
in FIG. 14, different compounds to the ones that are inhibiting C.
sporogenes and with very low polarity are inhibiting the growth of
vegetative cells of S. aureus and B. subtilis because disc
inhibition zones were observed for these microorganisms when discs
were inoculated with fractions of partition coefficient higher than
7, contrasting with the results of the inhibition of C. sporogenes
shown in Example 7.
[0133] Table 3, summarizes the antimicrobial results from previous
experiments obtained from the evaluation of the crude extracts of
Example 1, extracts partitioned as described in Example 5, and
unsaponifiable fractions from Example 6. As it can be observed,
interestingly, they did not showed any inhibitory effects on the
growth of S. aureus and very low disc inhibition zones when tested
against B. subtillis in comparison with the stronger inhibitory
effects observed for the enriched CPC fractions shown in FIG.
14.
TABLE-US-00003 TABLE 3 Evaluation of the antimicrobial activities
against the growth of vegetative cells of S. aureus and B. subtilis
of different crude extracts S. aureus Disc inhibition B. subtillis
zone Disc inhibition zone Fraction (cm) (cm) Acetone Extract -- 0.6
Hexane Extract (shaking) -- 0.6 Hexane Extract (without -- 0.7
shaking) Upper phase (hexane) of the -- 0.6 partitioned acetone
crude extract Lower phase (methanol) of the -- 0.7 partitioned
acetone crude extract Unsaponifiable compounds -- -- from acetone
extract Unsaponifiable compounds -- -- from hexane-methanol
partitioned acetone extract
Example 9
Effect of High Pressure and Temperature on the Stability of
Antimicrobial Activity
[0134] An acetone crude extract from avocado pit was obtained and
evaporated to dryness as described in Example 1. Then the acetone
extracted avocado solids were partitioned into a two-phase
hexane-methanol system as described in Example 5, followed by a
[then] second partitioning system of ethyl acetate:water used to
completely recover the active compounds present in the lower phase
(methanol) phase of the first partitioning system (also described
in Example 5). The hexane and the ethyl acetate phases were
recovered separately and evaporated to dryness using a Rotary
evaporator (35.degree. C., 22 in Hg). Both phases were then filled
in vials and exposed to high hydrostatic pressure (HHP) treatments
of 300 MPa and 600 MPa (43,511 and 87,022 psi, respectively),
during 3 and 6 minutes. No significant difference was observed in
the antibacterial properties of the extracts after the high
pressure treatments, indicating that the compounds responsible for
the observed antimicrobial properties are stable to HHP
treatments.
[0135] The thermal stability of the active compounds was also
tested at temperatures that ranged from 25 to 100.degree. C. for 60
min. The compounds with activity against the growth of vegetative
cells of C. sporogenes were the less sensitive to thermal treatment
(FIG. 15) than those responsible for the inhibitory properties
against the growth of native spores (FIG. 16). As it can be
observed in FIG. 15 the inhibitory properties against vegetative
cells were decreased by 20 and 23.5%, after a treatment of
100.degree. C. for 60 minutes of the ethyl acetate and hexane
extracts, respectively, and in reference to the inhibitory
properties of non-heated control extracts maintained at 25.degree.
C.
[0136] Heat shocked spores were more resistant to the action of the
thermally treated hexane and ethyl acetate crude extracts; the
inhibitory properties against heat-shocked spores were decreased by
50%, after exposure of the extracts to 100.degree. C. for 60
minutes, and in reference to the inhibitory properties observed for
the control extracts maintained at 25.degree. C.
Example 10
Identification of the Main Compounds Found in Bioactive
Fractions
[0137] The fractions with the highest disc inhibition zones (FIG.
12), obtained by the use of reverse phase Fast Centrifugal
Partition Chromatography (RP-FCPC), and that had a Kd between
0.19-0.35 were mixed together in order to form a "pool of active
fractions", as described in Example 7. Initially the fractions (13)
were adjusted at the same concentration of 192.3 mg/ml and equal
volumes of each of them (100 .mu.l) were taken and adjusted with
ethanol to a final concentration of 50 mg/ml.
[0138] FIG. 17 shows the progressive change in the chromatographic
profiles of the fractions present in the active pool, as their Kd
increases. Evaporated aliquotes of individual fractions were
adjusted to 1 mg/ml with HPLC grade methanol and 2 .mu.l were
injected. The column used was a Zorbax Extanded-C18 (100.times.3 mm
d.i., 3.5 .mu.m) column. The mobile phases included water 100% as
phase A and methanol 100% as phase B. The solvent gradient used is
described in Table 4, pumped at a flow rate of 0.38 ml/min and a
post equilibration time of 6 mins. The detector was set at a
wavelength of 220 nm.
TABLE-US-00004 TABLE 4 Solvent Gradient used to achieve the
chromatographic separation of the fractions collected after fast
centrifugal partition chromatography (A = water and B = Methanol).
Time (min) % A % B 0 30 70 4 15 85 22 10 90 24 0 100 26 0 100
[0139] The typical chromatograph obtained for the active pool of
antimicrobial compounds from avocado is shown in FIG. 17. The
numbers indicated in the chromatogram represent the common peaks
that absorb at 220 nm, labeled as Compounds I to 11, and the
information on their mass and molecular formula is presented in
Table 5. Some of these compounds have been previously reported in
avocado tissues, however some of them are being disclosed herein as
new chemical compounds since they were discovered by the inventors
in the antimicrobial fractions. In most of the bioactive fractions,
compounds such as 1, 2, 4 and 11 were in lower concentrations when
compared to compounds 7 and 9 (FIG. 17).
TABLE-US-00005 TABLE 5 Chemical characterization of the compounds
found in the antimicrobial fractions. [M + H].sup.+ Peak Number
Molecular (Common name).sup.a Formula Reference Compound 1 347.2279
None Compound 2 349.2418 None Compound 3 329.2708 Neeman et al.
1970, Bittner et al. 1971 C19H36O4 Brown 1972, Prusky et al. 1991b
Compound 4 329.2816 Kashman et al. 1969, Bittner et al. C19H36O4
1971, Brown 1972 Compound 5 353.2706 None C21H36O4 New compound
Compound 6 353.2708 None C21H36O4 New compound Compound 7 379.2864
Domergue et al., 2000, Kim et al., (Persenone A) C23H38O4 2000a
Compound 9A 355.2865 Kim, 2000a, 2000b and 2000c (Persenone B)
C21H38O4 Compound 9B 381.3022 Prusky et al. 1982, Oelrichs et al.,
1995 (Persin) C23H40O4 Sivanathan and Adikaram, 1989, Domergue et
al., 2000 .sup.aCommon name, where applicable
Example 11
Evaluation of Sporostatic and Sporicidal Activity of a Fraction
Enriched in Antimicrobial Compounds
[0140] In order to demonstrate that the pool of active fractions
described in Example 10 (partition coefficient 0.19-0.35) had
sporostatic or sporicidal activity, it was necessary to determine
its minimum inhibitory concentrations (MIC) and minimum
bactericidal concentrations (MBC). In general terms, MIC is defined
as the lowest concentration of an antimicrobial that will inhibit
the visible growth of a microorganism after overnight incubation.
While the MBC is the lowest concentration of the antimicrobial that
will prevent the growth of a microorganism after subculture to
fresh agar media free from the antibiotic or antimicrobial agent.
The pool of active the fractions was tested at concentrations
ranging from 0.005 to 2.5 mg/ml and nisin was used as control.
[0141] Table 6 shows that the pool of active fractions was much
better than nisin as an inhibitor of the growth of spores from C.
sporogenes since its MIC is almost one tenth of that obtained for
nisin. According to Smola (2007), if the ratio of the
MBC/MIC.ltoreq.4, the compound can be considered as sporocidal and
if the ratio of the MBC/MIC>4, it is only sporostatic. In this
example, both nisin and the pool of avocado active fractions
presented a sporocidal effect.
TABLE-US-00006 TABLE 6 Minimum Inhibitory Concentration (MIC),
Minimum Bactericidal Concentration (MBC) and MBC/MIC ratio, for
nisin and the pool of active fractions isolated from avocado seed,
against the growth of heat shocked spores from C. sporogenes.
Sample MIC (.mu.g/ml) MBC (.mu.g/ml) MBC/MIC.sup.a Nisin 234 156
1.5 Pool of active fractions 19.5 19.5 1 .sup.aRatios of the
MBC/MIC .ltoreq.4 indicate sporocidal activity. Ratios of the
MBC/MIC >4 indicate sporostatic activity.
Example 12
Antimicrobial Activities of Isolated Chemical Compounds from
Bioactive Fractions
[0142] In this example, the antimicrobial activities of the same
isolated compounds described in Example 10 (Table 5) were tested
against the growth of vegetative cells and heat shocked spores of
C. sporogenes, and on vegetative cells of S. aureus, P.
aeuroginosa, E. coli. and B. subtilis as previously described in
Example 1, and at a concentration of 0.5 mg/ml. As it can be
observed in Table 7, compound 6 (peak 6) and persenone B (peak 9A)
demonstrated greater antimicrobial properties when tested against
C. sporogenes, followed by persenone A (peak 7). Additionally, from
all the bioactive compounds, only persin (peak 9B) showed a lower
activity than nisin, although nisin a known antimicrobial was
tested at a 100-fold higher concentration. Since it has been
reported that persin is able to inhibit fungi spore germination
(Prusky et al., 1982), and in the present experiment it seems to
have the lowest activity, it can be assumed that the other
bioactive compound would have a higher activity against fungi
spore.
TABLE-US-00007 TABLE 7 Evaluation of the antimicrobial activities
of the active isolated compounds from FIG. 17 against the growth of
vegetative cells and heat shocked spores of Clostridium sporogenes
(ATCC 7955). Disc Inhibition Zone (cm) Vegetative Peak number
Common name Cells eat Shocked Spores Compound 3 1.1 1.0 Compound 5
1.0 1.1 Compound 6 1.9 1.7 Compound 7 Persenone A 1.6 1.5 Compound
9A Persenone B 1.9 1.7 Compound 9B Persin 1.0 0.6 Negative Control
0.0 0.0 Positive Control 1.1 1.0 (nisin at 50 mg/ml)
[0143] It is important to remark that, to our surprise, all the
compounds showing the highest activity against vegetative cells and
heat shocked spores of C. sporogenes (Compound 6, Persenone By
Persenone A mentioned from the highest to the lowest antimicrobial
activities reported in Table 7) contained a C5-C6 double bond (see
Table 8). Moreover, if the structures of the persin (compound 9B)
and persenone A (compound 7) are compared, the only difference is
the lack of the C5-C6 double bond in persin (compound 9B), and in
this example we demonstrate that its antimicrobial activity was
reduced by 37.5%. Additionally, the only structural difference
between persenone B (compound 9A) and compound 6 is that the later
also presents a C16-C17 double bond, but their inhibitory
activities were the same. This observation also supported the
finding that a C5-C6 double is a desirable structural feature to
improve the antimicrobial activities of the compounds described
herein, and that the C16-C17 double bond is also a preferred
structural feature, since it is the only unsaturation present in
compound 3, and it had a higher activity than persin (compound 9B)
that contains two instaurations and none between C16-C17.
TABLE-US-00008 TABLE 8 Chemical structures and common names of the
compounds responsible of the antimicrobial activities of avocado
seed. Peak/ Compound Number Chemical structure (Common name) Name
Compound 3 ##STR00006## Compound 5 ##STR00007## Compound 6
##STR00008## Compound 7 (Persenone A) ##STR00009## Compound 9A
(Persenone B) ##STR00010## Compound 9B (Persin) ##STR00011##
[0144] The most antibacterial compounds against C. sporogenes
(Compound 6, Persenone B and Persenone A) did not show inhibitory
activity against of S. aureus, P. aeuroginosa or E. coli (Table 9),
but compound 6 also presented the greatest inhibitory activities
against the growth of B. subtillis, followed by persenone A. Since
Compound 6 is a newly discovered compound that was not previously
reported as an avocado constituent, there are not previous reports
of its antimicrobial or any other biological activity. Persenone A
had been previously reported as antifungal but according to the
results of Table 7, its antibacterial activity is specific to spore
forming gram positive bacteria. The pool of active fractions
obtained as described in Example 10, and that presented
antibacterial properties against C. sporogenes in Example 10, in
the present example only resulted in inhibitory properties against
the spore forming bacteria B. subtilis.
TABLE-US-00009 TABLE 9 Disc inhibition zones of the bioactive
compounds and the pool of active fractions for vegetative cells and
B. Subtillis, S. aureus, P. aeuroginosa and E. coli Peak/Compound
Number Antibacterial Activity (Disc inhibition zone (cm)) (Common
name) B. Subtillis S. aureus P. aeuroginosa E. coli Compound 6 1.3
0.0 0.0 0.0 Compound 7 0.7 0.0 0.0 0.0 (Persenone A) Compound 9A
0.0 0.0 0.0 0.0 (Persenone B) Pool of active 0.9 0.0 0.0 0.0
fractions
[0145] The MICs for Compound 6, Persenone B (Compound 9A) and
Persenone A (Compound 7) was determined against the germination of
heat shocked spores from C. sporogenes as described in Example 11.
As can be seen in Table 10, the three compounds had MICs values
15-30 fold lower than nisin, demonstrating their efficacy against
bacterial spores. The MIC for the pool of active fractions was 19.5
.mu.g/ml (Example 11) and it was reduced to 7.8 .mu.g/ml for
persenone A and persenone B when isolated, but the antimicrobial
properties for Persenone B within the pool did not corresponded to
its lower concentration since it contained less .mu.g of that
compound but when combined with the other bioactive molecules its
activity appears to be potentiated. Interestingly, isolated
compounds presented only sporostatic activity against C. sporogenes
and did not showed the sporocidal bioactivity that was observed for
the pool of active fractions (Table 6).
TABLE-US-00010 TABLE 10 Minimum Inhibitory Concentrations (MIC),
for nisin, Compound 6, Persenone B y A, against heat shocked spores
from C. sporogenes. Peak/Compound Number (Common name) MIC
(.mu.g/ml) Compound 6 15.6 Compound 7 7.8 (Persenone A) Compound 9A
7.8 (Persenone B) Nisin 234
Example 13
Antibacterial Activities of Avocado Seed Extracts Combined with
Refrigeration Temperatures for the Control of Listeria
monocytogenes
[0146] The pool of active fractions described in Example 10 also
presents antibacterial effects against cold-stressed vegetative
cells of gram positive bacteria capable of growing under
refrigerated conditions, such as Listeria monocytogenes. At the
optimum growth temperature of 37.degree. C. for Listeria
monocytogenes the avocado pool extract enriched in bioactive
acetogenins was not particularly useful for the inhibition of the
growth of vegetative cells of the tested organism (Table 11).
Contrary to the expected we found that the avocado seed pool
extract was particularly useful for inhibiting the growth of
Listeria monocytogenes under refrigerated conditions. Furthermore,
in Table 12 we illustrate that when the antibacterial activities of
the avocado acetogenins isolated in the present disclosure, were
tested against the growth of vegetative cells of Listeria
monocytogenes, the compounds presenting the desirable feature of a
double bond between C5 and C6 can be used for the control of
Listeria monocytogenes in foods and biological matrixes stored
under refrigerated conditions.
TABLE-US-00011 TABLE 11 Antibacterial activities of avocado seed
extracts combined with low temperatures of storage against the
growth of vegetative cells of Listeria monocytogenes. Antibacterial
activity against vegetative cells of Listeria monocytogenes (Disc
inhibition zone (cm)) Incubation Incubation Temperature Temperature
Extract (4.degree. C.) (37.degree. C.) Antibacterial Concentration
Storage Time Storage Time Substance (mg/mL) (17 days) (48 hours)
Avocado Seed 50 1.0 0.0 (Persea americana) 25 1.1 0.0 12.5 1.1 0.0
6.25 0.0 0.0 3.125 0.0 0.0 Nisin 40 2.5 1.1 (positive control)
Methanol (negative 0.0 0.0 control)
TABLE-US-00012 TABLE 12 Antibacterial activities of the isolated
avocado compounds combined with refrigeration against the growth of
vegetative cells from Listeria monocytogenes. Antibacterial
Activity Peak/Compound (Disc inhibition zone (cm)) Number
Concentration 4.degree. C. 37.degree. C. (Common name) (mg/ml) 20
days 48 hours Compound 3 0.5 0.0 0.0 Compound 5 0.5 0.0 0.0
Compound 6 0.5 1.1 0.0 Compound 7 0.5 1.1 0.0 (Persenone A)
Compound 9A 0.5 1.0 0.0 (Persenone B) Compound 9B 0.5 0.0 0.0
(Persin) Nisin 40 2.6 1.1 (positive control) MeOH 0.0 0.0 (negative
control)
Example 14
Quantification of the Antimicrobial Compounds in Enriched Avocado
Extracts
[0147] The concentration of the antibacterial compounds present in
the pool of active fractions described in Table 7 (Example 10) is
presented in FIG. 18. Persenone A represents 36.32% of the dry
weight of the pool of active fractions, persenone B was only 0.20%
and compound 6 accounts for the lowest amount (0.05%). It seems
that the other components in the mixture do not affect the
inhibitory activity of Persenone A, and therefore no further
purification may be needed.
[0148] Table 13 shows that there is a very similar concentration of
the most bioactive compounds against C. sporogenes (Compound 6,
Persenone B and Persenone A) in fresh avocado pulp and seed, being
Persenone A the most concentrated. The information of this example
is relevant because if the bioactive compounds are also present on
the pulp they can be easily obtained from other parts of the fruit.
The present example also demonstrates that humans are being exposed
to the bioactive molecules, when eating the fruit, at the
concentrations required for achieving their antibacterial
properties; therefore establishing their commercial potential in
the food, medical and cosmetic arts.
TABLE-US-00013 TABLE 13 Concentrations of Compound 6, Persenone B
and Persenone A in fresh avocado pulp and seed (ug/g of fresh
weight). Avocado Pulp (ug/g of Avocado Seed (ug/g of Compound fresh
weight) fresh weight) Compound 6 18.59 .+-. 2.30 19.11 .+-. 3.45
Compound 7 74.86 .+-. 4.75 63.32 .+-. 6.34 (Persenone A) Compound
9A 42.42 .+-. 10.22 31.89 .+-. 2.87 (Persenona B)
[0149] Having thus described in detail various embodiments of the
present disclosure, it is to be understood that the disclosure
defined by the above paragraphs is not to be limited to particular
details set forth in the above description as many apparent
variations thereof are possible without departing from the spirit
or scope of the present disclosure.
LIST OF LITERATURE REFERENCES
[0150] Adikaram, N. K. B., Ewing, D. F., Karunaratne, A. M.,
Wijeratne, E. M. K. 1992. Antifungal compounds from immature
avocado fruit peel. Phytochemistry. 31:93-96. [0151] AOAC Official
Method 966.04 Sporicidal Activity of Desinfectants. Revised 2002.
[0152] Baratta et al. 1998. Chemical composition, antimicrobial and
antioxidant activities of laurel, sage, rosemary, oregano and
coriander essential oils. J. Essent. Oil Res. 10(6): 618-627.
[0153] Beltran, J. B. U. and Bonaventura, J. S. Use of cationic
preservative in food products. U.S. Pat. No. 7,862,842 B2. Jan. 4,
2011. [0154] Bevilacqua, A., Sinigaglia, M., Corbo, M. R. 2008.
Alicyclobacillus acidoterrestris: New methods for inhibiting spore
germination. International Journal of Food Microbiology.
125:103-110. [0155] Bittner, S., Gazit, S., Blumenfeld, A. 1971.
Isolation and identification of a plant growth inhibitor from
avocado. Phytochemistry, 10(7):1417-1421 [0156] Blackburn, P,
Gusik, S A., Polak, J., Rubino, S. D., Nisin compositions for use
as enhanced, broad range bactericides. U.S. Pat. No. 5,217,950.
Jun. 8, 1993. [0157] Brown, B. I. 1972. Isolation of Unpleasant
Flavor Compounds in the Avocado (Persea americana). J. Agr. Food
Chem. 20:753-757. [0158] Bull, S. D and Carman, R. M. Synthesis of
the Avocado Antifungal, (Z,Z)-2-hydroxy-4-oxohenicosa-12,
15-dien-1-yl acetate. Aust. J. Chem., 1994, 47, pp. 1661-1672.
[0159] Burt. S. 2004. Essential oils: their antibacterial
properties and potential applications in foods--a review.
International Journal of Food Microbiology. 94:223-253. [0160]
Chang, C. F., Isogai, A., Kamikado, T., Murakoshi, S., Sakurai, A.,
Tamura, S. Isolation and structure elucidation of growth inhibitors
for silkworm larvae from avocado leaves. Agr. Biol. Chem., 1975, 39
(5), pp. 1167-1168. [0161] Chia, T. W. R., Dykes, G. A. 2010.
Antimicrobial activity of crude epicarp and seed extracts from
mature avocado fruit (Persea Americana) of three cultivars.
Pharmaceutical Biology 48(7):753-756. [0162] Deans, S. G., Ritchie,
G. 1987. Antibacterial properties of plant essential oils.
International Journal of Food Microbiology. 5:165-180. [0163]
Domergue, F., Helms, G. L., Prusky, D., Browse, J. 2000. Antifungal
compounds from idioblast cells isolated from avocado fruits.
Phytochemistry 54: 183-189. [0164] Food Directorate, 2010.
Clostridium botulinum challenge testing of ready-to-eat foods. Food
Directorate Health Products and Food Branch. Health Canada. Version
Number 1, Issue: Nov. 24, 2010. [0165] Hashimura H, Ueda C,
Kawabata J, Kasai T. 2001. Acetyl-CoA carboxylase inhibitors from
avocado (Persea americana Mill.) fruits. Biosci Biotechnol Biochem.
65:1656-1658. [0166] Jensen, Lloyd B. 1951. Process for extraction
of antibiotic material. U.S. Pat. No. 2,550,254. SWIFT & CO.
United States. [0167] Kabuki, T., Nakajima, H., Arai, M., Ueda, S.,
Kuwabara, Y., Dosako, S. 2000. Characterization of novel
antimicrobial compounds from mango (Magnifera indica L.) kernel
seeds. Food Chem. 71:61-66. [0168] Kashman, Y., Neeman, I. and
Lifshitz, A. 1969. New Compounds from Avocado Pear. Tetrahedron.
25:4617-4631. [0169] Kim, O. K., Murakami, A., Nakamura, Y.,
Takeda, N., Yoshizumi, H., Ohigashi, H. 2000a. Novel nitric oxide
and superoxide generation inhibitors, persenone A and B, from
avocado fruit. Journal of Agricultural and Food Chemistry 48 (5),
pp. 1557-1563. [0170] Kim, O. K., Murakami, A., Nakamura, Y., Kim,
H. W., Ohigashi, H. 2000b. Inhibition by (-)-Persenone A-related
Compounds of Nitric Oxide and Superoxide Generation from
Inflammatory Leukocytes. Bioscience, Biotechnology and Biochemistry
64 (11), pp. 2500-2503. [0171] Kim, O. K., Murakami, A., Takahashi,
D., Nakamura, Y., Torikai, K., Kim, H. W., Ohigashi, H. 2000c. An
Avocado Constituent, Persenone A, Suppresses Expression of
Inducible Forms of Nitric Oxide Synthase and Cyclooxygenase in
Macrophages, and Hydrogen Peroxide Generation in Mouse Skin.
Bioscience, Biotechnology and Biochemistry 64 (11), pp. 2504-2507.
[0172] King, W., Ming, X. Antibacterial composition for control of
gram positive bacteria in food applications. U.S. Pat. No.
6,620,446B2. Sep. 16, 2003. [0173] Kobiler, I., Prusky, D.,
Midland, S., Sims, J. J., Keen, N. T. 1993. Compartmentation of
antifungal compounds in oil cells of avocado fruit mesocarp and its
effect on susceptibility to Colletotrichum gloeosporioides.
Physiol. Mol. Plant. Pathol. 43: 319-328. [0174] Maseko, R. B.
2006. Synthesis of authentic organic standards of antibacterial
compounds isolated from avocados. Master of Science Thesis. Tshwane
University of Technology, South Africa. [0175] MacLeod, J. K. and
Schaeffler, L. A Short Enantioselective Synthesis of a Biologically
Active Compound from Persea Americana. J. Nat. Prod., vol. 58., no.
8, pp. 1270-1273 (August 1995). [0176] Monticello, D. J. Control of
microbial growth with lantibiotic/lysozyme formulations. U.S. Pat.
No. 5,458,876. Oct. 17, 1995. [0177] Murakoshi, S., Isogai, A.,
Chang, C. F., Kamikado, T., Sakurai, A., Tamura, S. The effects of
two components from avocado leaves (Persea americana Mill.) and
related compounds on the growth of silkworm larvae. Bombyx mori L.
Nippon Oyo Dobutsu Konchu Gakkaishi 1976; 20:87-91. [0178] NCCLS
M100-S12: Performance Standards for Antimicrobial Susceptibility
Testing: Twelfth Informational Supplement (ISN 1-56238-454-6).
[0179] Neeman, I., Lifshitz, A., Kashman, Y. 1970. New
antibacterial agent isolated from the avocado pear. Applied
microbiology, 470-473. [0180] Oberlies, N. H., Rogers, L. L.,
Martin, J. M. and McLaughlin, J. L. 1998. Cytotoxic and
Insecticidal Constituents of the Unripe Fruit of Persea americana.
J. Nat. Prod. 61:781-785. [0181] Oelrichs, P. B., Ng, J. C.,
Seawright, A. A., Ward, A., Schaffeler, L., MacLeod, J. K. 1995.
Isolation and identification of a compound from avocado (Persea
americana) leaves which causes necrosis of the acinar epithelium of
the lactating mammary gland and the myocardium. Natural Toxins,
3(5):344-349 [0182] Perumalla, A. V. S., Hettiarachchy, N. S. 2011.
Green tea and grape seed extracts--Potential applications in food
safety and quality. Food Research International. 44(4): 827-839.
[0183] Prusky, D., Keen, N. T., Sims, J. J., Midland, S. L., 1982.
Possible involvement of an antifungal diene in the latency of
Colletotrichum gloeosporioides on unripe avocado fruits.
Phytopathol. 72 (12), 1578. [0184] Prusky, D., Plumbley, R. A.,
Koliber, I., 1991a. Modulation of natural resistance of avocado
fruits to Colletotrichum gloeosporioides by CO2. Plant Pathol. 40,
45. [0185] Prusky, D., Koliber, I., Fishman, Y., Sims, J. J.,
Midland, S. L., Keen, N. T., 1991b. Identification of an Antifungal
Compound in Unripe Avocado Fruits and its Possible Involvement in
the Quiescent Infections of Colletotrichum gloeosporioides. J.
Phytopathol. 132, 319. [0186] Ramos-Jerz, M. D. Phytochemical
analysis of avocado seeds (Persea americana Mill., c.v. Hass). [Ph.
D. Dissertation], Gottingen, Alemania, 2007. Technishen Universitat
Brauschweig. [0187] Rayman, M. K. 1981. Nisin: a possible
alternative or adjunct to nitrite in the preservation of meats.
Applied and Environmental Microbiology. 41(2):375-380. [0188]
Rodriguez-Carpena, J. G., Morcuende, D., Andrade, M. J., Kylli, P.
and Estevez, M. 2011. Avocado (Persea americana Mill.) phenolics,
in vitro antioxidant and antimicrobial activities and inhibition of
lipid and protein oxidation in porcine patties. J. Agric. Food
Chem. 59:5625-5635. [0189] Rodriguez-Saona, C., Millar, J. G.,
Trumble, J. T. 1997. Growth inhibitory, insecticidal, and feeding
deterrent effects of
(12Z,15Z)-1-acetoxy-2-hydroxy-4-oxo-heneicosa-12,15-diene, a
compound from avocado fruit, to Spodoptera exigua. Journal of
Chemical Ecology, 23(7):1819-1831 [0190] Seawright A. A., Oelrichs
P. B., Ng, J. C., MacLeod J. K., Ward, A., Schaffeler, L., Carman,
R. M. 1995. Method of treatment of cancer as well as method of
inhibition of lactition in mammals. Patent Coop. Treaty Int. Appl.
No WO 95/22969, Astralian National University, Australia. [0191]
Sivanathan, S., Adikaram, N. K. B., 1989. Biological Activity of
Four Antifungal Compounds in Immature Avocado. Journal of
Phytopathology, 125(2): 97-109 [0192] Smola, M. 2007. Contribution
al'etude de la formulation et de l' analyse physicochimique de
formulations pediatriques microemulsionnees. [Docteur in Sciences
Pharmaceutiques]. Universite Louis Pasteur Strasbourg. France.
[0193] Sugiyama, T., Sato, A. and Yamashita, K. Synthesis of all
four stereoisomers of antibacterial component of avocado. Agric.
Biol. Chem., 46(2), 481-485 (1982). [0194] Tang, Y., Shi, Y., Zhao,
W., Hao, G. and Le, G. 2008. Inhibition of Food-Borne Pathogens by
T1, a Novel Antimicrobial Peptide as a Potential Food Preservative.
International Journal of Food Engineering. Vol 4, Iss. 4, Art, 14.
p. 1-19. [0195] Ugbogu, O. C. & Akukwe, A. R. 2009. The
antimicrobial effect of oils from Pentaclethra macrophylla Bent,
Chrysophyllum albidum G. Don and Persea gratissima Gaerth F on some
local clinical bacteria isolates. African Journal of Biotechnology,
8(2): 285-287. [0196] Pollack S, Perez A, Plattner K. 2010. Fruit
and tree nuts outlook. Economic Research Service. United States
Department of Agriculture USDAFTS-341/Mar. 26, 2010. [0197] Valeri,
A., and N. Gimeno. 1954. Phytochemical and toxicological study of
pericarp of the avocado pear. Rev. Med. Vet. Parasitol (Maracay)
13:37. [0198] Wilhoit, D. Film and method for surface treatment of
foodstuffs with antimicrobial compositions. U.S. Pat. No.
5,573,797. Nov. 12, 1996. [0199] Wilhoit, D. Antimicrobial
composition for surface treatment of foodstuffs. U.S. Pat. No.
5,573,800. Nov. 12, 1996. [0200] Wilhoit, D. Surface treatment of
foodstuffs with antimicrobial compositions. U.S. Pat. No.
5,573,801. Nov. 12, 1996. [0201] Yang, H., Li, X., Tang, Y., Zhang,
N., Chen, J. and Cai, B. 2009. Supercritical fluid CO2 extraction
and simultaneous determination of eight annonaceous acetogenins in
Annona genus plant seeds by HPLC-DAD method. J Pharm Biomed Anal.
49:140-144.
* * * * *