THU LUMTIMULUTTUR US009743679B2

(12) United States Patent ( 10) Patent No.: US 9,743,679 B2

Perez et al. (45 ) Date of Patent: Aug. 29 , 2017
(54 ) PLANT EXTRACT COMPOSITIONS AND (56 ) References Cited
METHODS OF PREPARATION THEREOF
U .S . PATENT DOCUMENTS
(71 ) Applicant: aPEEL Technology, Inc., Santa
Ê Barbara , CA (US) 2,342, 063 A * 2/1944 Sells ........ A23B 7/ 16
239 /590
( 72 ) Inventors: Louis Perez , Santa Barbara , CA (US) ; 3,189,467 A * 6 /1965 Kalmar .................. A23B 7 /154
James Rogers , Santa Barbara , CA 426 / 308
(US ); Ronald C . Bakus , II, Santa ( Continued )
Barbara , CA (US); Chance Holland , FOREIGN PATENT DOCUMENTS
Goleta , CA (US) ; Jenny Du , Santa
Barbara , CA (US ) WO WOU 2004030455 AL
A 4 /2004
2004
WO WO 2012042404 A2 4 / 2012
( 73 ) Assignee : ?PEEL Technology , Inc ., Goleta, CA WO WO 2015028299 Al 3 / 2015
(US)
( * ) Notice: Subject to any disclaimer, the term of this OTHER PUBLICATIONS
patent is extended or adjusted under 35 Publication by Con J. Baker et al; “ Cutin Degradation by Plant
U . S .C . 154 (b ) by 0 days . Pathogenic Fungi ”; published May 15, 1978 by The American
(21) Appl. No.: 15 /254 ,263 Phytopathological Society .*
(Continued )
( 22 ) Filed : Sep . 1, 2016
(65 ) Prior Publication Data Primary Examiner — Joseph Drodge
US 2017 /0049119 A1 Feb . 23 , 2017 (74 ) Attorney , Agent, or Firm — Cooley LLP
Related U . S . Application Data
(63 ) Continuation of application No. PCT/US2016/ (57) ABSTRACT
ã 033617 , filed on May 20 , 2016 . Embodiments described herein relate generally to plant
(Continued ) extract compositions and methods to isolate cutin -derived
(51) Int . Cl. monomers, oligomers, and mixtures thereof for application
BOID 11 /02 ( 2006 .01) in agricultural coating formulations, and in particular, to
B01D 37 /00 ( 2006 .01) methods of preparing plant extract compositions that include
functionalized and non -functionalized fatty acids and fatty
(Continued ) esters (as well as their oligomers and mixtures thereof),
(52 ) U .S. CI. which are substantially free from accompanying plant- de
??? A23B 7/ 154 (2013.01 ); A23B 7/ 16 rived compounds ( e .g ., proteins, polysaccharides, phenols ,
( 2013 .01); A23L 3 /3517 ( 2013 .01); lignans , aromatic acids , terpenoids, flavonoids, carotenoids,
(Continued ) alkaloids, alcohols , alkanes, and aldehydes ) and can be used
(58 ) Field of Classification Search in agricultural coating formulations.
CPC .. A23B 7 / 154 ; A23B 7 /00 , A23B 7 / 14 ; A23B
7 / 153; A23B 7 / 16 ; A23L 3 /3517 ;
(Continued ) 30 Claims, 10 Drawing Sheets
109

epare tá ? otein Pet 1 tbs ana w 2

1 Openy
Optionality ,,Atechewan
a , Separate Coën Containing Portion from the
- - - - - Pant plantwatter
Matter
- - - - - -
Opony, Box Curan Comari Portion Obian Cutie Cuir" 106
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Content Poor

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-- - - - -
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Resolucity the intermediate Execb Ob

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te PlantExtract

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state
wwwx wwwwwwwwwwwwwwwwwwwwwwww!
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Optionally , Rocrystalline ile Exxacled Colin Mononen
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 US 9,743 ,679 B2
Page 2

Related U .S . Application Data 8 , 197 ,870

8,247, 609
B2
B2
6 / 2012
8 /2012
Krasutsky et al.
Roques et al .
(60 ) Provisional application No. 62/164,312 , filed on May 8,263 , 751 B2 9 / 2012 Peterson
8,424, 243 B1 4 / 2013 Narciso et al.
20 , 2015 . 8 , 501, 445 B2 8 /2013 Yoshikawa et al.
8, 546, 115 B2 10 / 2013 Butchert et al.
(51) Int. CI. 8, 609,169 B2 12 /2013 Chen et al .
A23B 716 ( 2006 .01) 8 ,752,328 B2 6 / 2014 Kaiser et al.
A23B 7 / 154 ( 2006 . 01) 8, 846,355 B2 9/2014 Yoshikawa et al.
A23L 3 /3517 9 , 095,152 B2 8 / 2015 Munger
( 2006 .01) 9 ,102, 125 B2 8 / 2015 Battersby et al.
(52 ) U .S . CI. 9 ,284 ,432 B23 / 2016 Yoshikawa et al.
CPC ..... BOID 11 /0284 (2013 . 01) ; B01D 11 /0288 2005/0249856 A1 * 11/2005 Marangoni ............ A23D 7 /013
( 2013 .01 ); B01D 37 /00 (2013 .01); A23V 426 /601
2002/ 00 (2013 .01) 2008/0038471 AL 2 /2008 Boger et al.
2008/0262190 A 10 /2008 Koskimies et al.
(58 ) Field of Classification Search 2009/0142453 AL 6 /2009 Lobisser et al.
CPC ... A23L 3 /00 , A23L 5 /30 ; A23L 19 /00 ; A23V 2009/0325240 A1 12 /2009 Daniell
2002 /00 ; B01D 11 /02 ; B01D 11 /028 ; 2013/0209617 A1 8 / 2013 Lobisser et al.
BO1D 11 /0284 ; B01D 11 /0288 ; BO1D 2014 / 0033926 A1 2 /2014 Fassel et al.
36 / 00 ; B01D 37 / 00 ; C07C 51/42 ; C07C 2014 /0199449 A1* 7/2014 Hernandez ........... A23B 7/0441
426 /310
51/47; C07C 51/48 ; C07C 51/487; C07C 2014/0234921 Al* 8/2014 Nyyssola D21C4355//005
51/493 : C11B 1 / 00 ; C11B 1 / 10 ; C11B 135
1/ 108 ; C11B 3/00 ; C11B 3 /006 ; C11B 2014 /0348945 AL 11/2014 Dong et al.
3 /008 ; C11B 3 / 02
USPC .... 210 /634 , 639, 806 ; 426 / 89 , 90 , 302 , 303 , OTHER PUBLICATIONS
426 /310 , 615 ; 554 /8 , 20 , 21, 29, 30 , 174 , Publication by Osman et al, “ Preparation , Isolation , and Character
554 / 175
See application file for complete search history . ization of Cutin Monomers and Oligomers from Tomato Peels” ,
Journal of Agric. Food Chem ., vol. 47 , Published 1999 , pp . 799
(56 ) References Cited 802. *
Andrade, R . D . et al., 2012 , “ Atomizing spray systems for appli
U . S . PATENT DOCUMENTS cation of edible coatings ” , Comprehensive Reviews in Food Science
and Food Safety , 11 ( 3 ): 323 -337 .
4 ,421, 775 A 12 / 1983 Chan Ayala -Zavala, J. F . et al., 2008 , “ High Relative Humidity In -Package
4 ,710 , 228 A 12 / 1987 Seaborne et al. of Fresh -Cut Fruits and Vegetables: Advantage or Disadvantage
4 ,732,708 A * 3 / 1988 Ekman . . .. . . .. . . ... . ... C07C 51/09 Considering Microbiological Problems and Antimicrobial Deliver
554 / 138 ing Systems? ” J. Food Science 73 :R41-R47 .
5 , 110 ,509 A 5 / 1992 Peter et al. Ben - Yehoshua , S . et al., 1998 , “ Modified -atmosphere packaging of
5 ,354, 573 A * 10 / 1994 Gross ...... ........... A23D 7 / 0053 fruits and vegetables : reducing condensation of water in bell pep
106 / 244 pers and mangoes” , Acta Hort ( ISHS ) 464 : 387 - 92 .
5 ,658 , 768 A 8/ 1997 Quinlan Bourtoom , T ., 2008, “ Edible films and coatings: characteristics and
5 ,827,553 A 10 / 1998 Dimitroglou et al. properties” , International Food Research Journal, 15 ( 3 ): 237 - 248 .
5 ,925 ,395 A 7 / 1999 Chen et al. Deell, J. R . et al., 2006 , “ Addition of sorbitol with KMnO4 improves
5 ,939 ,117 A 8 / 1999 Chen et al. broccoli quality retention in modified atmosphere packages ” , J .
6,254 , 645 B1 7 / 2001 Kellis et al. Food Qual. 29 :65-75.
6,255,451 B1 7 / 2001 Koch et al. Roy, S . et al., 1996 , “ Modified atmosphere and modified humidity
6 , 348, 217 B1 2 / 2002 Santos et al. packaging of fresh mushrooms” , J. Food Sci. 61: 391 -7 .
7 ,785 , 897 B2 8 /2010 Agnes et al.
7 ,851, 002 B2 12 / 2010 Hekal et al. Shirazi, A ., and Cameron , A ., 1992 , “ Controlling relative humidity
7, 931 ,926 B2 4 /2011 Lidster et al. in modified atmosphere packages of tomato fruit ." HortScience
7 , 943,336 B2 5 / 2011 Viksoe -Nielsen et al. 27: 336 - 9 .
8,101 ,221 B2 1 / 2012 Chen et al.
8 ,119,178 B2 2 / 2012 Lidster et al. * cited by examiner
U , S , Patent Aug. 29, 2017 _ Sheet1of 10 US 9,743 ,679 B2

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U . S . Patent Aug. 29, 2017 Sheet 2 of 10 US 9 ,743 ,679 B2

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U . S . Patent Aug. 29, 2017 Sheet 3 of 10 US 9 ,743 ,679 B2

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U . S . Patent Aug. 29, 2017 Sheet 4 of 10 US 9 ,743 ,679 B2

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U . S . Patent Aug. 29 , 2017 Sheet 5 of 10 US 9 ,743 ,679 B2

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U . S . Patent Aug. 29 , 2017 sheet 6 of 10 US 9 ,743,679 B2

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U . S . Patent Aug. 29, 2017 Sheet 7 of 10 US 9 ,743 ,679 B2

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 US 9 ,743,679 B2
PLANT EXTRACT COMPOSITIONS AND can also contain additional compounds (e.g., epicuticular
METHODS OF PREPARATION THEREOF waxes , phenolics, antioxidants , colored compounds, pro
teins, polysaccharides , etc .). This variation in the cutin
CROSS -REFERENCE TO RELATED composition as well as the thickness and density of the cutin
APPLICATIONS 5 layer between plant species and / or plant organs and / or a
given plant at different stages of maturation can lead to
This application is a continuation of PCT/US2016 / varying degrees of resistance between plant species or plant
033617, filed May 20 , 2016 , which claims priority to and the organs to attack by environmental stressors (i.e ., water loss ,
benefit of U . S . Provisional Patent Application Ser . No. oxidation , mechanical injury , and light ) and /or biotic stress
62 / 164, 312 , entitled “ Plant Extract Compositions and Meth - 10 ors (e . g ., fungi, bacteria , viruses , insects, etc . ).
ods of Preparation Thereof," filed May 20 , 2015 , the dis
closure of which are hereby incorporated by reference in SUMMARY
their entirety .
Embodiments described herein relate generally to plant
BACKGROUND 15 extract compositions and methods to isolate cutin - derived
monomers , oligomers , and mixtures thereof for application
The present disclosure relates to plant extract composi- in agricultural coating formulations, and in particular, to
tions , and to methods of forming the same. Common agri- methods of preparing plant extract compositions that include
cultural products are susceptible to degradation and decom - functionalized and non - functionalized fatty acids and fatty
position (i.e ., spoilage ) when exposed to the environment. 20 esters (as well as their oligomers and mixtures thereof),
Such agricultural products can include, for example , eggs , which are substantially free from accompanying plant- de
fruits, vegetables , produce, seeds , nuts , flowers, and/ or rived compounds ( e . g ., proteins, polysaccharides, phenols ,
whole plants ( including their processed and semi-processed lignans , aromatic acids, terpenoids, flavonoids, carotenoids,
forms). Non -agricultural products ( e. g ., vitamins, candy, alkaloids, alcohols, alkanes , and aldehydes ) and can be used
etc . ) are also vulnerable to degradation when exposed to the 25 in agricultural coating formulations.
ambient environment. The degradation of the agricultural In some embodiments, a method for preparing a plant
products can occur via abiotic means as a result of evapo - extract composition includes thermally and /or mechanically
rative moisture loss from an external surface of the agricul- and / or enzymatically and / or chemically treating the plant
tural products to the atmosphere and /or oxidation by oxygen matter to at least partially separate a cutin - containing portion
that diffuses into the agricultural products from the environ - 30 from the plant matter. In some embodiments, the plant
ment and /or mechanical damage to the surface and / or light matter is subjected to elevated temperature and /or pressure
induced degradation (i. e ., photodegradation ). Furthermore , in an aqueous medium ( e . g ., as in pressure cooking ) to
biotic stressors such as, for example , bacteria , fungi, viruses, partially separate a cutin - containing portion from the plant
and/ or pests can also infest and decompose the agricultural matter. Alternatively, the plant matter may be subjected to
products . 35 lower temperatures ( e . g ., as in freezing ) to partially separate
Conventional approaches to preventing degradation , a cutin - containing portion from the plant matter. In some
maintaining quality, and increasing the life of agricultural embodiments , the plant matter is subjected to sonication in
products include refrigeration and/or special packaging an aqueous medium to partially separate a cutin -containing
Refrigeration requires capital- intensive equipment, demands portion from the plant matter. Optionally , the cutin -contain
constant energy expenditure , can cause damage or quality 40 ing portion is heated in a mixture of ammonium oxalate and
loss to the product if not carefully controlled , must be oxalic acid to aid separation of the cutin from the non - cutin
actively managed , and its benefits are lost upon interruption containing portion (i.e ., the remainder of the cuticle and
of a temperature - controlled supply chain . Special packaging unwanted plant matter). Optionally , this separation can be
can also require expensive equipment, consume packaging achieved (or assisted ) enzymatically using enzymes capable
material, increase transportation costs, and require active 45 of hydrolyzing ester bonds and / or alternatively using
management. Despite the benefits that can be afforded by enzymes capable of breaking down polysaccharides that
refrigeration and special packaging , the handling and trans- comprise the non - cutin - containing portion of the plant.
portation of the agricultural products can cause surface Optionally , the cutin - containing portion is refluxed in at least
abrasion or bruising that is aesthetically displeasing to the one organic solvent ( such as chloroform and /ormethanol) to
consumer and serves as points of ingress for bacteria and 50 remove residual waxes and /or any remaining soluble polar
fungi. Moreover, the expenses associated with such components from the cutin . Alternatively, removal of
approaches can add to the cost of the agricultural product. residual waxes and remaining soluble components can be
The cells that form the aerial surface ofmost plants ( such achieved using supercritical CO , or supercritical H2O . The
as higher plants ) include an outer envelope or cuticle , which cutin is then refluxed in a solvent having a high pH (e.g ., in
provides varying degrees of protection against water loss , 55 the range of about 10 to 14 , and typically in the range of 12
oxidation , mechanical damage , photodegradation , and/ or to 14 ), for example a solvent in which metal alkoxide or
biotic stressors, depending upon the plant species and the metal hydroxide (or alternative source of alkoxide or
plant organ (e. g., fruit , seeds, bark , flowers , leaves , stems
18,, hydroxide) is dissolved, to at least partially depolymerize
etc .). Cutin , which is a biopolyester derived from cellular the cutin and obtain an intermediate extract including a
lipids, forms the major structural component of the cuticle 60 plurality of esterified or fatty acid cutin monomers , their
and serves to provide protection to the plant against envi- oligomers, or mixtures thereof. In cases where the interme
ronmental stressors ( both abiotic and biotic ). The thickness , diate extract is obtained from (metal) alkoxide -mediated
density, as well as the composition of the cutin (i.e., the depolymerization , the pH of the intermediate extract is then
different types of monomers that form the cutin and their adjusted to be in the range of about 6 .5 to 9. 0 . In cases where
relative proportions) can vary by plant species, by plant 65 the intermediate extract obtained from (metal) hydroxide
organ within the same or different plant species, and by stage mediated depolymerization , the pH of the intermediate
of plant maturity . The cutin -containing portion of the plant extract is then adjusted to be in the range of about 1.0 to 6 .0 .
 US 9 ,743,679 B2
Alternatively, the cutin can be at least partially depolymer- vonoids, carotenoids, alkaloids, alcohols, alkanes, alde
ized under acidic conditions to obtain an intermediate hydes, and waxes. The method further includes at least
extract including a plurality of fatty acid cutin monomers, partially depolymerizing the cutin in a first solvent to obtain
oligomers , or mixtures thereof. The intermediate extract is a first solution comprising a first intermediate extract dis
then precipitated and / or extracted and purified (such as by 5 solved in the first solvent, wherein the first solution has a pH
washing with one or more selective solvents ) to obtain the in the range of 10 to 14, and the first intermediate extract
plant extract such that the plant extract is substantially free includes a plurality of cutin -derived monomers, oligomers ,
from accompanying plant-derived compounds ( e . g ., pro
teins, polysaccharides , phenols, lignans , aromatic acids, or ing
combinations thereof. The method also includes acidify
the first intermediate extract and obtaining the plant
terpenoids, flavonoids, carotenoids , alkaloids , alcohols , 10
alkanes, and aldehydes ). Further purification by chromatog extract composition , wherein the plant extract composition
comprises a second solution comprising the monomers,
raphy or recrystallization in a selective solvent may also be oligomers, or combinations thereof dissolved in a second
carried out after washing to obtain the final extract.
In some embodiments, a method for preparing a plant solvent.
extract composition includes obtaining cutin from a cutin - 15 In some embodiments , a method for preparing a plant
containing portion of a plant matter, the cutin -containing extract composition includes obtaining cutin from a cutin
portion being at least partially separated from a non-cutin - containing portion of a plant matter, the cutin - containing
containing portion of the plant matter, and at least partially portion being at least partially separated from a non - cutin
depolymerizing the cutin in a first solvent to obtain a first containing portion of the plant matter, and at least partially
solution comprising a first intermediate extract dissolved in 20 depolymerizing the cutin in a first solventhaving a pH in the
the first solvent, wherein the first solution has a pH in the range of 10 to 14 to obtain a first solution comprising a first
range of 10 to 14 , and the first intermediate extract includes intermediate extract dissolved in the first solvent, wherein
a plurality of cutin -derived monomers, oligomers , or com - the first intermediate extract includes a plurality of cutin
binations thereof. The method further includes evaporating derived monomers, oligomers, or combinations thereof. The
at least a portion of the first solvent, causing the first 25 method further includes acidifying the first solution , selec
intermediate extract to solidify, dissolving the solidified first tively filtering the first solution to remove impurity constitu
intermediate extract in a polar solvent to obtain a second ents , at least partially evaporating the first solvent to cause
solution , and acidifying the second solution , causing the first the first intermediate extract to solidify, and dissolving the
intermediate extract to resolidify.
In some embodiments, a method for preparing a plant 30 firstIn intermediate
some
extract in a second solvent.
embodiments , a method of preparing a plant
extract composition includes obtaining cutin from a cutin extract composition includes
containing portion of a plant matter, the cutin -containing containing portion of a plantobtaining cutin from a cutin
matter, the cutin - containing
portion being at least partially separated from a non - cutin
containing portion of the plant matter, and at least partially 5 portion being at least partially separated from a non -cutin
containing portion of the plant matter and at least partially
depolymerizing the cutin in a first solvent to obtain a first 35 depolymerizing
containing por the cutin in a first solvent to obtain a first
solution comprising a first intermediate extract dissolved in solution comprising a first intermediate extract in the first
the first solvent, wherein the first intermediate extract
includes a plurality of cutin - derived monomers , oligomers, solvent, the first intermediate extract including a plurality of
or combinations thereof. The method further includes acidi - cutin -derived monomers , oligomers , or combinations
fying the first intermediate extract, selectively filtering the 40 thereof. The method further includes selectively filtering the
first intermediate extract to obtain a second intermediate first intermediate extract to obtain a second intermediate
extract having a higher purity than the first intermediate extract having a higher purity than the first intermediate
extract , and dissolving the second intermediate extract in a extract, the second intermediate extract including at least
second solvent to obtain the plant extract composition . one of the cutin -derived monomers , oligomers , or combi
In some embodiments , a method for preparing a plant 45 nations thereof, and functionalizing the cutin -derived mono
extract composition includes obtaining cutin from a cutin - mers , oligomers , or combinations thereof of the second
containing portion of a plant matter, the cutin - containing intermediate extract to form the plant extract composition .
portion being at least partially separated from a non -cutin - In some embodiments, a method of protecting an agri
containing portion of the plant matter, and at least partially cultural product from biotic stress includes obtaining a plant
depolymerizing the cutin in a first solvent to obtain a first 50 extract composition comprising a plurality of cutin -derived
solution comprising a first intermediate extract dissolved in monomers , oligomers , or combinations thereof, functional
the first solvent, wherein the first solution has a pH in the izing the cutin -derived monomers , oligomers , or combina
range of 10 to 14 , and the first intermediate extract includes tions thereof by esterifying the cutin -derived monomers ,
a plurality of cutin -derived monomers, oligomers, or com - oligomers , or combinations thereof with a glycerol mol
binations thereof. The method further includes evaporating 55 ecule , and causing the agricultural product to be coated with
at least 25 % of a volume of the first solvent from the the functionalized cutin - derived monomers , oligomers , or
solution , adding a polar solvent to the first solution to obtain combinations thereof.
a second solution , and acidifying the second solution , caus In some embodiments , a method of prolonging the life of
ing the first intermediate extract to precipitate . an agricultural product includes obtaining a plant extract
In some embodiments , a method for preparing a plant 60 comprising a plurality of cutin -derived monomers , oligom
extract composition includes obtaining cutin from a cutin - ers , or combinations thereof, functionalizing the cutin -de
containing portion of a plant matter, the cutin -containing rived monomers, oligomers, or combinations thereof by
portion being at least partially separated from a non -cutin - esterifying the cutin - derived monomers, oligomers, or com
containing portion of the plant matter, and exposing the binations thereof with a glycerol molecule , and causing the
compound to supercritical carbon dioxide to selectively 65 agricultural product to be coated with the functionalized
reduce a concentration of at least one of proteins , polysac - cutin - derived monomers , oligomers , or combinations
charides , phenols, lignans, aromatic acids, terpenoids, fla - thereof.
 US 9 ,743,679 B2
BRIEF DESCRIPTION OF THE FIGURES deposited coatings ) which are on the outer surface of the
products . The applied coatings can , for example , serve to
FIG . 1 is a schematic flow diagram of an exemplary protect the products from biotic stressors such as bacteria ,
method for preparing a plant extract composition , according fungi, viruses , and / or pests . The applied coatings can also
to an embodiment. 5 ( or alternatively ) serve to increase the shelf life of produce
FIG . 2 shows high resolution time lapse photographs of without refrigeration , and /or to control the rate of ripening
lemons, both with and without coatings formed of com - of produce .
pounds described herein . In some embodiments, in order to achieve one or more of
FIG . 3 is a normalized plot of the cross -sectional areas of the benefits described above for applications in which an
the lemons coated with and without compounds described 10 agricultural coating is subsequently formed from a plant
herein as a function of time. extract composition derived from plant matter, it may be
FIG . 4 shows high resolution time lapse photographs of necessary for the plant extract composition to be highly
strawberries , both with and without coatings formed of purified , e . g ., to include the monomer and /or oligomer units
compounds described herein . that form the structural building blocks of the coatings and
FIG . 5 is a plot of the percent mass loss of blueberries 15 to simultaneously be substantially free of other accompa
with and without coatings formed of the compounds nying plant-derived compounds such as proteins, polysac
described herein as a function of time. charides, phenols, lignans, aromatic acids, terpenoids, fla
FIG . 6 shows high resolution photographs of blueberries , vonoids, carotenoids, alkaloids , alcohols, alkanes , and
both with and without coatings formed of compounds aldehydes . In particular, plant extracts to be used in agri
described herein after five days . 20 cultural coating applications typically need a substantially
FIG . 7 shows plots of purity data of an extract composi - higher level of purity than when extracted for other appli
tion described herein . cations ( e.g ., characterization of plant matter). In some
FIG . 8 is an elemental analysis of an extract composition cases, achieving the required level of purity requires a
described herein . number of additional steps thatmay not be advantageous or
FIG . 9 shows high resolution photographs of uncoated 25 desirable in other applications.
key limes . The plant extract compositions described herein can be
FIG . 10 shows high resolution photographs of key limes derived or obtained in a number of ways . For example , in
coated with compounds of a first extract. some embodiments the compositions are derived directly
FIG . 11 shows high resolution photographs of key limes from a cutin - containing portion of plant matter , such as by
coated with compounds of a second extract . 30 depolymerizing (or at least partially depolymerizing ) the
cutin to obtain a plurality of cutin - derived monomers, oli
DETAILED DESCRIPTION gomers , or combinations thereof, and optionally function
alizing ( e . g ., modifying ) the cutin - derived monomers or
The biopolyester cutin forms the main structural compo - oligomers. Alternatively , in other embodiments the plant
nent of the cuticle that composes the aerial surface of most 35 extract compositions are obtained by decomposition of palm
land plants and plays a significant role in providing plants a oil, and optionally subsequently functionalizing ( e.g ., modi
protective barrier against both abiotic and biotic stressors . fying) the monomers or oligomers obtained by the palm oil
The thickness, density , as well as the composition of the decomposition . In still other embodiments , the plant extract
cutin (i.e., the different types of monomers that form the compositions are obtained from commercial sources, for
cutin and their relative proportions ) can vary by plant 40 example by purchasing monomers , oligomers , or combina
species , by plant organ within the same or different plant tions thereof which were derived or formed by one of the
species, and by stage of plantmaturity . These variations can methods described above, or by other methods .
define the amount, degree , or quality of protection ( and As described above , the compositions of the present
degree of plasticity ) offered by the cutin layer to the plant or disclosure can be derived or obtained from commercial
plant organ against environmental and/ or biotic stressors . 45 sources. For instance , compositions can be made by func
Cutin is formed from a mixture of polymerized mono - tionalizing a commercially available fatty acid (e . g ., palm
and / or polyhydroxy fatty acids and embedded cuticular itic acid or 10 , 16 -dihydroxyhexadecanoic acid ). The fatty
waxes . Among the hydroxy fatty acids, polyhydroxy fatty acids thus obtained can be functionalized to improve their
acids (e . g ., dihydroxy fatty acids or trihydroxy fatty acids), properties as coatings for agricultural products . For
once esterified , can form tightly bound networks with high 50 example , in some embodiments , a fatty acid ( e . g ., a com
crosslink density and lower permeability as compared to mercially available fatty acid or a fatty acid extracted from
monohydroxy fatty acids and can thereby provide better plant cutin ) is esterified . The esterification can be completed
protection against environmental stressors. directly via the reaction of the free carboxylic acid with an
Embodiments described herein relate generally to plant appropriate alcohol. Alternatively , the esterification can be
extract compositions and , in particular, to methods of pre - 55 completed by transesterification of a fatty acid ester ( e. g.,
paring plant extract compositions that include hydroxy fatty methyl palmitate ) with an appropriate alcohol.
acids and hydroxy fatty esters (aswell as their oligomers and In some embodiments, the cutin - derived monomers , oli
mixtures thereof), which are substantially free from accom gomers, or combinations thereof (e.g ., the fatty acid com
panying plant- derived compounds (e .g., proteins, polysac positions ) of the present disclosure are functionalized to
charides , phenols, lignans , aromatic acids, terpenoids, fla - 60 their corresponding glycerol esters ( e. g ., 1 - glycerol or
vonoids , carotenoids, alkaloids, alcohols, alkanes, and 2 -glycerol esters ). That is , a fatty acid of the present dis
aldehydes ) and can be used in agricultural coating formu- closure (e .g ., palmitic acid ) can be esterified using glycerol
lations . Specifically , plant extract compositions described or a derivative thereof to give a fatty acid ester of glycerol
herein that are formed from plantmatter can subsequently be (e . g ., 1 ,3 - dihydroxypropan - 2 - yl palmitate or 2 , 3 - dihydroxy
applied to other plant or agricultural products in order to 65 propyl palmitate ).
form a protective coating over the products, or to enhance or As set forth in the present disclosure , functionalized fatty
modify existing coatings (either naturally occurring or acids (e.g., fatty acid - glycerol esters ) can have advantageous
 US 9, 743,679 B2
properties that help to preserve agricultural produce when plurality of fatty acid or esterified cutin monomers, oligom
applied to the surface of the produce ( e . g ., can prolong the ers , or combinations thereof. The first solvent (and therefore
shelf life of agricultural produce ). In some embodiments , the resulting first solution ) typically has a pH in the range of
substantially all of the fatty acids in the compositions 10 to 14 , in order to enable the depolymerization process.
described herein are esterified with a glycerolmolecule . For 5 The high pH indicates that the extract is substantially
instance, both commercially derived fatty acids as well as deprotonated . In some embodiments , the first solvents and
fatty acids extracted from plant compositions can be func - or the first solution has a pH in the range of 12 to 14 .
tionalized as a glycerol ester. Following depolymerization , the extract including the
In some embodiments , the monomer and /or oligomer monomer and/or oligomer units is acidified in order for the
units of the plant extract compositions described herein can 10 monomer and/ or oligomer units to be protonated or rendered
be applied to the surface of an agricultural product that does neutral (i.e ., to generate the corresponding free fatty acids
not naturally produce the monomer and / or oligomer units . and /or fatty esters and to be in a state that enables them to
For instance , the monomer and /or oligomer units can com - subsequently form an agricultural coating ). Finally , the
prise a plurality of fatty acids that can be commercially extract including the free fatty acid and /or free fatty ester
available or extracted from plantmatter . These compositions 15 monomer and /or oligomer units is dissolved in another
can be applied to agricultural products that do not naturally solvent, thereby resulting in a plant extract composition
produce , for instance , the same types of fatty acids. In one suitable for agricultural coating applications.
example , a composition of monomers and /or oligomers Because the cutin obtained from the cutin -containing
derived from palmitic acid can be applied to an agricultural portion is typically intermixed with many of the other
product that does not naturally produce palmitic acid . 20 constituents described above , the extract obtained from the
Additionally , in some embodiments, the monomer and/or depolymerization process may have a higher level of impu
oligomers units of the compositions described herein can be rity constituents than can be tolerated in agricultural coating
functionalized and applied to agricultural products that do applications . As such , the cutin and /or the extract composi
not naturally produce such functionalization . For instance , a tion and /or the solution containing the extract composition
glycerol - ester of a fatty acid ( e . g ., 1, 3 -dihydroxypropan - 2 -yl 25 can be purified by selectively removing or filtering out the
palmitate or 2 , 3 - dihydroxypropyl palmitate or a combina - impurity constituents. Selective filtering can occur either
tion thereof) can be applied to the surface of an agricultural before or after the depolymerization process , or both before
product that does not naturally produce glycerol- function and after depolymerization . Selective filtering may include
alized fatty acids . one or more of the following processes :
In some embodiments , the addition of non -naturally 30 (a ) Prior to depolymerizing or partly depolymerizing the
occurring ( e. g., functionalized ) monomer and /or oligomer cutin , washing and/or heating the cutin in a selective
units of the plant extract (e . g ., fatty acids ) can have benefi solvent for which the solubility of impurity constituents
cial properties when applied to produce to form a coating. in the selective solvent is higher than the solubility of
For instance , the coatings can reduce moisture loss from the the cutin . In this case , impurities are dissolved into the
produce and/ or reduce microbial growth on the surface of 35 selective solvent , thereby resulting in fewer impurities
the produce . in the first intermediate extract . In some embodiments ,
In some embodiments, the monomer and / or oligomer impurities are dissolved into the selective solvent
units of the plant extract compositions are obtained from immediately after depolymerization . Examples of such
cutin found in plant matter ( e .g ., extracted from plant matter a solvent can include chloroform , diethyl ether, dichlo
such as plant waste from other applications ). Extracting the 40 romethane, hexane, petroleum ether, ethyl acetate ,
monomer and /or oligomer units of plant extract can be acetone, isopropanol, ethanol, methanol, supercritical
performed instead of purchasing fatty acids from commer carbon dioxide, supercriticalwater , water , and mixtures
cial sources, or as a supplement to fatty acids from com thereof.
mercial sources. Plant matter typically includes some por (b ) After depolymerizing or partly depolymerizing the
tions that contain cutin and/ or have a high density of cutin 45 cutin to obtain the first solution comprising the first
( e . g ., fruit peels , leaves , shoots , etc . ), as well as other intermediate extract dissolved in the first solvent, and
portions that do not contain cutin or have a low density of prior to acidifying the first intermediate extract, wash
cutin ( e . g ., fruit flesh , seeds , etc . ). The cutin - containing ing and /or heating the first intermediate extract in a
portions can be formed from the monomer and /or oligomer selective solvent ( e . g ., acetonitrile ) for which the solu
units and can also include other constituents such as pro - 50 bility of impurity constituents in the selective solvent is
teins , polysaccharides , phenols, lignans , aromatic acids, lower than the solubility of the monomers and /or
terpenoids , flavonoids, carotenoids, alkaloids , alcohols, oligomers . In this case , themonomers and /or oligomers
alkanes, and aldehydes. The non -cutin - containing portions are dissolved into the selective solvent while the impu
typically lack the monomer and/or oligomer units , or oth rities are not. The impurities can then be filtered out,
erwise include a much lower ratio of monomer and /or 55 resulting in a second intermediate extract dissolved in
oligomer units to other constituents as compared to the the selective solvent, whereby the second intermediate
cutin -containing portions. extract has a higher purity than the first intermediate
Methods described herein for forming plant extract com extract. The second intermediate extract may subse
positions from cutin can generally include first separating quently be solidified , e .g ., by evaporating the selective
( or at least partially separating ) cutin - containing portions of 60 solvent.
plantmatter from non -cutin -containing portions, and obtain (c) After depolymerizing or partly depolymerizing the
ing cutin from the cutin - containing portions ( e . g ., when the cutin to obtain the first solution comprising the first
cutin -containing portion is a fruit peel, the cutin is separated intermediate extract dissolved in the first solvent, and
from the peel ). The cutin is then depolymerized ( or at least prior to acidifying the first intermediate extract, wash
partially depolymerized ) in a first solvent to obtain a first 65 ing and/or heating the first intermediate extract in a
solution comprising a first intermediate extract dissolved in selective solvent ( e .g ., chloroform or hexane ) for which
the first solvent, the first intermediate extract including a the solubility of impurity constituents in the selective
 US 9 ,743,679 B2
10
solvent is higher than the solubility of the monomers controlled by the chemical composition and degree of cross
and /or oligomers . In this case , impurities are dissolved linking. Polyhydroxy fatty acids ( as well as their monomeric
into the selective solvent, thereby removing the impu esters and oligomers) are multifunctional and thus capable
rities from the extract, and a second intermediate of forming highly crosslinked polymers . However, such
extract having a higher purity than the first intermediate 5 polyhydroxy fatty acids are not currently commercially
extract is obtained. available . Thus, such hydroxy fatty acids can either be
(d ) After depolymerizing or partly depolymerizing the synthesized or obtained from a suitable biological source .
cutin to obtain the first solution comprising the first Since the cutin ofmany plants and /or plant organs (e. g ., fruit
intermediate extract dissolved in the first solvent, and peels, juice sac , leaves, shoots , etc .) is composed ofmono
after subsequently acidifying the first intermediate 10 and polyhydroxy fatty acids (or can be decomposed into
extract , washing and / or heating the first intermediate mono - or polyhydroxy fatty acids ), an extract composition
extract in a selective solvent (e .g ., acetonitrile ) for from such plants and/or plant organs can provide a suitable
which the solubility of impurity constituents in the biological source of mono - and /or polyhydroxy fatty acids .
selective solvent is lower than the solubility of the In some embodiments , the cutin monomers included in
monomers and/or oligomers . In this case , the mono - 15 the plant extract compositions described herein can be
mers and /or oligomers are dissolved into the selective mono - and/ or polyhydroxy fatty acids and / or esters thereof
solvent while the impurities are not. The impurities can in a combination suitable for use in coating formulations ,
then be filtered out, resulting in a second intermediate e .g . for protecting plant species against environmental and /
extract dissolved in the selective solvent, whereby the or biotic stressors . Since bacteria , fungi, and pests all
second intermediate extract has a higher purity than the 20 identify food sources via recognition of specific molecules
first intermediate extract. The second intermediate on the surface of agricultural products, coating agricultural
extract may subsequently be solidified , e . g ., by evapo - products with a formulation that includes embodiments of
rating the selective solvent. the plant extract compositions described herein can render
( e ) After depolymerizing or partly depolymerizing the the agricultural products unattractive or unrecognizable to
cutin to obtain the first solution comprising the first 25 said bacteria , fungi, and pests . Such formulations can , for
intermediate extract dissolved in the first solvent, and example , also alter the chemical environment of the surface
after subsequently acidifying the first intermediate of agricultural products , making the surface unfavorable for
extract, washing and / or heating the first intermediate bacteria , fungi, or pests to grow . Moreover, such formula
extract in a selective solvent ( e . g ., chloroform or t ions can also be configured to simultaneously prevent
hexane ) for which the solubility of impurity constitu - 30 moisture loss from agricultural products and / or their oxida
ents in the selective solvent is higher than the solubility tion by ambient air and /or impart resistance to mechanical
of the monomers and / or oligomers . In this case , impu - damage and / or photodegradation and / or delay ripening,
rities are dissolved into the selective solvent, thereby thereby minimizing decomposition and increasing the life of
removing the impurities from the extract, and a second the agricultural products by aiding in resistance to abiotic
intermediate extracthaving a higher purity than the first 35 stressors .
intermediate extract is obtained . Embodiments of the plant extract compositions described
(f) After obtaining a compound comprising cutin from a herein can be synthesized or extracted from plant matter,
cutin -containing portion of plant matter and prior to including agriculturalwaste products , and can be included in
depolymerizing or partly depolymerizing the cutin , coating formulations for protecting agricultural products
exposing the compound to supercritical carbon dioxide 40 from environmental and biotic stressors . Embodiments of
to selectively reduce a concentration of at least one of the plant extract compositions described herein provide
proteins , polysaccharides , phenols, lignans, aromatic several advantages, including, for example: (1 ) formation of
acids, terpenoids, flavonoids, carotenoids, alkaloids , agricultural coating formulations that can protect the agri
alcohols, alkanes, aldehydes, and waxes. cultural products from biotic stressors (i.e ., bacteria , viruses ,
( g ) After depolymerizing or partly depolymerizing the 45 fungi, or pests ); (2 ) formation of agricultural coating for
cutin to obtain the first solution comprising the first mulations that can prevent evaporation of water and /or
intermediate extract dissolved in the first solvent, and diffusion of oxygen and /or other gaseous species (e.g.,
either prior to or after acidifying the first intermediate carbon dioxide and ethylene ); (3 ) extension of the shelf life
extract, filtering the solution to remove undissolved of agricultural products, for example , postharvest produce ,
impurity constituents , for example by passing the solu - 50 without refrigeration ; ( 4 ) introduction ofmechanical stabil
tion through a filter. ity to the surface of the agricultural products , helping to
In some embodiments , at least a portion of the plant prevent the types of bruising and surface rupture that accel
extract can include monomers that include multiple func - erate spoilage ; (5 ) reduction of photodegradation of the
tional groups ( e. g ., one of at least hydroxyl, carboxyl, epoxy , agricultural products; (6 ) use of agricultural waste materials
olefin , etc . ). For example , in some embodiments , the mono - 55 to obtain the plant extract compositions, which in turn help
mers can include multiple functional groups of the same eliminate the breeding environments of bacteria , fungi, and
type ( e . g ., dihydroxy - and trihydroxy -substituted ). In some pests , and divert materials away from landfills ; ( 7 ) use in
embodiments, the monomers can include multiple functional place of pesticides to protect plants , thereby minimizing the
groups , at least one of which is different than the remaining harmful impact of pesticides to human health and the
functional group ( s ) ( i.e ., includes more than one type of 60 environment; and ( 8 ) extraction from natural and /or edible
functional group ). These functional groups can be chemi- waste products, making the plant extract compositions safe
cally reactive , allowing the formation of highly crosslinked for human consumption .
networks, unlike their monofunctional analogs (which form A method for preparing a plant extraction composition
dimers ), or their di- functional analogs (which can polymer - can include thermally and / or mechanically and /or enzymati
ize into linear chains ). The barrier properties (i.e., the ability 65 cally and /or chemically treating the plantmatter to at least
of such extracts to form an effective agricultural coating partially separate the cutin - containing portion from the
barrier against biotic or abiotic stressors ) are generally non -cutin -containing portion of the plant matter. In some
 US 9 ,743,679 B2
11 12
embodiments , the plant matter is subjected to elevated powder. It has been found that agricultural coatings formed
temperature and / or pressure in an aqueous medium ( e . g ., as from plant extract compositions with low coloration and
in pressure cooking ) to partially separate the cutin - contain with the powdery consistency described above can be sub
ing portion from the non -cutin - containing portion of the stantially undetectable to the human eye, withoutmodifying
plantmatter. Alternatively , the plantmatter may be subjected 5 the appearance of the agricultural product to which they are
to lower temperatures (e .g., as in freezing) to partially applied . On the other hand , coatings formed from extracts
separate the cutin - containing portion from the non -cutin with higher levels of coloration and/or sticky cannot be
containing portion of the plant matter . In some embodi- deposited as uniformly and may cause the coated agricul
ments, the plant matter is subjected to sonication in an tural product to appear discolored and / or damaged .
aqueous medium to partially separate the cutin - containing 10 The reasons for the substantial difference in color and
portion from the non - cutin - containing portion of the plant consistency of the resulting solidified compositions when
matter. Optionally , the cutin -containing portion is heated in water- containing and non water - containing (or low water
a mixture of ammonium oxalate and oxalic acid to aid content ) solvents are used for depolymerization are unclear.
separation of the cutin from the non - cutin -containing portion However, without wishing to be bound by theory , it is
(i.e ., the remainder of the cuticle and unwanted plant mat- 15 hypothesized that the steric bulk of the nucleophile dictates
ter ). Optionally , this separation can be achieved ( or assisted ) the species that are depolymerized , including trace compo
enzymatically using enzymes capable of hydrolyzing ester n ents , and that an undesirable mix of depolymerized species
bonds and / or alternatively using enzymes capable of break - and trace components resulting from depolymerization in
ing down polysaccharides that comprise the non -cutin water containing solvents gives rise to color and/ or sticky
containing portion of the plant. Optionally , the cutin - con - 20 consistency in the extract composition .
taining portion is refluxed in at least one organic solvent Next, the solution comprising the intermediate extract
(such as chloroform and /or methanol) to remove residual dissolved in the high pH solvent is acidified in order to cause
waxes and/or any remaining soluble polar components from the monomer and /or oligomer units to be protonated or
the cutin . Alternatively , removal of residual waxes and rendered neutral (i. e., to generate the corresponding free
remaining soluble components can be achieved using super - 25 fatty acids and / or fatty esters and to be in a state that enables
critical CO2 or supercritical H2O . them to subsequently form an agricultural coating ). In cases
The cutin (or cutin - containing component) obtained from where the intermediate extract is obtained from (metal )
the plant matter is then refluxed in a solvent having a high alkoxide-mediated depolymerization , the pH of the solvent
pH ( e . g ., in the range of about 10 to 14 , and typically in the and intermediate extract can be adjusted to be in the range
range of 12 to 14 ), for example a solvent which is alkalized 30 of about 6 .5 to 9 .0 . In cases where the intermediate extract
with metal alkoxide or metal hydroxide ( or alternative is obtained from (metal) hydroxide -mediated depolymeriza
source of alkoxide or hydroxide ) to at least partially depo - tion , the pH of the solvent and intermediate extract can be
lymerize the cutin and obtain an intermediate extract includ - adjusted to be in the range of about 1 . 0 to 6 .5 . Alternatively ,
ing a plurality of esterified or fatty acid cutin monomers, rather than depolymerizing in an alkalized solvent and
their oligomers, or mixtures thereof. The high pH solvent 35 subsequently acidifying, the cutin can be at least partially
can be one in which the monomers and/ or oligomers are depolymerized under acidic conditions to obtain an inter
highly soluble , but one or more of the other constituents of mediate extract including a plurality of fatty acid cutin
the cutin - containing compound (e . g ., proteins, polysaccha - monomers , oligomers, or mixtures thereof.
rides, phenols, lignans , aromatic acids, terpenoids, fla - The intermediate extract is then precipitated and/or
vonoids, carotenoids, alkaloids, alcohols , alkanes , and alde - 40 extracted and purified ( such as by washing with one or more
hydes) is insoluble or is less soluble than the monomers selective solvents ) to obtain the plant extract such that the
and/ or oligomers . As such , after the depolymerization pro - plant extract is substantially free from accompanying plant
cess, the non -dissolved impurities can be filtered from the derived compounds (e.g., proteins, polysaccharides , phe
solution . In some embodiments, the high -pH solvent nols, lignans, aromatic acids, terpenoids, flavonoids, caro
includes ethanol,methanol , or another alcohol having metal 45 tenoids, alkaloids , alcohols, alkanes , and aldehydes ). Further
(e .g ., sodium metal), metal alkoxide , or metal hydroxide purification by chromatography or recrystallization in selec
dissolved within . tive solvent may also be carried out after washing to obtain
In some embodiments, the high -pH solvent is substan - the final extract.
tially free of water (e . g ., less than 5 % water by mass ) or has In some embodiments, after depolymerization in the high
a relatively low water content ( e . g ., less than 50 % , less than 50 pH solvent to obtain the intermediate extract, instead of
40 % , less than 35 % , less than 30 % , less than 25 % , less than directly acidifying the high pH solvent and the dissolved
20 % , less than 15 % , or less than 10 % water by mass ). intermediate extract, the intermediate extract is first precipi
Without wishing to be bound by theory , the use of non tated ( i.e ., solidified ), for example by evaporating at least a
water-containing or low water content solvents has been portion (and in some cases substantially all of) the high pH
found to cause the resulting plant extract compositions, 55 solvent. The solidified intermediate extract is then dissolve
when solidified ( e . g ., after removal of the solvent from the in a polar solvent such as water in order to obtain a second
composition ), to be much more tractable (e . g ., to be a solid solution , and the second solution is then acidified to a pH in
powder rather than a sticky mass ), and to have substantially the range of 1 to 6 .5 . Acidifying the second solution com
reduced coloration . That is, even after subsequent purifica - prising the polar solvent and the intermediate extract may
tion steps, solidified plant extract compositions that were 60 cause the intermediate extract to resolidify , after which the
formed via depolymerization of cutin in a high -pH solvent intermediate extract can be filtered out of the second solu
containing a substantial amount of water ( e .g ., greater than tion . The resolidified intermediate extract can then be dis
50 % water by mass ) were found to result in a sticky mass solved in another solvent, for example ethanol, to form the
with a substantial amount of coloration , whereas extract final plant extract composition . Alternatively , the resolidi
compositions formed via cutin depolymerization in a non 65 fied intermediate extract can be selectively filtered to
water -containing or low water content solvent were able to remove additional impurities and thereby obtain a second
be purified into a yellow /orange to colorless free flowing intermediate extract having a higher purity than the inter
 US 9 ,743,679 B2
13 14
mediate extract. For example, the second intermediate The method 100 can optionally include mechanically
extract can be obtained by washing the intermediate extract processing the plant matter to at least partially separate the
in acetonitrile , as previously described . Finally , the second cutin - containing portion from the plant matter, at 104 . The
intermediate extract can be dissolved in another solvent, for mechanical process can be performed before and /or after
example ethanol, to form the final plant extract composition . 5 thermal treatment of the plantmatter ( i. e., 102 ) ( e. g ., boiling
In still other embodiments , after depolymerization in the of the plant matter in water) to facilitate separation of the
high pH solvent to obtain the intermediate extract, instead of cutin -containing portion from the plant matter. Suitable
directly acidifying the high pH solvent and the dissolved mechanical processes can include, for example , centrifuga
intermediate extract, at least 25 % of the high pH solvent is110 tion , (ultra )sonication , pressing, ball milling, grinding , etc .
first evaporated , a polar solvent such as water is then added In some embodiments, mechanical separation can include
separating a fruit peel from the fruit pulp . In some embodi
to form a second solution , and the second solution is then ments , mechanical removal of the pulp might not be per
acidified to a pH in the range of 1 to 6 .5. Acidifying the formed and the fruit skins (e . g ., waste fruit skins leftover
second solution comprising the polar solvent and the inter after processing of the fruit ) may be macerated , blended , cut,
mediate extract may cause the intermediate extract
extract toto pre
pre-- 1515 Shredded
shredded , food
food pro
processed , or otherwise subjected to some
cipitate (i.e., solidify ), after which the intermediate extract other mechanical treatment operation to physically break
can be filtered out of the second solution . The solidified down the fruit skins into smaller or finer pieces. In some
intermediate extract can then be dissolved in another sol embodiments , a plurality of intermediate mechanical pro
vent, for example ethanol, to form the final plant extract cesses can be used to obtain the plant extract composition .
composition . Alternatively, the solidified intermediate 20 For example , a mechanical step can be used to separate the
extract can be selectively filtered to remove additional cutin from the non -cutin - containing portion , as described
impurities and thereby obtain a second intermediate extract herein , or be used to augment any other operation included
having a higher purity than the intermediate extract. For in the method 100 . Such mechanical processes can include
example , the second intermediate extract can be obtained by any of the mechanical processes described herein such as ,
washing the intermediate extract in acetonitrile , as previ- 25 for example , centrifugation , sonication , (ultrasonication ,
ously described. Finally, the second intermediate extract can milling , grinding, filtration , etc .
be dissolved in another solvent, for example ethanol, to form The cutin -containing portion is then optionally heated in
the final plant extract composition . a mixture of ammonium oxalate and oxalic acid to separate
As used herein , " plant matter ” refers to any portion of a the cutin from the non - cutin -containing portion , at 106 .
plant that contains cutin including, for example , fruits ( in the 30 Optionally this process can also be achieved (or assisted )
botanical sense , including fruit peels and juice sacs ), leaves, using enzymes capable of breaking down polysaccharides or
stems, barks, seeds, flowers , or any other portion of the pectin . For example , the cutin can include the cuticular layer
plant. of the plant matter. The heating in the ammonium oxalate
As used herein , the terms " about” and “ approximately ” and oxalic acid mixture disrupts the pectinaceous glue that
generally mean plus or minus 10 % of the value stated . For 35 attaches the cuticle to the underlying cells of the plant matter
example , " about 250 um ” would include 225 um to 275 um , and helps release the cuticle . Furthermore , this step disrupts
and " about 1, 000 um ” would include 900 um to 1, 100 um . the pectinaceous glue that is found within primary cell walls
FIG . 1 shows a schematic flow diagram of a method 100 and between plant cells ( e.g ., in the middle lamella that
for preparing a plant extract composition . The method 100 binds neighboring cells), aiding in the isolation of a cutin
includes thermally treating a plantmatter to at least partially 40 containing portion . In this manner, the ammonium oxalate
separate a cutin - containing portion from the plantmatter , at and oxalic acid solution can facilitate at least partial chemi
102. The thermal treating can include , for example , heating cal detachment of remaining debris from the cutin - contain
the plant matter ( e . g ., with steam , in water or in another ing portion of the plant ( e . g ., removal of any remaining pulp
solvent), freezing the plant, or subjecting the plant matter to from the fruit peel). The heating can be performed at any
cyclic thermal treatments . The plant matter can include any 45 suitable temperature (e . g ., 35 degrees Celsius, 50 degrees
suitable plant matter or otherwise agricultural product such Celsius, 55 degrees Celsius, 60 degrees Celsius , 65 degrees
as, for example , fruits (including fruit peels and juice sacs ), Celsius, 70 degrees Celsius , 75 degrees Celsius , 80 degrees
leaves, stem , barks, seeds, and flowers. In some embodi- Celsius, 85 degrees Celsius, 90 degrees Celsius , 95 degrees
ments, the plant matter can include agricultural waste prod Celsius, or 100 degrees Celsius, inclusive of all ranges and
ucts such as, for example , tomato peel, grape skins, apple 50 values therebetween and for any suitable time (this process
peel, pepper peel, lemon peel, lemon leaf, lime peel, lime can be accelerated if carried out under elevated pressure ).
leaf, orange peel, orange leaf, orange fruit , clementine leaf, For example , in some embodiments, the cutin -containing
clementine fruit, mandarin leaf, mandarin fruit, pea seeds, portion can be heated in the mixture of ammonium oxalate
grapefruit peel, grapefruit leaf, grapefruit seed , papaya peel, and oxalic acid at a temperature of about 75 degrees Celsius
cherry fruits , cranberry skins , coffee cherries, grass clip - 55 for about 24 hours. In some embodiments , the portion of the
pings , or any other plants or portions ofplants that can yield plant, for example , the fruit peel, after treatment with the
any embodiment of the plant extract compositions described ammonium oxalate and oxalic acid solution , can be isolated
herein . In some embodiments , the plant matter can be a fruit by filtration and dried ( e . g ., air - dried under ambient condi
( e . g ., a tomato , cranberry , or grape) and the cutin - containing tions, oven - dried or freeze- dried ) to remove any residual
portion can be a peel of the fruit ( e . g ., a tomato peel or 60 water.
cranberry skin or grape skin ) such that the boiling can at In some embodiments , the cutin can optionally be treated
least partially separate the peel from the fruit. The fruit can with an enzyme, at 108 . For example , the cutin can be
be washed to remove surface residue, waxes , or otherwise treated with an enzyme such as a carbohydrate -hydrolyzing
debris before operation 102 . Furthermore , the fruit can be enzyme to digest or otherwise remove carbohydrates ( e. g .,
cut into halves, quarters , or small pieces or ground to finer 65 cellulose or pectin ) attached to or embedded within the
pieces and then boiled until the peels or otherwise skins are cutin . Such enzymes can include, for example , naturally
visibly separated from the fruit pulp . derived or synthetic cellulases, pectinases, and hemicellu
 US 9 ,743,679 B2
15 16
lases . The enzymatic degradation can be used before , after, monomeric and/ or oligomeric fatty cutin esters and / or acids
or otherwise in place of operation 106 to obtain the cutin (or combinations thereof), at 112 . The metal alkoxide can
from the non - cutin -containing portion . In some embodi- include, for example , sodium methoxide , sodium ethoxide ,
ments, the reverse process may be employed wherein the sodium iso -propoxide , sodium n -propoxide , sodium iso
cutin can be treated with an enzyme that can at least partially 5 butoxide, sodium n -butoxide, potassium methoxide, potas
depolymerize the cutin to yield cutin oligomers or any sium ethoxide, potassium iso -propoxide , potassium
combination of cutin oligomers and cutin monomers to leave n -propoxide, potassium iso -butoxide , or potassium n -butox
behind the non -cutin -containing components , which could ide . The metal hydroxide can include , for example , Group I
be filtered out or otherwise separated . Such enzymes can or Group II metal hydroxides, such as lithium , sodium ,
include, for example , cutinases, esterases , or lipases . 10 potassium , calcium , rubidium , or cesium hydroxide. Also
Optionally, the cutin is refluxed in at least one suitable included are precursors or compounds that will generate
solvent ( e . g ., chloroform and / or methanol) to remove alkoxide or hydroxide in a suitable reaction medium ( such as
soluble waxes or polar impurities from the cutin , at 110 . For neat metals ( e . g ., sodium metal) or oxides in methanol, or
example , the cutin can be refluxed only in chloroform , ammonia in water). Refluxing of the cutin in the presence of
refluxed in chloroform followed by refluxing in methanol, 15 the metal alkoxide or metal hydroxide can be performed at
refluxed only in methanol, or refluxed in a mixture of any suitable temperature and for any suitable length of time
chloroform and methanol, or any other suitable solvent(s ) such as, for example , at about 65 degrees Celsius for about
( or combinations thereof) in which the wax and /or polar 24 hours . In some embodiments , refluxing of the cutin in the
components are soluble . In some embodiments , the cutin can presence of the metal alkoxide to obtain fatty esters (or
be refluxed in a dilute solution of a strong base ( e . g ., 20 oligomers thereof) can be carried out in anhydrous reagents ,
potassium hydroxide in water or in alcoholic solvent), or a anhydrous solvents, a closed atmosphere , and /or a nitrog
solution of a moderately strong or weak base ( e . g ., potas - enous atmosphere in order to favor ester formation over
sium carbonate in water or in alcoholic solvent) to remove saponification and acid formation . In some embodiments ,
soluble pigmented impurities. Alternatively , removal of the temperature and /or the refluxing time can be such that
residual waxes and remaining soluble components can be 25 the cutin is only partially depolymerized to yield a prede
achieved using supercritical CO , or supercritical H , O . The termined combination of oligomers and monomers . In some
refluxing can be performed at any suitable temperature and embodiments, the temperature and/ or the refluxing time can
for any suitable length of time. For example , in some be adjusted such that the cutin is mostly depolymerized by
embodiments , the cutin can be refluxed in chloroform at the metal alkoxide or metal hydroxide into a plurality of
about 60 -65 degrees Celsius for about 24 - 36 hours to 30 monomeric cutin -derived fatty esters or fatty acids, respec
remove any wax and / or non -polar compounds embedded in tively . In some embodiments , the refluxing in the metal
the cutin . This can be followed by refluxing in methanol at alkoxide or metal hydroxide can be performed in a mixture
65 - 70 degrees Celsius for about 4 - 12 hours , for example, to of the metal alkoxide or metal hydroxide and a solvent, for
remove any polar organic components (e .g ., flavonoids and example , methanol, ethanol, hexane, toluene , etc . In the case
flavonoid glycosides ) present in the cutin . The completion of 35 of cutin depolymerization with metal hydroxide, water can
the operation can be determined by the clarity of solvents . also be used as a solvent to obtain cutin - derived fatty acids .
For example , the process can be monitored with instrumen - In some embodiments, the solvent can include methanol.
tation (e . g ., NMR , GC -MS, React -IR , FTIR , spectropho - The concentration ofmetal alkoxide, solvent, and/ or the pH
tometry , etc .) configured to analyze the clarity of the sol- of the solution can , for example , facilitate the preservation
vents and can continue until a predetermined clarity is 40 of the depolymerized cutin components in monomeric ester
achieved . Each of the chloroform and/or methanol extrac - or acid form , which can prevent oligomerization or repo
tion processes can be performed in any apparatus capable of lymerization of the liberated cutin monomers included in the
refluxing ( i. e ., recirculating and / or recycling ) the solvents intermediate extract . In some embodiments , the depolymer
such as, for example , a reaction flask equipped with a ization and transesterification of the cutin to obtain esterified
condenser, a Soxhlet apparatus, a Kumagawa extractor , an 45 cutin monomers can be performed by refluxing in a solution
ultrasound assisted extractor, a robot automated extractor, or of methanol containing boron trifluoride and/ or a metal
any other suitable extraction apparatus. Such an apparatus alkoxide ( e . g ., sodium methoxide ) .
can , for example , reduce the amount of solvent used in the In some embodiments , after depolymerization , any unde
extraction process . Any other solvent or combinations polymerized solids can be removed from the reaction mix
thereof (i . e ., a binary or ternary mixture ) can be used to wash 50 ture by, for example , filtration or centrifugation . The filtrate
out undesired impurities . Suitable solvents can include , for containing solubilized cutin monomers is then subjected to
example , diethyl ether, dichloromethane , hexane, petroleum pH adjustment. The pH of the intermediate extract obtained
ether, ethyl acetate , acetone , isopropanol, ethanol, acetoni- after depolymerization with metal alkoxide is adjusted to be
trile , supercritical carbon dioxide , supercritical water,water , in the range of about 6 .5 to 9 .0 at 114 , inclusive of all ranges
and mixtures thereof. In some embodiments , multiple 55 and values therebetween . In some embodiments , the pH of
extraction steps in one or more solvents can also be per - the intermediate extract obtained after depolymerization
formed . In some embodiments, intermediate enzymatic with metal hydroxide is adjusted to be in the range of about
treatment steps can also be performed between the solvent 1 .0 to about 6 .5 , at 114 . In some embodiments, the pH
extraction processes, for example, to liberate undesired adjustment can be directly carried out in the depolymeriza
compounds from the cutin . The solution obtained after 60 tion reaction medium (e . g ., methanolic KOH ) . Optionally ,
operation 110 can include a relatively pure sample of the the reaction solvent can be removed , for example , by
cutin included in the portion of the plant along with any evaporation to obtain a crude , concentrated extract that can
residually attached or embedded polysaccharides (e.g ., cel then be reconstituted in a solvent different from the depo
lulose ), plant metabolites (e . g ., flavonoids ), and /or proteins. lymerization reaction ( e. g ., evaporation of methanol and
The cutin is then heated in a solution of metal alkoxide or 65 reconstitution with water ) prior to pH adjustment. For
metal hydroxide to at least partially depolymerize the cutin example , the pH can be adjusted to be in the range of 1 .5 to
and obtain an intermediate extract including a plurality of about 6 .5 , about 2 to about 6 .5 , about 2 .5 to about 6 .5 , about
 US 9 ,743,679 B2
17 18
3 to about 6 .5 , about 3 .5 to about 6 .5 , about 4 to about 6 .5 , 80 - 85 degrees Celsius for about 24 - 36 hours to solubilize
about 4 .5 to about 6 .5 , about 5 to about 6 .5 , or about 5 .5 to monomeric components , leaving behind a partly soluble
about 6 .5 , inclusive of all ranges and values therebetween . and /or insoluble residue of oligomeric , phenolic , and col
The adjusting of the pH can be performed using any suitable ored impurities. The completion of the operation can be
acid such as , for example , citric acid , acetic acid , hydro - 5 determined by the clarity of the monomer -enriched solution .
chloric acid , sulfuric acid , phosphoric acid , hydrobromic For example , the process can be monitored with instrumen
acid , ascorbic acid , tartaric acid , formic acid , gluconic acid , tation ( e. g ., NMR, GC -MS, React -IR , FTIR , spectropho
lactic acid , oxalic acid , boric acid , any other suitable acid , tometry, etc .) configured to analyze the clarity of the sol
any other suitable pH adjusting agent, or a combination vents and can continue until a predetermined clarity is
thereof. In some embodiments , the adjusting of the pH can 10 achieved . The post -depolymerization extraction processes
be sufficient to protonate a secondary and/ or a primary can be performed in any apparatus capable of refluxing (i. e.,
hydroxyl group of theplurality of esterified cutin monomers, recirculating and / or recycling ) the solvents such as , for
but insufficient to protonate a carboxylate group present as example , a Soxhlet apparatus , a Kumagawa extractor, an
a result ofdepolymerization at alkaline pHs. For example , in ultrasound assisted extractor , a robot automated extractor, or
some embodiments , the liberated cutin monomers can 15 any other suitable extraction apparatus. Such an apparatus
include esters of a fatty acid , a hydroxy fatty acid , a can , for example , reduce the amount of solvent used in the
dihydroxy fatty acid , and /or a trihydroxy fatty acid . In some extraction process. Any other solvent or combinations
embodiments , the liberated cutin monomers can include a thereof ( i.e ., a binary or ternary mixture ) can be used to wash
fatty acid , a hydroxy fatty acid , a dihydroxy fatty acid , out undesired impurities. Suitable solvents can include , for
and /or a trihydroxy fatty acid . In some embodiments , the 20 example, diethyl ether, dichloromethane, hexane, petroleum
liberated cutin monomers can include the salts of a fatty ether, ethyl acetate , acetone , isopropanol , ethanol, acetoni
acid , a hydroxy fatty acid , a dihydroxy fatty acid , and/ or a trile , supercritical carbon dioxide, supercritical water, water,
trihydroxy fatty acid . Upon lowering the pH of the inter and mixtures thereof. In some embodiments , multiple
mediate extract that includes one or more of these hydroxy extraction steps in one or more solvents can also be per
fatty esters or acids, the secondary hydroxyl groups are 25 formed . In some embodiments, intermediate enzymatic
protonated first (included in dihydroxy fatty acids and treatment steps can also be performed between the solvent
trihydroxy fatty acids ), followed by the primary hydroxyl extraction processes, for example , to liberate undesired
groups (included in trihydroxy fatty acids, dihydroxy fatty compounds from the crude extract. The resulting solution
acids , and monohydroxy fatty acids ). Any further lowering can be resolidified . The resolidification can include, for
of the pH of the intermediate extract obtained from hydrox - 30 example , precipitation of the cutin -derived fatty esters or
ide-mediated cutin depolymerization can also protonate the fatty acids from the solution by evaporation of the solvent( s)
carboxylate group of the obtained cutin monomers , which or addition of an agent that induces precipitation (such as
will modify solubility of the monomers in a polar reaction salts, unfavorable solvents , or co -precipitation agents ). The
medium . Therefore , the pH can be adjusted such that the plant extract obtained using the method 100 can be substan
degree of protonation can be controlled so as to tune 35 tially free of accompanying plant-derived compounds ( e . g .,
downstream methods ofmonomer isolation . For example , in proteins, polysaccharides , phenols , lignans , aromatic acids ,
some embodiments, the pH of a solution of the intermediate terpenoids, flavonoids, carotenoids, alkaloids , alcohols,
extract can be adjusted so as to preferentially promote alkanes, and aldehydes ).
precipitation of the fatty acid cutin monomers (and oligom - In some embodiments, the plant extract composition
ers ) from the solution and facilitate recovery of the mono- 40 obtained using the method 100 or any other method
mers ( and oligomers ) by, for example, filtration or centrifu - described herein , can include any suitable combination of
gation . In this example , the pH of a solution of the cutin monomers including, for example , C16 - w -hydroxy
intermediate extract in water can be adjusted to be about pH fatty acids, and /or C16 hydroxy fatty acids, and /or C16
1 -3 such that fatty cutin monomers ( and oligomers ) precipi- dihydroxy fatty acids, and /or C16 trihydroxy fatty acids,
tate from the solution and the solids can be recovered by 45 and / or C16 - w -hydroxy -oxo fatty acids, and / or Cis - w -hy
filtration or centrifugation . droxy fatty acids, and /or Cie hydroxy fatty acids , and/ or Cis
The intermediate extract is resolidified at 116 . The reso dihydroxy fatty acids, and /or C18 trihydroxy fatty acids,
lidification can include , for example , precipitation of the and/ or C18 - w -hydroxy - oxo fatty acids, and / or Cis -w -hy
cutin -derived fatty esters or fatty acids from the solution by d roxy -epoxy fatty acids , and residual fatty acids, and /or
evaporation of the solvent( s ) or addition of an agent that 50 esters of said families of C18 hydroxy fatty acids and / or Cis
induces precipitation (such as salts , acids, unfavorable sol hydroxy fatty acids and residual fatty acids . For example , in
vents , or co -precipitation agents ) . some embodiments , the plant extract composition can
Optionally , the crude extract can be purified by washing include a first fraction including C , hydroxy fatty acids or
of the precipitated extract with a selective solvent (e .g ., C18 hydroxy fatty esters in the range of about 15 wt % to
acetonitrile ) or combinations of selective solvents , at 118 , 55 about 100 wt % , a second fraction including Cis hydroxy
and / or recrystallization of the extracted cutin monomers , at fatty acids or C , hydroxy fatty esters in the range of 0 wt
120, and /or chromatographic separation and isolation of the % to about 90 wt % , and a third fraction including residual
cutin monomers, at 122. fatty acids or residual fatty esters in the range of about 0 wt
In some embodiments , the crude extract is refluxed in at % to about 75 wt % . In some embodiments , the plant extract
least one suitable solvent or ( e . g ., acetonitrile , acetone, etc .) 60 ?omposition can include a first fraction including C16
or combinations thereofto remove oligomeric , phenolic , and hydroxy fatty acids or C hydroxy fatty esters in the range
colored impurities, at 118 . Alternatively , removal of oligo - of about 50 wt % to about 100 wt % , a second fraction
meric , phenolic, and colored impurities can be achieved including C18 hydroxy fatty acids or C18 hydroxy fatty esters
using supercritical CO , or supercritical H , O . The refluxing in the range of 0 wt % to about 35 wt % , and a third fraction
can be performed at any suitable temperature and for any 65 including residual fatty acids or residual fatty esters in the
suitable length of time. For example , in some embodiments , range of about 0 wt % to about 23 wt % . In some embodi
the crude extract can be refluxed in acetonitrile at about ments , the combination and relative concentration of the C16
 US 9,743 ,679 B2
20
hydroxy fatty acids (or C18 hydroxy fatty esters ), the C18 clementine leaf, clementine fruit, mandarin leaf, mandarin
hydroxy fatty acids (or C18 hydroxy fatty esters), and the fruit, pea seeds, grapefruit peel, grapefruit leaf, grapefruit
residual fatty acids ( or residual fatty esters) can be con - seed , papaya peel, cherry fruits , cranberry skins, coffee
trolled by adjusting the process parameters ( e . g ., depolymer - cherries , grass clippings , or any other plants or portions of
ization conditions, and choice of post- depolymerization 5 plants that can yield any embodiment of the plant extract
extraction , resolidification , wash , and purification condi compositions described herein . For example, in some
tions) included in the method 100, or by blending the embodiments, the plant extract can be a tomato peel extract.
extracts from multiple plant sources . The Cohydroxy fatty
acids can include, but are not limitedTheto , 16at hydroxy
least onelany
of Infor such embodiments , the tomato peel extract can include ,
example, about 65 wt % to about 85 wt % of a
16 -hydroxyhexadecanoic acid , 9 ( 10 ), 16 -dihydroxyhexade - 10 9 ( 10 ),16 -dihydroxyhexadecanoic acid ( or the ester thereof),
canoic acid , 9 , 10 , 16 -trihydroxyhexadecanoic acid , and about 3 wt % to about 7 wt % of a 16 - hydroxyhexadecanoic
16 -hydroxy - 10 - oxohexadecanoic acid . The C16 hydroxy acid
fatty esters can include, but are not limited to , at least one of a 9 , 10( or, 18the
-
ester thereof), about 1 wt % to about 4 wt % of
trihydroxyoctadecanoic acid (or the ester thereof),
methyl 16 -hydroxyhexadecanoate , methyl 9 ( 10 ), 16 -dihy
droxyhexadecanoate, methyl 9 ,10 ,16 -trihydroxyhexade - 155 the and about 20 wt % to about 25 wt % residual fatty acids (or
esters thereof) . For example , the tomato peel extract
canoate , and methyl 16 -hydroxy - 10 - oxohexadecanoate . The
C , hydroxy fatty acids can include at least one of 18 -hy- composition can include about 66 wt % , about 67 wt % ,
droxyoctadecanoic acid , 9 (10 ), 18 -dihydroxyoctadecanoic about 68 wt % , about 69 wt % , about 70 wt % , about 71 wt
acid , 9 , 10 , 18 -trihydroxyoctadecanoic acid , and 18 -hydroxy % , about 72 wt % , about 73 wt % , about 74 wt % , about 75
9 , 10 - epoxy - octadecanoic acid . The C , hydroxy fatty esters 20 wt % , about 76 wt % , about 77 wt % , about 78 wt % , about
can include at least one ofmethyl 18 -hydroxyoctadecanoate , 79 wt % , about 80 wt % , about 81 wt % , about 82 wt % ,
methyl 9 ( 10 ), 18 - dihydroxyoctadecanoate, methyl 9 , 10 , 18about 83 wt % or about 84 wt % of the 9 ( 10 ), 16 -dihydroxy
trihydroxyoctadecanoate, and methyl 18 -hydroxy - 9, 10 -ep hexadecanoic acid (or the ester thereof), inclusive of all
oxy -octadecanoate . ranges therebetween . Moreover, the tomato peel extract
For example , in some embodiments, the first fraction can 25 composition can include about 4 wt % , about 5 wt % , or
include about 50 wt % to about 100 wt % of a 9 ( 10 ), 16 - about 6 wt % of the 16 - hydroxyhexadecanoic acid (or the
dihydroxyhexadecanoic acid (or the ester thereof), about 2 ester thereof), inclusive of all ranges therebetween . The
wt % to about 27 wt % of a 16 -hydroxyhexadecanoic acid 9,10 ,18 - trihydroxyoctadecanoic acid (or the ester thereof)
(or the ester thereof ), and I wt % to about 35 wt % of a can be about 2 wt % , or about 3 wt % , inclusive of all ranges
16 -hydroxy - 10 -oxohexadecanoic acid (or the ester thereof) . 30 therebetween , and the residual fatty acids (or the esters
In some embodiments, the second fraction can include at thereof) can be about 21 wt % , about 22 wt % , about 23 wt
least 0 wt % to about 100 wt % of a 9, 10 , 18 -trihydroxyoc - % , or about 24 wt % , inclusive of all ranges therebetween .
tadecanoic acid ( or the ester thereof). In some embodiments , In some embodiments , the plant or portion of the plant
the second fraction can also include ( wt % to about 8 wt % selected for obtaining the plant extract composition can
of a 9 , 10 , 18 - trihydroxyoctadecanoic acid ( or the ester 35 naturally include higher proportions of the Co and/ or Cie
thereof), at least 0 wt % to about 9 wt % of a w -hydroxyoc - hydroxy fatty acids (or the esters thereof). For example , the
tadecanoic acid (or the ester thereof), and / or at least 0 wt % portions of the plant selected for obtaining the plant extract
to about 33 wt % of a w -hydroxy - 9 , 10 - epoxy -octadecanoic compositions described herein can include a higher propor
acid ( or the ester thereof). The residual fatty acids ( and /or tion of the 9 ( 10 ), 16 - dihydroxyhexadecanoic acid (or the
fatty esters ) can include any other fatty acids (or fatty esters ) 40 ester thereof) such that the plant extract composition
excluding C16 and C18 hydroxy fatty acids ( or fatty esters ) in obtained from the portion of the plant has a concentration of
any proportion or combination such as , for example , hexa- the 9( 10 ), 16 -dihydroxyhexadecanoic acid (or the ester
decanoic acid , dicarboxylic acids, 9 , 10 - dihydroxyhexade thereof ) as described herein (e .g., about 50 wt % to about
cane- 1 , 16 -dioic acid , 9 , 12 -octadecadienoic acid , trans - 9 . 100 wt % of the first fraction , i.e ., theCohydroxy fatty acid
octadecenoic acid , cis - 9 -octadecenoic acid , octadecanoic 45 ( or the ester thereof) fraction ). In some embodiments , vari
acid , tetradecanoic acid , pentadecanoic acid , heptadecanoic o us combinations of different plant species and/or portions
acid , 22 -hydroxydocosanoic acid , dihydroxyhexadecane , of plants (i. e., plant organs) can be selected to adjust the
methyl- n , w - dihydroxyoctadecanoate , coumaric acid , ferulic relative concentrations of the Co and / or Cie hydroxy fatty
acid , other Cloor C18 fatty acids, esters thereof, or any other acids (or the esters thereof) obtained in the plant extract
<C18 fatty acid (or esters thereof) included in a portion of a 50 compositions described herein . Dihydroxy fatty acids (e. g.,
plant from which the plant extract composition is prepared . 9 ( 10 ), 16 - dihydroxyhexadecanoic acid ) (or the esters
Furthermore, the plant extract compositions described thereof ) and trihydroxy fatty acids ( e. g ., 9 , 10 , 18 - trihy
herein can be substantially free from accompanying plant- droxyoctadecanoic acid ) ( or the esters thereof) can form
derived compounds (e.g ., proteins , polysaccharides, phe- tightly bound networks with high crosslink density and low
nols, lignans, aromatic acids, terpenoids, flavonoids, caro - 55 permeability relative to monohydroxy fatty acids (or the
tenoids, alkaloids, alcohols, alkanes, and aldehydes ). esters thereof). Thus , the plant extract compositions
The relative concentrations of the various cutin monomers described herein can include a relative proportion of the
in the extract can be dependent on the source plantmatter or dihydroxy fatty acids ( or the esters thereof ) and the trihy
otherwise agricultural product used to prepare the plant droxy fatty acids (or the esters thereof), which can allow the
extract . In some embodiments , a plant or a portion of a plant 60 plant extract compositions to be used in agricultural coating
can be selected such that the composition of the cutin formulations. Such coatings can be used for coating various
monomers forming the cuticular layer in the portion of the portions of the plants such as, for example , plant stems,
plant yields the plant extract compositions described herein . shoots , flowers, fruits, leaves , seeds, roots , etc ., and pre
In some embodiments , the plant matter can include agricul- venting moisture loss, oxygen diffusion (which leads to
tural waste products such as , for example , tomato peel, grape 65 oxidation ), biotic stressors, surface scratching or damage,
skins , apple peel, pepper peel, lemon peel, lemon leaf, lime etc . Such coatings can also be used to delay ripening. Since
peel, lime leaf, orange peel, orange leaf, orange fruit, the plant extract compositions are obtained from natural
 US 9 ,743,679 B2
21
sources, the plant extract compositions can be safe for hexadecanoic acid from tomato skins can be converted to
human consumption . Furthermore, the plant extract compo - hexadecane -1,7 (8 ), 16 - triol with LiAlH4.
sitions can be biodegradable . Functionalization of Compounds
In some embodiments , a method of protecting a portion of In some embodiments, functionalizing the cutin - derived
a plant from environmental and biotic stressors can include 5 monomers and /or oligomers includes carrying out one or
coating the portion of the plant with a coating including a more of the following reactions:
plant extract composition . The plant extract composition can
include at least about 50 wt % to about 100 wt % of C16
hydroxy fatty acids (and / or esters thereof), at least 0 wt %
to about 35 wt % of Cis hydroxy fatty acids ( and / or esters 10
thereof) , and at least about 0 wt % to about 23 wt % of
residual fatty acids ( and/ or esters thereof). The plant extract
can be substantially free of accompanying plant-derived
OH
*** R - OH
R DR
Ester
compounds ( e .g ., proteins, polysaccharides, phenols, lign
ans, aromatic acids , terpenoids , flavonoids, carotenoids , 15
alkaloids, alcohols , alkanes , and aldehydes ). In some
embodiments, theC hydroxy fatty acids (or esters thereof)
can include about 50 wt % to about 100 wt % of a
9 ( 10 ), 16 -dihydroxyhexadecanoic acid (or an ester thereof),
R
in line without
OH
LiAlH .

where R represents the fatty acid radical.

R
Alcohol
OH

about 2 wt % to about 27 wt % of a 16 -hydroxyhexadecanoic In some embodiments, the monomers and /or oligomers
acid (or an ester thereof ), and I wt % to about 35 wt % of can be glycerated to form monoacylglyceride ( e. g .,
a 16 -hydroxy - 10 -oxohexadecanoic acid ( or an ester thereof). 1 -monoacylglyceride and /or 2 -monoacylglyceride) mono
In some embodiments , the Cie hydroxy fatty acids (or esters mers and /or oligomers . For example , the monomers and/or
thereof) can include 0 wt % to about 100 wt % of a oligomers can be functionalized or modified to one or more
9 , 10 , 18 -trihydroxyoctadecanoic acid (or an ester thereof), 0
wt % to about 8 wt % of a 9 (10 ). 18 -dihydroxvoctadecanoic 25 compounds of Formula 1:
acid (or an ester thereof), O wt % to about 9 wt % of a
w -hydroxyoctadecanoic acid ( or an ester thereof), and /or 0
wt % to about 33 wt % of a w -hydroxy - 9 , 10 -epoxy - octade (Formula I)
canoic acid ( or an ester thereof) . In some embodiments , the / R RS 1 R4 R3 0
residual fatty acids (and /or fatty esters) can include for 30 R13 R12
example, hexadecanoic acid , dicarboxylic acids, 9 , 10 - dihy
droxyhexadecane - 1, 16 -dioic acid , 9 ,12 -octadecadienoic
acid , trans - 9 - octadecenoic acid , cis - 9 -octadecenoic acid ,
RULY
RIO R RO RS R2R
octadecanoic acid , tetradecanoic acid , pentadecanoic acid ,
heptadecanoic acid , 22 -hydroxydocosanoic acid , dihydroxy- 3
hexadecane, methyl-n ,w -dihydroxyoctadecanoate , coumaric wherein :
acid , ferulic acid , other C16 or C18 fatty acids, esters thereof, R is a 2- glyceryl optionally substituted with one or more
or any other < C19 fatty acid (or esters thereof). The plant C , -C6 alkyl or hydroxy ;
extract composition included in the coating can be extracted R ?, R ?, RS, R®, R ", R19 , R1, R12 and R13 are each
from a suitable plant or portion of a plant such as , for independently — H , OR14, - NR14R15 , SR14, halogen ,
example , tomato skin , grape skin , lemon peel, lemon leaf, * C -C alkyl, C2- C . alkenyl, C2- C . alkynyl, - C3- C ,
lime peel, lime leaf, orange peel, orange leaf, orange fruit, cycloalkyl, C . - C . aryl, or 5 to 10 -membered ring het
clementine leaf, clementine fruit, pea seed, grapefruit leaf, eroaryl, wherein each - C . - C . alkyl, C . - C . alkenyl,
cranberry skins, and cherry fruit. Furthermore , the plant C2- C . alkynyl, Cz-C , cycloalkyl, Co -C10 aryl, or 5 to
extract composition included in the coating can be extracted 10 -membered ring heteroaryl is optionally substituted with
using method 100 or any methods described herein . 45 OR14, NR14R15 , SR14 , or halogen ;
In some embodiments , the cutin - derived monomers and / R3, R4, R7 and R8 are each independently - H , - OR14,
or oligomers resulting from the depolymerization of cutin
are functionalized ( e . g ., modified , such as by conversion of NR14R15 , SR14 , halogen, C .-C . alkyl, C2-C6 alk
the carboxylic acid to an ester or alcohol) prior to being enyl, C2- C6 alkynyl, C3 -C , cycloalkyl, C8 -C10 aryl,
applied to the surface of an agricultural product to form a 50 or 5 to 10 -membered ring heteroaryl wherein each - C1- C6
protective coating. Functionalizing the monomers and /or alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C3- C ,
oligomers can in some cases impart desirable properties cycloalkyl, C .-C10 aryl , or 5 to 10 -membered ring het
such as modified solubility or hydrophobicity, which can eroaryl is optionally substituted with OR14, - NR14R15,
result in improved properties of the subsequently formed - SR14, or halogen ; or
protective coatings . Fatty acids can be converted to esters by 55 R3 R and
and R4
R ' can
can combine
combine with the carbon
with the carbon atoms
atoms toto which
which
several chemical transformations, for example via Fischer they are attached to form a 3 to 6 -membered ring, such as a
esterification with an alcohol and a catalytic amount of acid cyclic ether ;
( e.g . methanol, ethanol, butanol, etc .) to form the corre R7 and R8 can combine with the carbon atoms to which
sponding ester (methyl ester, ethyl ester, butyl ester ). For they are attached to form a 3 to 6 -membered ring, such as a
example , 9 ( 10 ), 16 - dihydroxyhexadecanoic acid from cyclic ether;
tomato skins can be converted to ethyl 9 ( 10 ), 16 - dihydroxy - 60 R14 and R15 are each independently — H , - C , - C alkyl,
hexadecanoate by adding excess ethanol and a catalytic C -Coalkenyl, or C -Co alkynyl;
amount of acid . As another example, 9 ( 10 ), 16 - dihydroxy - the symbol represents an optionally single or cis or
hexadecanoic acid from tomato skins can be converted to trans double bond ;
methyl 9 ( 10 ), 16 -dihydroxyhexadecanoate by adding excess n is an integer between 0 and 8 ;
methanol and a catalytic amount of acid . Alternatively, fatty 65 m is an integer between 0 and 3 ;
acids could be converted to alcohols by using a reduction is an integer between 0 and 5 ; and
agent such as LiAlH4. For example, 9 ( 10 ), 16 -dihydroxy r is an integer between 0 and 8 .
 US 9 ,743,679 B2
23 24
In some implementations , the monomers and /or oligom The modified / functionalized (e.g., esterized or glycer
ers are modified to form compounds of Formula I and/ or ated ) monomers and/or oligomers that make up the resulting
compounds of Formula II: plant extract composition can then be applied to the surface
of an agricultural product to form a protective coating. As
5 seen in FIGS. 2 - 6 and their associated descriptions below ,
(Formula II) protective coatings formed from compositions that comprise
/ R7 R8 1 R4 R3 monoacylglycerides ( e . g ., compounds of Formula I and / or
R13 0
R !3 R12 Formula II , as described above) have been found to reduce
the rate of post-harvestmass loss in a variety of agricultural
RIX xmla in o 10 products , and in some cases to reduce the rate of post
R10 R9 RO RS R2 R1 harvest molding and /or spoilage.
The compounds of Formula I and /or Formula II may be
prepared by methods known in the art of organic synthesis
wherein : as set forth in part by the following synthetic schemes and
R ' is a 1- glyceryl optionally substituted with one or more 15 examples . In the schemes described below , it is well under
CZ -C6 alkyl or hydroxy; stood that protecting groups for sensitive or reactive groups
R1, R2, RS, R " , R9, R19, R1, R12 and R13 are each are employed where necessary in accordance with general
independently - H , - OR14, NR14R15, - SR14, halogen , principles or chemistry . Protective groups are manipulated
C .- C . alkyl, C . -C . alkenyl , C2- Co alkynyl, C3- C , according to standard methods of organic synthesis ( T . W .
cycloalkyl, C . - C . aryl, or 5 to 10 -membered ring het - Greene and P . G . M . Wuts , “ Protective Groups in Organic
eroaryl, wherein each - C ,- C6 alkyl, C2-C6 alkenyl, 20 Synthesis” , Third edition , Wiley, New York 1999 ). These
- C2- C . alkynyl, - C3- C , cycloalkyl, C .-C1o aryl, or 5 to groups are removed at a convenient stage of the compound
10 -membered ring heteroaryl is optionally substituted with synthesis using methods that are readily apparent to those
OR14 , NR14R15, SR14, or halogen ; skilled in the art. The selection processes , as well as the
R3, R4, R7 and R8 are each independently — H , OR14 , reaction conditions and order of their execution , shall be
- NR14R15 , - SR14, halogen , C , -C6 alkyl, C2- C6 alk - 25 consistent with the preparation of compounds of Formula I.
enyl, C2- C . alkynyl, C3- C , cycloalkyl, C6-C10 aryl, Those skilled in the art will recognize if a stereocenter
or 5 to 10 -membered ring heteroaryl wherein each - C -Co exists in the compounds of Formula I and /or Formula II .
alkyl, C2 -C6 alkenyl, C2 -C6 alkynyl, C3- C , Accordingly , the present disclosure includes both possible
cycloalkyl, C8 -C10 aryl, or 5 to 10 -membered ring het stereoisomers (unless specified in the synthesis ) and
eroaryl is optionally substituted with OR14, - NR14RS, 30 includes not only racemic compounds but the individual
- SR14 , or halogen ; or enantiomers and /or diastereomers as well. When a com
R3 and R can combine with the carbon atoms to which pound is desired as a single enantiomer or diastereomer, it
they are attached to form a 3 to 6 -membered ring , such as a may be obtained by stereospecific synthesis or by resolution
cyclic ether; of the final product or any convenient intermediate . Reso
R7 and R8 can combine with the carbon atoms to which w lution of the final product, an intermediate , or a starting
they are attached to form a 3 to 6 -membered ring , such as a > material may be affected by any suitable method known in
cyclic ether ; the art. See , for example , " Stereochemistry of Organic
R14 and R15 are each independently - H , - C , -Co alkyl, Compounds” by E . L . Eliel , S . H . Wilen , and L . N . Mander
- C -C , alkenyl, or — C -C6 alkynyl; (Wiley -Interscience , 1994 ).
the symbol represents an optionally single or cis or The compounds described herein may be made from
trans double bond ; 40 commercially available starting materials or synthesized
n is an integer between 0 and 8 ; using known organic , inorganic , and /or enzymatic pro
m is an integer between 0 and 3 ; cesses . In some embodiments , a compound of Formula I can
q is an integer between 0 and 5 ; and be prepared from a corresponding acid or ester according to
ris an integer between 0 and 8 . Scheme 1.
Scheme 1. Preparation of 2- glycero - 10 ,16 -dihydroxy Palmate from 10 ,16 -dihydroxy Palmitic Acid

Hom ??

OH
o ?? eren,met"
TBS - CI
Imidazole, DMF

150mm
TBSO
OTBS
odore ??
OH
DMAP, DCC , DCM
 25
25 US9,743,679B2
US 9, 743,679 B2
26

TBSO

mwego OTBS
-continued
20 % aq HF
ACN , 0° C .

HO

OH

HO
?? ???? ? ? 1 atm H2
Pd(OH ) / C , THF

OH

??

25
As shown in Scheme 1, compounds of Formula I can be suitable protecting group (e. g., -TBS), whereas in some
prepared by protecting any hydroxy groups that may be embodiments the hydroxy groups on the fatty acid are not
present in the acid . As shown above in Scheme 1, hydroxy protected .
groups can be protected with a - TBS ( tert-butyldimethylsi- FIGS. 2 -6 illustrate the effects of coating a variety of
lyl ) protecting group . Esterification of the protected acid 30 agricultural products with plant extract compositions
with an appropriately protected glycerol derivative (e .g., described herein . The coatings formed on the agricultural
2 -phenyl- 1 , 3 - dioxan - 5 -ol) can be accomplished with the aid products shown in FIGS. 2 , 3 , 5 , and 6 were each formed
of DMAP and DCC . Deprotection of the silyl protecting from compositions that included a mixture of 2 -monoacyl
groups can be accomplished with an appropriate agent such glyceride compounds (i.e., compounds of Formula I) and an
as hydrofluoric acid . Finally , the glycerol group can be 35 additive that included 1 -monoacylglyceride compounds
deprotected by, for instance , hydrogenation . (i.e ., compounds of Formula II), wherein a mass ratio of the
A skilled artisan will understand the chemical synthesis additive to the compound of Formula I was in a range of 0 . 1
procedures set forth herein can be adjusted as necessary . For to 1 . To form the coatings , the solid mixtures of the
instance , other protecting groups can be used to protect, e .g ., compositions were first fully dissolved in ethanol at a
the alcohol groups as will be understood by one of skill in 40 concentration of 10 mg/mL to form a solution . The solution
the art. was then applied to the agricultural products either by
In some embodiments, the cutin -derived monomers, oli- spraying or dip coating, as detailed for each of the cases
gomers , and combinations thereof comprise fatty acids, and below . The agricultural products were then dried on drying
the converting step comprises esterifying the fatty acids . In racks under ambient conditions ( temperature in the range of
some embodiments , the cutin -derived monomers , oligom - 45 23 - 27° C ., relative humidity in the range of 40 % - 55 % ) until
ers , and combinations thereof comprise esters , and the all of the solvent had evaporated , allowing the coatings to
converting step comprises transesterifying the esters. In form over the substrates. The resultant coatings each had a
some embodiments, the cutin -derived monomers , oligom - thickness in the range of 0 . 1 um to 1 um .
ers , and combinations thereof comprise amides, and the FIG . 2 shows the effects of mass loss over time observed
converting step comprises transesterifying the amides . In 50 in lemons over the course of 3 weeks , both for uncoated
some embodiments , the converting step comprises treating lemons and for lemons that were coated with the composi
the cutin -derived monomers , oligomers, and combinations tions described above . In order to form the coatings , the
thereofwith an appropriate alcohol and base or acid . In some lemons were placed in a bag , and the solution containing the
embodiments, the base is sodium hydroxide , lithium composition was poured into the bag . The bag was then
hydroxide , potassium hydroxide, sodium carbonate , lithium 55 sealed and lightly agitated until the entire surface of each
carbonate , or potassium carbonate . In some embodiments, lemon was wet . The lemonswere then removed from the bag
the acid is hydrochloric , sulfuric , or acetic acid . and allowed to dry on drying racks under ambient room
In some embodiments , compounds of the present disclo - conditions at a temperature in the range of about 23 - 27° C .
sure can be prepared by direct esterification using a suitable and relative humidity in the range of about 40 - 55 % . The
solvent, e . g ., methanol. For instance, an appropriate fatty 60 lemons were held at these same temperature and relative
acid (e .g ., 10 , 16 -dihydroxyhexadecanoic acid ) can be con - humidity conditions for the entire duration of the time they
verted to a corresponding ester ( e.g., methyl 10, 16 -dihy - were tested . 202 is a high resolution photograph of an
droxyhexadecanoate ) by treatment with an excess of a uncoated lemon immediately after being picked (Day 1 ), and
suitable solvent (e . g ., methanol). In some embodiments , the 204 is a high resolution photograph of a lemon immediately
esterification takes place in the presence of a suitable acid 65 after being picked and coated on the same day. 212 and 214
such as sulfuric acid . In some embodiments, the hydroxy are photographs of the uncoated and coated lemons, respec
groups on the fatty acid , if present are protected using a tively , taken on Day 22, 21 days after photographs 202 and
 US 9 ,743,679 B2
27 28
204. In order to better visualize the cross -sectional area loss mass loss for blueberries coated with the 20 mg/mL solution
(which is directly related to mass loss ), an overlay 222 of the was less than 10 % after 5 days, thereby indicating reduced
outline of the untreated lemon on Day 1 is shown around mass loss observed for coated blueberries as compared to
212 , and an overlay 224 of the outline of the untreated lemon uncoated blueberries stored under the same conditions .
on Day 1 is shown around 214 . The coated lemons had a 5 FIG . 6 shows high -resolution photographs of the uncoated
cross sectional area greater than 90 % of their original area blueberries (602 ) and of the blueberries coated with the 10
( e. g., greater than 92 % of their original area ), whereas the mg/mL solution (604 ) at Day 5 . The skins of the uncoated
uncoated lemons had a cross sectional area less than 80 % of blueberries 602 were highly wrinkled as a result of mass loss
their original area , thereby indicating reduced mass loss of the blueberries , whereas the skins of the coated blueber
observed for coated lemons as compared to uncoated lemons 10 ries remained very smooth .
stored under the same conditions . In some embodiments , the depolymerization of the cutin
FIG . 3 shows plots for both coated ( 302 ) and uncoated and /or forming of the plant extract composition is carried
( 304 ) lemons indicating the reduction in cross sectional area out by a first party , while the application of the plant extract
as a function of time over a period of 20 days. Specifically, composition to an agricultural product to form a protective
on each day, high resolution images of each of the lemons 15 coating over the agricultural product is carried out by a
were taken and analyzed with image processing software (as second party different from the first party . For example, a
in FIG . 2 ) to determine the ratio of the cross sectional area manufacturer of the plant extract compositions (i.e ., a first
on the particular day to the initial cross sectional area of the party ) can form the compositions by one or more of the
lemon . As seen in FIG . 3 , after 20 days , the coated lemons methods described herein . Themanufacturer can then sell or
had a cross sectional area greater than 90 % of their original 20 otherwise provide the resulting plant extract composition to
area (e.g., greater than 92 % of their original area ), whereas a second party, for example a farmer, shipper , distributor, or
the uncoated lemonshad a cross sectional area less than 80 % retailer of produce , and the second party can apply the
of their original area , thereby indicating the reduced mass composition to one or more agricultural products to form a
loss observed for coated lemons as compared to uncoated protective coating over the products . Alternatively , the
lemons stored under the same conditions. 25 manufacturer can sell or otherwise provide the resulting
FIG . 4 shows high -resolution photographs of 4 coated and plant extract composition to an intermediary party , for
4 uncoated strawberries over the course of 5 days . The example a wholesaler, who then sells or otherwise provides
coated strawberries were spray coated according to the the plant extract composition to a second party such as a
following procedures . First, the strawberries were placed on farmer, shipper, distributor, or retailer of produce , and the
drying racks . A solution containing the coating composition 30 second party can apply the composition to one or more
was placed in a spray bottle that generated a fine mist spray . agricultural products to form a protective coating over the
The spray head was held approximately six inches from the products .
strawberries, and the strawberries were sprayed and then In some cases where multiple parties are involved , the
allowed to dry on the drying racks. The strawberries were first party may optionally provide instructions or recommen
kept under ambient room conditions at a temperature in the 35 dations about the extract composition , either written or oral,
range of about 23 -27° C . and relative humidity in the range indicating one or more of the following: (i ) that the com
of about 40 - 55 % while they dried and for the entire duration position is intended to be applied to a product for the
of the time they were tested . As seen , the uncoated straw - purpose of coating or protecting the product, to extend the
berries began to exhibit fungal growth and discoloration by life of the product, to reduce spoilage of the product, or to
Day 3 , and were mostly covered in fungus by Day 5 . In 40 modify or improve the aesthetic appearance of the product ;
contrast, the coated strawberries did not exhibit any visible (ii) conditions and /or methods that are suitable for applying
fungal growth by Day 5 and were largely similar in overall the compositions to the surfaces of products ; and/or ( iii)
color and appearance on Day 1 and Day 5 , indicating a potential benefits ( e .g ., extended shelf life , reduced rate of
reduction in molding and spoilage for coated strawberries as mass loss, reduced rate ofmolding and /or spoilage , etc.) that
compared to uncoated strawberries stored under the same 45 can result from the application of the composition to a
conditions.
?? product. While the instructions or recommendations may be
FIG . 5 shows plots of the percent mass loss over the supplied by the first party directly with the plant extract
course of 5 days in uncoated blueberries (502 ), blueberries composition (e . g ., on packaging in which the composition is
coated using a solution of 10 mg/mL of compounds dis - sold or distributed ), the instructions or recommendations
solved in ethanol (504 ), and blueberries coated using a 50 may alternatively be supplied separately, for example on a
solution of 20 mg/mL of compounds dissolved in ethanol website owned or controlled by the first party , or in adver
(506 ). To form the coatings over the blueberries, the fol- tising or marketing material provided by or on behalf of the
lowing dip coating procedures were used . Each blueberry first party .
was gently picked up with a set of tweezers and individually In view of the above , it is recognized that in some cases ,
dipped in the solution for approximately 1 second or less , 55 a party that manufactures a plant extract composition
after which the blueberry was placed on a drying rack and according to one or more methods described herein (i.e ., a
allowed to dry . The blueberries were kept under ambient first party ) may not directly form a coating over a product
room conditions at a temperature in the range of about from the extract composition , but can instead direct ( e . g .,
23 -27° C . and relative humidity in the range of about can instruct or request ) a second party to form a coating over
40 - 55 % while they dried and for the entire duration of the 60 a product from the extract composition . That is , even if the
time they were tested . Mass loss was measured by carefully first party does not coat a product by the methods and
weighing the blueberries each day , where the reported compositions described herein , the first party may still cause
percentmass loss was equal to the ratio of mass reduction to the plant extract composition to be applied to the product to
initial mass . As shown , the percent mass loss for uncoated form a protective coating over the product by providing
blueberries was almost 20 % after 5 days , whereas the 65 instructions or recommendations as described above .
percent mass loss for blueberries coated with the 10 mg/mL Accordingly , as used herein , the act of applying a plant
solution was less than 15 % after 5 days, and the percent extract composition to a product (e .g., a plant or agricultural
 US 9, 743,679 B2
30
product) also includes directing or instructing another party can be added to reduce or increase the melting temperature
to apply the plant extract composition to the product, or or the glass-transition temperature of the coating . In some
causing the plant extract composition to be applied to the embodiments , the additive can be configured to reduce the
product. diffusivity of water vapor, oxygen , CO2, or ethylene through
In some embodiments , the plant extract composition can 5 the coating or enable the coating to absorb more ultra violet
be applied directly to a portion of a plant. In some embodi - (UV ) light, for example to protect the agricultural product
ments, the plant extract composition can be heated to modify (e . g ., any of the products described herein ). In some embodi
the physical and /or chemical properties of the composition ments , the additive can be configured to provide an inten
prior to and/ or during and /or after the application process . In tional odor, for example a fragrance ( e .g ., smell of flowers ,
some embodiments , the plant extract composition can be 10 fruits , plants, freshness , scents , etc .) . In some embodiments ,
dissolved and /or suspended in a solvent, in aqueous solu - the additive can be configured to provide color and can
tions, or in a carrier liquid to form the coating . The solvent include, for example , a dye or a US Food and Drug Admin
can include any polar, non - polar, protic , or aprotic solvents, istration (FDA ) approved color additive. In some embodi
including any combinations thereof. Examples of solvents ments, the additives can include sweeteners , color additives,
that can be used to dissolve the plant extract compositions 15 flavors , spices , flavor enhancers , fat replacers , and compo
described herein include water, methanol, ethanol, isopro - nents of formulations used to replace fats , nutrients , emul
panol, butanol, acetone, ethyl acetate, chloroform , acetoni- sifiers , bulking agents, cleansing agents , stabilizers, emul
trile , tetrahydrofuran , diethyl ether, methyl tert- butyl ether, sion stabilizers, thickeners, flavor or fragrance , an ingredient
any other suitable solvent or a combination thereof. Aqueous of a flavor or fragrance , binders, texturizers, humectants , pH
solutions of such plant extract compositions can be suitable 20 control agents , acidulants , leavening agents , anti- caking
for coating on agricultural products, for example , forming a agents, antifungal agents, antimicrobial agents, antioxidants ,
coating on the agricultural product. In some embodiments , and/ or UV filters . In some embodiments, the coating can
the coatings can be configured to change the surface energy include a photoinitiator, which can initiate crosslinking of
of the agricultural product. Various properties of coatings the coating on exposure to an appropriate light source , for
described herein can be adjusted by tuning the crosslink 25 example , UV light.
density of the coating, its thickness , or its composition . This In some embodiments , any of the plant extract composi
can , for example , be used to control the ripening of post- tion coatings described herein can be flavorless or have high
harvest fruit or produce . For example , plant extract compo - flavor thresholds, e . g . above 500 ppm , and can be odorless
sition coatings that primarily include bifunctional or poly - or have a high odor threshold . In some embodiments , the
functional cutin monomer units can , for example , have 30 materials included in any of the coatings described herein
higher crosslink densities than those that include monofunc - can be substantially transparent . For example , the plant
tional or bifunctional cutin monomer units . Thus, the plant extract composition , the solvent, and /or any other additives
extract composition coatings that primarily include bifunc included in the coating can be selected so that they have
tional or polyfunctional cutin monomer units can result in substantially the same or similar indices of refraction . By
slower rates of ripening . 35 matching their indices of refraction , they may be optically
In some embodiments, an acid or a base can be added to matched to reduce light scattering and improve light trans
the coating formulation to achieve a desired pH suitable for mission . For example , by utilizing materials that have simi
coating the plant extract composition coating on the agri- lar indices of refraction and have a clear, transparent prop
cultural product. In some embodiments, additives such as, erty, a coating having substantially transparent
for example , surfactants , emulsifiers , thickening agents , 40 characteristics can be formed .
nonionic polymers, waxes, or salts can be included in the Any of the coatings described herein can be disposed on
coating formulation . In some embodiments , weak acids, the external surface of an agricultural product using any
ions, or non - reactive molecules can be included in the suitable means. For example , in some embodiments , the
coating formulation to control or adjust the properties of the agricultural product can be dip -coated in a bath of the
resulting films or coatings . In some embodiments , pH sta - 45 coating formulation ( e . g ., an aqueous or mixed aqueous
bilizers or modifiers can also be included in the coating organic or organic solution of the plant extract composition ).
formulation . In some embodiments, the coating formulation The deposited coating can form a thin layer on the surface
can include additional materials that are also transported to of an agricultural product, which can protect the agricultural
the surface with the coating , or are deposited separately and product from biotic stressors , water loss, and/ or oxidation .
are subsequently encapsulated by the coating ( i. e ., the coat- 50 In some embodiments , the deposited coating can have a
ing is formed at least partially around the additional mate - thickness of less than about 1500 nm , such that the coating
rial), or are deposited separately and are subsequently sup - is transparent to the naked eye. For example, the deposited
ported by the coating ( i. e ., the additional material is coating can have a thickness of about 10 nm , about 20 nm ,
anchored to the external surface of the coating). Examples of about 30 nm , about 40 nm , about 50 nm , about 100 nm ,
such additional materials can include cells, biological sig - 55 about 150 nm , about 200 nm , about 250 nm , about 300 nm ,
naling molecules , vitamins, minerals , pigments, aromas, about 350 nm , about 400 nm , about 450 nm , about 500 nm ,
enzymes, catalysts, antifungals, antimicrobials , and /or time about 550 nm , about 600 nm , about 650 nm , about 700 nm ,
released drugs . The additionalmaterials can be non - reactive about 750 nm , about 800 nm , about 850 nm , about 900 nm ,
with surface of the agricultural product and /or coating , about 950 nm , 1 ,000 nm , about 1 , 100 nm , about 1, 200 nm ,
and / or reactive with the surface and / or coating . 60 about 1, 300 nm , about 1, 400 nm , or about 1 ,500 nm ,
In some embodiments , the coating can include an additive inclusive of all ranges therebetween . In some embodiments ,
configured , for example , to modify the viscosity , vapor the deposited coating can be uniformly deposited over the
pressure , surface tension , or solubility of the coating. In agricultural product and free of defects and/or pinholes. In
some embodiments , the additive can be configured to some embodiments, the dip - coating process can include
increase the chemical stability of the coating. For example , 65 sequential coating of the agricultural product in baths of
the additive can be an antioxidant configured to inhibit coating precursors that can undergo self -assembly or cova
oxidation of the coating. In some embodiments the additive lentbonding on the agricultural product to form the coating .
 US 9 ,743,679 B2
31 32
In some embodiments , the coating can be deposited on mally deposited over the agricultural product and is free of
agricultural products by passing the agricultural products defects and /or pinholes . In some embodiments , the dip
under a stream of the coating formulation ( e . g ., a waterfall coating process can include sequential coating of the agri
of the liquid coating ). For example , the agricultural products cultural product in baths of precursors that can undergo
can be disposed on a conveyor that passes through the 5 self -assembly or covalent bonding on the agricultural prod
stream of the coating formulation . In some embodiments, uct to form the coating . In some embodiments, the coatings
the coating can be misted , vapor- or dry vapor-deposited on can be deposited on agricultural products by passing the
the surface of the agricultural product. In some embodi- agricultural products under a stream of the coating ( e . g ., a
ments, the coating can be configured to be fixed on the waterfall of the liquid coating). For example, the agricultural
surface of the agricultural product by UV crosslinking or by 10 products can be disposed on a conveyor that passes through
exposure to a reactive gas , for example , oxygen . the stream of the coating. In some embodiments, the coating
In some embodiments, the plant extract composition can be vapor deposited on the surface of the agricultural
coating can be spray - coated on the agricultural products. product. In some embodiments , the coating can be formu
Commercially available sprayers can be used for spraying lated to be fixed on the surface of the agricultural product by
the coating or precursors of the coating onto the agricultural 15 UV cross - linking or by exposure to a reactive gas, for
product. In some embodiments, the coating formulation can example , oxygen . In some embodiments, the coating can be
be electrically charged in the sprayer before spray -coating applied in the field before harvest as an alternative to
on to the agricultural product, such that the deposited pesticides .
coating electrostatically and/or covalently bonds to the exte - In some embodiments , the functionalized cutin -derived
rior surface of the agricultural product. 20 monomers, oligomers , or combinations thereofare dissolved
The coating can be configured to prevent water or other in a suitable solvent (e. g ., water, ethanol, or a combination
wise moisture loss from the coated portion of the plant, thereof ) prior to coating the agricultural product.
delay ripening , and/ or prevent oxygen diffusion into the In some embodiments the process of disposing the com
coated portion of the plant, for example , to reduce oxidation position on the agricultural product comprises dip -coating
of the coated portion of the plant . The coating can also 25 the agricultural product in a solution comprising the plural
protect the coated portion of the plant against biotic stress - ity of cutin - derived monomers , oligomers , or combinations
ors , such as, for example , bacteria , fungi, viruses, and/ or thereof . In some embodiments the process of disposing the
pests that can infest and decompose the coated portion of the composition on the agricultural product comprises spray
plant. Since bacteria , fungi and pests all identify food coating the produce with a solution comprising the plurality
sources via recognition of specific molecules on the surface 30 of cutin -derived monomers , oligomers , or combinations
of the agricultural product , coating the agricultural products thereof.
with the coating containing the plant extract compositions In some embodiments, any of the coatings can be spray
can deposit molecularly contrasting molecules on the sur- coated on the agricultural products . Commercially available
face of the portion of the plant, which can render the sprayers can be used for spraying the coating or precursors
agricultural products unrecognizable . Furthermore , the coat- 35 of the coating onto the agricultural product. In some embodi
ing can also alter the physical and / or chemical environment ments , the coatings can be electrically charged in the sprayer
of the surface of the agricultural productmaking the surface before spray coating on the agricultural product, such that
unfavorable for bacteria , fungi or pests to grow . The coating the coating covalently bonds to the exterior surface of the
can also be formulated to protect the surface of the portion agricultural product.
of the plant from abrasion , bruising, or otherwise mechani- 40 In some embodiments , the coating can be deposited on the
cal damage , and /or protect the portion of the plant from agricultural product such that the coating is unbound to the
photodegradation . The portion of the plant can include, for surface of the agricultural product. In some embodiments ,
example , a leaf, a stem , a shoot, a flower, a fruit, a root, etc . one or more components of the coating, for example , the
In some embodiments , the coating can be used to coat fruits hydrogen - bonding organic molecule, can be covalently (or
and , for example , delay ripening of the fruit . 45 hydrogen ) bonded to at least a portion of the surface of the
Application of Coatings to Produce agricultural product . This can result in improved coating
Any of the coatings described herein can be disposed on properties such as , for example , higher durability , tighter
the external surface of an agricultural product using any control of coating permeability and thickness . In some
suitable means. For example , in some embodiments , the embodiments , multiple layers of the coating can be depos
agricultural product can be dip coated in a bath of the 50 ited on the surface of agricultural product to achieve a
coating (e.g., an aqueous solution of hydrogen -bonding durable coating .
organic molecules ). The coating can form a thin layer on the In some embodiments the plant extract composition com
surface of agricultural product, which can protect the agri - prises a plurality of cutin -derived monomers , oligomers , or
cultural product from biotic stressors , water loss , and /or combinations thereof and a solvent selected from water,
oxidation . In some embodiments , the deposited coating can 55 ethanol, and combinations thereof. In some embodiments
have a thickness of less than about 2 microns, for example the method causes one or more of the cutin monomers to
less than 1 micron , less than 900 nm , less than 800 nm , less cross -link on the surface of the produce .
than 700 nm , less than 600 nm , less than 500 nm , less than Any of the coatings described herein can be used to
400 nm , less than 300 nm , less than 200 nm , or less than 100 protect any agricultural product. In some embodiments, the
nm , such that the coating is transparent to the naked eye . For 60 coating can be coated on an edible agricultural product, for
example , the deposited coating can have a thickness ofabout example , fruits , vegetables , edible seeds and nuts, herbs ,
50 nm , 60 nm , 70 nm , 80 nm , 90 nm , 100 nm , 110 nm , 120 spices, produce ,meat, eggs , dairy products, seafood, grains,
nm , 130 nm , 140 nm , 150 nm , 200 nm , 250 nm , 300 nm , 350 or any other consumable item . In such embodiments, the
nm , 400 nm , 450 nm , 500 nm , 600 nm , 700 nm , 800 nm , 900 coating can include components that are non -toxic and safe
nm , or about 1 ,000 nm inclusive of all ranges therebetween . 65 for consumption by humans and / or animals. For example ,
The deposited coating can have a high degree of crystallinity the coating can include components that are U . S . Food and
to decrease permeability , such that the coating is confor - Drug Administration ( FDA ) approved direct or indirect food
 US 9, 743,679 B2
33 34
additives, FDA approved food contact substances , satisfy losses of agricultural products due to decomposition by
FDA regulatory requirements to be used as a food additive biotic and / or environmental stressors .
or food contact substance, and /or is an FDA Generally The following examples describe plant extract composi
Recognized as Safe (GRAS ) material. Examples of such tions obtained using the methods described herein . These
materials can be found within the FDA Code of Federal 5 examples are only for illustrative purposes and are notmeant
Regulations Title 21, located at " http ://www .accessdata . fda - to limit the scope of the present disclosure .
. gov /scripts /cdrh / cfdocs / cfcfr /cfrsearch . cfm " , the entire
contents of which are hereby incorporated by reference EXAMPLES
herein . In some embodiments, the components ofthe coating
can include a dietary supplement or ingredient of a dietary 10 outAllfurther
reagents and solvents were purchased and used with
purification unless specified . Palmitic acid ( 98 % )
supplement. The components of the coating can also include
an FDA approved food additive or color additive. In some was purchased from Sigma- Aldrich . p - TsOH and MTBE
embodiments, the coating can include components that are were purchased from Alfa - Aesar. Toluene, Et20 , and EtOAC
were purchased from VWR . Lipozyme® TL IM lipase was
naturally derived , as described herein . In some embodi- 1115 purchased from Novozymes. 10 wt % Pd / C was purchased
ments, the coating can be flavorless or have a high flavor from Strem Chemicals and used as received . All reactions
threshold of below 500 ppm , are odorless or have a high were carried out under an atmosphere of air with non -dried
odor threshold , and /or are substantially transparent. In some solvents unless otherwise stated . Yields refer to chromato
embodiments , the coating can be configured to be washed graphically and spectroscopically ('H NMR ) homogeneous
off an edible agricultural product , for example, with water . 20 materials , unless otherwise stated . Reactions were moni
In some embodiments, the coating described herein can tored by thin layer chromatography ( TLC ) carried out on
be coated on an inedible agricultural product. Such inedible 0 . 25 mm E . Merck silica gel plates (60 Å , F - 254 ) using UV
agricultural products can include , for example , inedible light as the visualizing agent and an acidic mixture of
flowers , seeds, shoots , stems, leaves, whole plants , and the anisaldehyde , ceric ammonium molybdate , or basic aqueous
likes . In such embodiments, the coating can include com - 25 potassium permanganate (KMnO2), and heat as developing
ponents that are non - toxic but the threshold level for non - agents .NMR spectra were recorded on a Bruker Avance 500
toxicity can be higher than that prescribed for edible prod MHz and/or Varian VNMRs 600 MHz instruments and
ucts. In such embodiments , the coating can include an FDA calibrated using residual un - deuterated solvent as an internal
approved food contact substance, an FDA approved food reference (CHCl3 @ 7 . 26 ppm ' H NMR , 77 .16 ppm 13C
additive , or an FDA approved drug ingredient, for example , 30 NMR ). The following abbreviations (or combinations
any ingredient included in the FDA ' s database of approved thereof) were used to explain the multiplicities: s = singlet,
drugs, which can be found at " http ://www .accessdata .fda . d = doublet, t - triplet, q - quartet, m =multiplet, br = broad .
. gov /scripts /cder/ drugsatfda/index .cfm " , the entire contents Mass spectra (MS) were recorded on a time of- flight mass
of which are hereby incorporated herein by reference . In spectrometer by electrospray ionization (ESI) or field des
some embodiments, the coating can include materials that 35 orption ( FD ) at the UC Santa Barbara mass spectrometry
satisfy FDA requirements to be used in drugs or are listed facility . 1 ,3 -bis (benzyloxy )propan - 2 - ol was synthesized
within the FDA ' s National Drug Discovery Code Directory, according to the procedure ofNemoto et al. ( J. Org. Chem .,
" http ://www .accessdata . fda . gov/scripts /cder/ndc/default - 1992 , 57 , p . 435 ).
. cfm ” , the entire contents of which are hereby incorporated The following abbreviations are used in the following
herein by reference . In some embodiments , the materials can 40 examples and throughout the specification :
include inactive drug ingredients of an approved drug prod - DCC = N ,N '-Dicyclohexylcarbodiimide
uct as listed within the FDA ' s database , “ http ://www .access - DCM - dichloromethane
data . fda.gov/scripts/ cder /ndc/default .cfm ” , the entire con DMAP = Dimethylamino pyridine
tents of which are hereby incorporated herein by reference . DMF = N ,N -dimethylformamide
Embodiments of the coatings described herein provide 45 MBTE = BME = tert- butylmethyl ether
several advantages, including, for example : ( 1) the coatings p - TsOH = para toluenesulfonic acid
can protect the agricultural products from biotic stressors , TBS = TBDPS = tert-butyldimethyl silyl
i. e. bacteria , viruses, fungi, or pests ; ( 2 ) the coatings can
prevent evaporation of water and/ or diffusion of oxygen ; (3 ) Example 1 : Plant Extract Composition Obtained
coating can help extend the shelf life of agricultural prod - 50 from Tomato Skin /Peel-I
ucts, for example , post-harvest produce , without refrigera
tion ; (4 ) the coatings can introduce mechanical stability to Whole tomatoes were cut in to halves or quarters and
the surface of the agricultural products eliminating the need boiled in water until the skins were visibly loosened . The
for expensive packaging designed to prevent the types of skins were mechanically separated from the tomato flesh
bruising which accelerate spoilage ; (5 ) use of agricultural 55 using a food mill to remove as much of the fruit flesh and
waste materials to obtain the coatings can help eliminate the seeds from the peels as possible . The separated peels were
breeding environments of bacteria, fungi, and pests ; (6 ) the then heated in an aqueous solution of about 16 grams per
coatings can be used in place of pesticides to protect plants , liter ammonium oxalate and about 4 grams per liter oxalic
thereby minimizing the harmful impact of pesticides to acid at 75 degrees Celsius for about 16 hours to disrupt the
human health and the environment; ( 7 ) the coatings can be 60 pectinaceous glue present between the cuticular layer and
naturally derived and hence , safe for human consumption . the underlying tomato flesh . The remaining peel including
Since the components of the coatings described herein can the cutin was isolated by filtration , rinsed with water, and
in some embodiments be obtained from agricultural waste , oven - dried at 65 degrees Celsius to remove residual mois
such coatings can be made at a relatively low cost. There - ture prior to depolymerization . The skin (i. e ., the cutin
fore , the coatings can be particularly suited for small scale 65 containing portion ) was depolymerized in a methanolic
farmers, for example , by reducing the cost required to solution including a stoichiometric excess of potassium
protect crops from pesticides and reducing post -harvest hydroxide (relative to tomato skin ) at about 65 degrees
 US 9 ,743,679 B2
35 36
Celsius for about 24 hours . The resulting solution was extraction with chloroform for 2 days followed by a second
filtered to remove any leftover solids and the filtrate was Soxhlet extraction with methanol for 1 day . The cutin -rich
collected . Any excess solvent was removed from the filtered skins were subsequently depolymerized in a methanolic
solution by rotary evaporation . The crude solid was redis solution including a stoichiometric excess of potassium
solved in distilled water and chilled in an ice bath , and the 5
pH of the resulting solution was adjusted to about 2 by hydroxideCelsius for
(relative to cranberry skin ) at about 65 degrees
about 24 hours. The resulting solution was
adding aqueous HCl to the solution . The depolymerized filtered to remove
multifunctional fatty acid monomers and oligomers of the collected . Any excessanysolvent leftover solids and the filtrate was
was removed from the filtered
cutin were solidified by precipitating from solution or by
evaporation and the monomers were isolated by filtration to solution by rotary evaporation . The crude solid was redis
yield the tomato peel extract. The tomato peel extract was solved in distilled water and chilled in an ice bath , and the
purified using column chromatography using a 3 :1 volumet- pH of the resulting solution was adjusted to about 2 by
ric ratio of hexane:acetone. The tomato peel extract included adding aqueous HCl to the solution . The depolymerized
70 wt % 10 , 16 -dihydroxyhexadecanoic acid , 5 wt % 16 -hy - 15 multifunctional fatty acid monomers and oligomers of the
droxyhexadecanoic acid , 3 wt % 9 ,10 , 18 - trihydroxyoctade cranberry cutin were solidified by precipitating from solu
canoic acid ,and 22 wt % residual fatty acids and other plant tion or by evaporation and the monomers were isolated by
by- products. filtration to yield the cranberry skin extract. The cranberry
Example 2 : Plant Extract Composition Obtained O
extract was purified using column chromatography using a
from Tomato Skin / Peel-II 3:1 volumetric ratio of hexane:acetone .
Dried tomato peels and pomace obtained directly from an Example 4 : Plant Extract Composition Obtained
industrial tomato processing facility were ground to a fine from Grape Berry
powder using an electric grinder and then directly subjected 23 White grape pressings leftover from winemaking were
to depolymerization . The waste (i.e ., cutin - containing peels
and pomace ) was depolymerized in a methanolic solution boiled in water until their skins were visibly loosened . The
including a stoichiometric excess of potassium hydroxide aqueous mixture containing grape flesh and grape skins was
(relative to tomato waste ) at about 65 degrees Celsius for 30 then
grape
subjected to sonication to mechanically detach the
flesh from the grape skins. The mixture was sonicated
about 24 hours . The resulting solution was filtered to remove for 5 - 10 minutes at a time. Sonication was applied in
any leftover solids and the filtrate was collected . Any excess alternation with filtration of the aqueous berry mixture
solvent was removed from the filtered solution by rotary through a coarse filter whereby the filter cake was washed
evaporation . The crude solid was redissolved in distilled with distilled water to eliminate seeds and fine debris and
water and chilled in an ice bath , and the pH of the resulting » isolate a filter cake increasingly enriched in grape skin . The
solution was adjusted to about 2 by adding aqueous HC1 to sonication and filtration steps were continued in alternation
the solution . The depolymerized multifunctional fatty acid until approximately half of the berry flesh (volumetrically )
monomers and oligomers of the cutin were solidified by was removed from the final grape skin -enriched filter cake.
precipitating from solution or by evaporation and the mono - 10 The filter cake was then subjected to enzymatic treatment to
mers were and isolated by filtration to yield the crude tomato detach any remaining grape flesh from the grape skins . The
peel extract. The dried crude tomato peel extract was dry - filter cake was dispersed into an aqueous buffer solution (0 .2
loaded onto Celite and subjected to Soxhlet extraction with mM acetate , pH 4 ) containing 0 . 1 wt % cellulase and 0 . 1 wt
acetonitrile for 1 day. The solution in the solvent reservoir % pectinase and shaken on an orbital shaker at 40 degrees
below the Soxhlet apparatus was collected and excess sol- 45 Celsius and 200 rpm for 24 hours. The aqueous mixture was
vent was removed by rotary evaporator to vield a purified . then filtered through a coarse filter and washed with distilled
resolidified , tomato peel extract. water to obtain a filter cake enriched with cutin - containing
grape skin . After oven -drying at 65 degrees Celsius to
Example 3 : Plant Extract Composition Obtained remove residual moisture , the grape skins were next sub
from Cranberry 50 jected to a first Soxhlet extraction with chloroform for 2 days
followed by a second Soxhlet extraction with methanol for
1 day. The cutin -rich skins were subsequently depolymer
Cranberries were diced and boiled in water until their ized in a methanolic solution including a stoichiometric
skins were visibly loosened . The aqueous mixture contain excess of potassium hydroxide (relative to grape skin ) at
ing berry flesh and berry skins was then subjected to 55 about 65 degrees Celsius for about 24 hours. The resulting
sonication to mechanically detach the berry flesh from the solution was filtered to remove any leftover solids and the
skins. The mixture was sonicated for 5 - 10 minutes at a time. filtrate was collected . Any excess solvent was removed from
Sonication was applied in alternation with filtration of the the filtered solution by rotary evaporation . The crude solid
aqueous berry mixture through a coarse filter whereby the wa
filter cake was washed with distilled water to eliminate fine 60 and the pH of the resulting solution was adjusted to about 2
by adding aqueous HCl to the solution . The depolymerized
debris and isolate a filter cake increasingly enriched in berry multifunctional fatty acid monomers and oligomers of the
skin . The sonication and filtration steps were continued in cutin were solidified
alternation until the majority of the berry flesh was removed
by precipitating from solution or by
evaporation and the monomers were isolated by filtration to
from the berry skin -enriched final filter cake. After oven - 65 yield the grape skin extract. The grape skin extract was
drying at 65 degrees Celsius to remove residual moisture , purified using column chromatography using a 3 : 1 volumet
the cranberry skins were next subjected to a first Soxhlet ric ratio of hexane:acetone.
 US 9 ,743,679 B2
38
Example 5 : Plant Extract Composition Obtained dihydroxypalmitate, while a second portion was functional
from Tomato Skin / Peel and Agricultural Coating ized to form 2 ,3 - dihydroxypropyl 10 , 16 -dihydroxypalmitate
Formed Thereof (i.e ., the 1-monoacylglycerideester of 10 , 16 -dihydroxyp
almitic acid ). The ethyl 10 , 16 - dihydroxypalmitate was pre
Dried tomato peels or pomace obtained directly from an 5 pared by Fisher esterification of the 10 , 16 -dihydroxypalm
industrial tomato processing facility were ground to a fine itic acid in ethanol using sulfuric acid as a catalyst . The
powder using an electric grinder and then directly subjected 2 ,3 -dihydroxypropyl 10 ,16 - dihydroxypalmitate was pre
pared by
to depolymerization . The cutin -containing peels or pomace pared reaction ofo the 10 ,16 -dihydroxypalmitic acid with
by under
reaction
were depolymerized in two batches of an ethanolic mixture 10 819The ethyl 10 ,basic
glycidol
16 -
catalysis.
dihydroxypalmitate was then dissolved in
( e.g ., a first solvent) that were each substantially free of substantially pure ethanol at a concentration
water (e. g., less than 5 % water by mass ). The ethanolic form a first extract composition , and the 2,3of 67 mg/mL to
-dihydroxypro
mixture was formed by adding sodium metal to ethanol pyl 10 , 16 -dihydroxypalmitate was dissolved in substantially
under inert conditions , generating sodium ethoxide within
the mixture . The cutin -containing peels and pomace were 15 pure ethanol at a concentration of 37 mg/mL to form a
second extract composition . Finally, agricultural coatings
then added to the first and second batches of the ethanolic were formed from the first extract composition on a first set
mixture. The resulting solutions were heated to and held at of key limes and from the second extract composition on a
reflux under inert atmosphere . The reaction proceeded for 24 second set ofkey limes using the following process . The first
hours for the first batch and 48 hours for the second batch , 20 and second extract compositions were poured into respective
resulting in respective first and second solutions which each first and second plastic bags. 10 key limes were then placed
included crude sodium 10 , 16 - dihydroxyhexadecanoate . in each bag , and the bags were each agitated for about 10
The solutions were each then cooled to room temperature seconds to ensure that the extract compositions covered the
and acidified to a pH of about 7 by addition of 3M HCl. The entirety of each key lime. The key limes were then removed
acidified solutions were then filtered to remove any 25 and placed on dryings racks, and the solvent allowed to
insoluble salts, unreacted material, and other impurities , and evaporate . This resulted in about 20 mg of the ethyl 10 , 16
the filtrates were evaporated to dryness. The resulting mate dihydroxypalmitate extract being deposited over each key
rials were each then re -dissolved in methanol and absorbed lime of the first set of key limes, and about 11 mg of the
onto 3x their respective mass of Celite by evaporating the 2 , 3 - dihydroxypropyl 10 , 16 - dihydroxypalmitate extract
solvent. The adsorbed mixtures were then Soxhlet extracted 30 being deposited over each key lime of the second set of key
sequentially by heptane, ethyl acetate , and methanol. Sepa limes .
ration of the 10 , 16 -dihydroxypalmitic acid from other spe - FIGS. 9 - 11 are high resolution photographs of untreated
cies and impurities within the adsorbed mixtures was moni- key limes. FIG . 9 shows untreated key limes 900 . FIG . 10
tored by UPLC -MS (ESI+ ). The 10 , 16 - dihydroxypalmitic shows key limes coated with the plant extracts of the first
acid preferentially partitioned into the ethyl acetate, with 35 extract composition ( 1000 ). FIG . 11 showskey limes coated
less polar impurities extracting from the Celite into the with the plant extracts of the second extract composition
heptane wash , and the more polar impurities extracting into ( 1100 ). Each photograph was taken approximately 24 hours
the methanol wash . The obtained extract composition from after the coatings were formed . The coatings formed from
each of the two batches was a yellow to colorless free both extract compositions were translucent and substantially
flowing powder after evaporation of the solvent. The pow - 40 undetectable to the human eye . However, the coating formed
ders from each of the two batches were then combined into from the second extract composition reduced the glossiness
a single composition . FIG . 7 shows purity data , obtained of the key limes , whereas the glossiness of key limes coated
using mass spectrometry and FIG . 8 shows elemental analy - with the first extract composition was maintained .
sis, of the resulting powder, indicating a high level of purity .
A first portion of the 10 , 16 - dihydroxypalmitic acid of the 45 Example 6 : Synthesis of 1 ,3 -dihydroxypropan - 2 -yl
composition was then functionalized to form ethyl 10 , 16 Palmitate

Bno -
Bn0
OH
Bn0
"OH cat. p -TsOH , Tol
OBn

OBn

N /H2
10 %*Pec 2016
10 wt % Pd /C EtoAc

LOH

COH
 US 9 ,743,679 B2
39 40
Step 1. 1,3 -bis(benzyloxy )propan -2 -yl palmitate 7 .66 g ( 15 .00 mmoles ) of 1,3 -bis (benzyloxy )propan - 2-yl
palmitate, 79.8 mg (0 .75 mmole ) of 10 wt % Pd /C and 100
mL of EtoAc were charged to a 3 neck round bottom flask
OBn 5
equipped with a Teflon coated magnetic stir bar . A cold
finger, with a bubbler filled with oil attached to it, and a
OBn
bubbling stone connected to a 1 :4 mixture of H /N2 gas tank
were affixed to the flask . Hy/N , was bubbled at 1.2 LPM into
the flask until the disappearance of both starting material and
70 .62 g (275.34 mmole ) of palmitic acid , 5. 24 g (27 .54' 10 mono - deprotected substrate as determined by TLC (~ 60
mmole ) of p - TsOH , 75 g (275 .34 mmole ) of 1,3 -bis (benzy
loxy )propan -2 -ol, and 622 mL of toluene were charged into min ). Once complete , the reaction mixture was filtered
a round bottom flask equipped with a Teflon coated magnetic through a plug of Celite , which was then washed with 100
stir bar. A Dean -Stark Head and condenser were attached to mL of EtOAc. The filtrate was placed in a refrigerator at 4°
the flask and a positive flow of N2 was initiated . The flask 15 C . for 24 hrs. The precipitate from the filtrate ( white and
was heated to reflux in a heating mantle while the reaction transparent needles) was filtered and dried under high
mixture was stirred vigorously until the amount of water
collected ( ~ 5 mL ) in the Dean - Stark Head indicated full vacuum yielding (2. 124 g , 6 .427 mmole, yield = 42 .8 % ) of
ester conversion (~ 8 hr ). The flask was allowed to cool down 1,3 -dihydroxypropan -2 -yl palmitate .
to room temperature and the reaction mixture was poured HRMS (FD - TOF) (m /z ): calcd . for C19H3:04, 330 .2770 ;
into a separatory funnel containing 75 mL of a saturated - found , 330 .2757 ;
aqueous solution of NaCO2 and 75 mL of brine. The toluene H NMR (600 MHz, CDC1, ): 8 4 .93 (p . J = 4 .7 Hz. 1H ) .
fraction was collected and the aqueous layer was extracted 3 . 84 (t. J = 5 . 0 Hz, 4H ) , 2 . 37 (t. J = 7 .6 Hz, 2H ), 2 . 03 ( t. J = 6 . 0
with 125 mL of Et 0 . The organic layers were combined and Hz, 2H ), 1 .64 (p , J = 7 .6 Hz, 2H ), 1.38 - 1. 17 (m , 26H ), 0 . 88
washed with 100 mL of brine, dried over MgSO4, filtered as (t, J= 7 .0 Hz, 3H ) ppm .
and concentrated in vacuo . The crude colorless oil was dried 13C NMR (151 MHz, CDC1. ): 8 174 . 22 . 75 .21. 62 .73 ,
under high vacuum providing (135 .6 g, 265 .49 mmole , 34.51, 32. 08 , 29 .84, 29. 83 , 29 .81, 29. 80 , 29 .75 , 29.61,
crude yield = 96 .4 % ) of 1 ,3 -bis (benzyloxy ) propan - 2 -yl 29.51 , 29.41, 29.26 , 25 .13, 22.85 , 14 .27 ppm .
palmitate .
HRMS (ESI- TOF) (m /z ): calcd. for C33H500 Na, Example 7 : Synthesis of 1 ,3 -dihydroxypropan - 2 -yl
[M +Na]*, 533.3607 ; found , 533. 3588 ; Palmitate by Enzyme Catalysis

cat. Lipozyme(R) TL IM TE
EtOH
LOH

OH

'H NMR (600 MHz, CDC13 ): 8 7.41-7 .28 (m , 10H ), 5.28 50 3.66 g (4 .50 mmoles) of tripalmitin , 7 .26 mg of
(p . J = 5 .0 Hz, 1H ), 4 . 59 (d , J = 12 . 1 Hz, 2H ), 4 . 54 ( d , J = 12 . 1 Lipozyme® TL - IM lipase , 2 .65 mL of EtOH , and 363 mL of
Hz, 2H ), 3 .68 (d , J = 5 .2 Hz, 4H ), 2 .37 (t, J = 7 .5 Hz, 2H ), 1.66 MTBE were charged
MTBE were charged to a round bottom flask equipped with
(p , J = 7 .4 Hz, 2H ), 1.41 - 1. 15 (m , 24H ), 0 . 92 (t, J= 7 .0 Hz, 3H ) a Teflon coated magnetic stir bar. The reaction mixture was
ppm .
28..43
13C NMR ( 151 MHz, CDC12 ): 8 173 . 37 , 138.09, 128 43 , 5555 Stil
stirred for 15 min at room temperature , filtered , and con
127.72 , 127.66 , 73. 31 , 71.30 , 68. 81 , 34 .53 , 32 .03 , 29.80 , centrated in vacuo . 15 mL ofhexanes was added to the crude
29 .79, 29. 76 , 29 .72 , 29. 57 , 29 .47 , 29 .40 , 29 .20 , 25 . 10 , product and the product/hexanes mixture was stored in a
22 .79, 14 . 23 ppm . refrigerator at 4° C . for 24 hrs . The crude mixture was
Step 2 . 1,3 -dihydroxypropan -2 -yl palmitate filtered , washed with 30 mL of cold hexanes, and dried
under high vacuum yielding 1.256 g (3 .8 mmole,
yield = 84.4 % ) of 1,3 -dihydroxypropan -2 -yl palmitate .
(Note : yield is based on total mass being from 1, 3 -dihy
LOH droxypropan - 2- yl palmitate , however it contains 12 . 16 mole

m i LotVOH 65 %% HRMS
65 o( 20 wt (%FD) -ofTOF
diacylglycerol palmitate .)
) (m /z): calcd . for C19H3304, 330 .2770 ;
found, 330 .2757;
 US 9 ,743,679 B2
41
'H NMR (600 MHz, CDC13 ): 8 4 .93 (p , J = 4 .7 Hz, 1H ), at least partially depolymerizing the cutin in a first sol
3 .84 (t, J = 5 .0 Hz, 4H ), 2 .37 (t, J = 7 .6 Hz, 2H ), 2.03 (t, J= 6 .0 vent, the first intermediate extract including a plurality
Hz, 2H ), 1 .64 (p , J= 7 .6 Hz, 2H ) , 1 .38 - 1 . 17 ( m , 26H ), 0 . 88 of cutin - derived monomers, oligomers , or combina
( t, J = 7 .0 Hz, 3H ) ppm . tions thereof;
13C NMR ( 151 MHz, CDC1z): 8 174 .22, 75 . 21 , 62 .73 , 5 selectively filtering the first intermediate extract to obtain
34 .51, 32 .08 , 29 .84 , 29 . 83 , 29 .81 , 29 .80 , 29 .75 , 29 .61 , a second intermediate extract having a higher purity
29 .51, 29 .41, 29. 26 , 25 .13, 22 .85 , 14 .27 ppm . than the first intermediate extract , the second interme
While various embodiments of the system , methods and diate extract including at least one of the cutin -derived
devices have been described above , it should be understood monomers, oligomers, or combinations thereof; and
that they have been presented by way of example only , and functionalizing the cutin -derived monomers, oligomers,
not limitation . Where methods and steps described above or combinations thereof of the second intermediate
indicate certain events occurring in certain order, those of extract by esterification to form the plant extract com
ordinary skill in the art having the benefit of this disclosure position .
would recognize that the ordering of certain steps may be 16 9 . The method of claim 8 , further comprising:
modified and such modification are in accordance with the causing the plant extract composition to be applied to a
variations of the invention . Additionally, certain of the steps plant or agricultural product to form a protective coat
may be performed concurrently in a parallel process when ing over the plant or agricultural product.
possible , as well as performed sequentially as described 10 . The method of claim 9 , wherein the plant or agricul
above . The embodiments have been particularly shown and 20 tural product to which the plant extract composition is
described, but it will be understood that various changes in applied is different from the plant from which the cutin is
form and details may be made . obtained .
11 . The method of claim 8 , wherein functionalizing the
The invention claimed is : cutin - derived monomers , oligomers , or combinations
1 . A method of preparing a plant extract composition , 25 thereof comprises glycerating the cutin -derived monomers ,
comprising: oligomers , or combinations thereof to form monoacylglyc
obtaining cutin from a cutin -containing portion of a plant eride compounds .
matter , the cutin -containing portion being at least par - 12 . The method of claim 8 , wherein functionalizing the
tially separated from a non - cutin -containing portion of cutin - derived monomers , oligomers , or combinations
the plant matter; 30 thereof comprises esterifying the cutin -derived monomers ,
at least partially depolymerizing the cutin in a first solvent oligomers , or combinations thereof to form ethyl esters .
to obtain a first solution comprising a first intermediate 13. A method of preserving an agricultural product , com
extract dissolved in the first solvent, the first interme- prising :
diate extract including a plurality of cutin -derived obtaining a plant extract composition comprising a plu
monomers , oligomers, or combinations thereof: 35 rality of cutin - derived monomers , oligomers , or com
acidifying the first intermediate extract: binations thereof;
selectively filtering the first intermediate extract to obtain functionalizing the cutin -derived monomers, oligomers ,
a second intermediate extract having a higher purity or combinations thereof by esterifying the cutin - de
than the first intermediate extract; rived monomers, oligomers, or combinations thereof
functionalizing the cutin -derived monomers, oligomers, 40 with a glycerol molecule ; and
or combinations thereof of the second intermediate causing the agricultural product to be coated with the
extract by esterification ; and functionalized cutin -derived monomers, oligomers, or
dissolving the functionalized second intermediate extract combinations thereof.
in a second solvent to obtain the plant extract compo - 14 . The method of claim 13 , wherein the cutin -derived
sition . 45 monomers , oligomers , or combinations thereof comprise
2 . The method of claim 1, wherein obtaining the second fatty acids.
intermediate extract includes removing the second interme 15 . The method of claim 14 , wherein the fatty acids are
diate extract from the first solvent. palmitic acid or 10 - 16 - dihydroxyhexadecanoic acid .
3 . The method of claim 2 , wherein the first solvent 16 . The method of claim 13, wherein obtaining the plant
includes at least one of chloroform , hexane , acetonitrile , and 50 extract composition comprises at least partially depolymer
ethyl acetate . izing cutin derived from plant matter.
4 . Themethod of claim 1 , wherein selectively filtering the 17 . The method of claim 13 , wherein the functionalized
first intermediate extract includes solidifying the first inter - cutin -derived monomers , oligomers , or combinations
mediate extract and washing the solidified first intermediate thereof are dissolved in a solvent prior to coating the
extract in a selective solvent. 55 agricultural product.
5 . The method of claim 1 , wherein the first solution has a 18 . The method of claim 17 , wherein the solvent is water,
pH in the range of 10 to 14 . ethanol, or a combination thereof.
6 . The method of claim 1, wherein the first solvent is less19 . The method of claim 13, wherein the process of
than 5 % water by mass. coating the agricultural product with the functionalized
7. The method of claim 6 , wherein the first solvent 60 cutin - derived monomers, oligomers, or combinations
comprises at least one of ethanol and methanol. thereof comprises dip - coating or spray -coating of the agri
8 . A method of preparing a plant extract composition , cultural product with a solution comprising the functional
comprising: ized cutin - derived monomers, oligomers , or combinations
obtaining cutin from a cutin - containing portion of a plant thereof .
matter, the cutin -containing portion being at least par- 65 20 . The method of claim 13 , wherein the process of
tially separated from the non -cutin -containing portion coating the agricultural product with the functionalized
of the plant matter; cutin - derived monomers, oligomers, or combinations
 US 9,743,679 B2
43 44
thereof causes one or more of the functionalized cutin 25 . The method of claim 23 , wherein the functionalized
derived monomers or oligomers to cross - link on the surface monomers , oligomers , or combinations thereof are dissolved
of the product. in a solvent prior to coating the agricultural product.
21. The method of claim 13 , wherein the cutin -derived 26 . The method of claim 25 , wherein the solvent is
monomers, oligomers , or combinations thereof are function - 5 ethanol, water, or a combination thereof.
alized by esterifying as a 1 - glycerol or a 2 - glycerol ester . 27 . The method of claim 23 , wherein the process of
22 . The method of claim 13 , wherein the functionalized coating the agricultural product with the functionalized
cutin -derived monomers, oligomers, or combinations monomers, oligomers , or combinations thereof comprises
thereof are not naturally occurring on the surface of the dip -coating or spray -coating of the agricultural product with
agricultural product.
23 . A method of treating an agricultural product, com a solution comprising the functionalized monomers , oligom
prising : ers , or combinations thereof.
obtaining a plant extract composition comprising a plu 28 . The method of claim 23 , wherein the process of
rality of monomers , oligomers, or combinations coating the agricultural product with the functionalized
thereof, wherein the monomers, oligomers or combi- 15is monomers
or more
, oligomers, or combinations thereof causes one
of the functionalized monomers or oligomers to
nations thereof comprise fatty acids or esters ;
functionalizing the monomers , oligomers , or combina cross - link on the surface of the agricultural product .
29 . The method of claim 23 , wherein the monomers ,
tions thereof by esterification , and
causing the agricultural product to be coated with the oligomers , or combinations thereof are functionalized by
functionalized monomers , oligomers , or combinations 20 esterifying as a 1- glycerol or a 2 -glycerol ester.
30 . The method of claim 23 , wherein the functionalized
thereof.
24 . The method of claim 23 , wherein obtaining the plant monomers , oligomers , or combinations thereof are not natu
extract composition comprises at least partially depolymer - rally occurring on the surface of the agricultural product.
izing cutin derived from plant matter. * * * * *