United States Patent: Weir Jan - 30, 2018

US009877991B2
(12) United States Patent ( 10 ) Patent No.: US 9 ,877, 991 B2

Weir (45) Date of Patent: Jan . 30 , 2018
( 54 ) REACTION PLATFORM AND METHOD FOR A61K 47 /14 (2013.01); A61K 47 /20 (2013.01 );
MAKING POLLEN BASED MATERIALS IN A61K 47/26 (2013.01 ); A61K 47/44 (2013.01)
COMBINATION WITH BEESWAX AND USES (58 ) Field of Classification Search
THEREOF ??? .............. A61K 36 / 00
See application file for complete search history .
(71) Applicant: Decima Health Limited , Christchurch
(NZ ) (56 ) References Cited
(72 ) Inventor: Iona Weir , Auckland (NZ ) U . S . PATENT DOCUMENTS
(73 ) Assignee: Decima Health Limited , Christchurch 4 ,746 ,071 A 5 / 1988 Grunhoff et al.
6 ,270 ,811 B18 /2001 Fregonese
(NZ ) 6 ,482 ,442 B1 11/2002 Dado
7 ,288, 265 B1 10 /2007 Rolf
( * ) Notice : Subject to any disclaimer, the term of this 2004 /0258765 A1 12 / 2004 Gee
patent is extended or adjusted under 35 2006 /0172022 AL 8 /2006 Szanzer
U .S .C . 154 (b ) by 136 days. 2007/0098671 AL 5 /2007 Martin
2007/0141168 A1 6 / 2007 Alkazemi
(21) Appl. No.: 2009/ 0291122 AL 11/2009 Vandeputte
14 /911,652 (Continued )
( 22 ) PCT Filed : Aug. 12 , 2014 FOREIGN PATENT DOCUMENTS
(86 ) PCT No.: PCT/IB2014 /002786 CN 1165637 A * 11 /1997
$ 371 (c )( 1), CN 103053893 A 4 /2013
( 2 ) Date: Feb . 11 , 2016 (Continued )
(87) PCT Pub. No.: WO2015 /028892 OTHER PUBLICATIONS
PCT Pub. Date: Mar. 5 , 2015 Gaze , P.D ., et al., Honeydew and Its Importance to Birds in Beech
(65) Prior Publication Data Forests of South Island, New Zealand . New Zealand Journal of
Ecology . 1983 ; 6 : 33 -37 .
US 2016 /0193263 Al Jul. 7 , 2016 O 'Brien , IE , et al., Annexin - V and TUNEL Use in Monitoring the
Progression of Apoptosis in Plants. Cytometry . Sep . 1, 1997 ;
29 : 28 -33.
Related U .S . Application Data O ' Brien , IE , et al., Early Stages of the Apoptotic Pathway in Plant
Cells are Reversible . The Plant Journal . 1998; 13 (6 ), 803 -814 .
(60 ) Provisional application No. 61/865 ,011 , filed on Aug. O 'Brien , IE , et al., Protoplasts to Plants of Gentianaceae. Regen
12 , 2013 . eration of Lisianthus (Eustoma grandiflorum ) is Affected by Cal
cium Ion Preconditioning, Osmolality and pH of the Culture Media .
(51) A61K
Int. CI.36 / 18 Apr. 1993 ; 33 ( 1): 31-37. Abstract Only.
( 2006 .01) (Continued )
A61K 38 / 16 ( 2006 .01)
A61K 9 /00 ( 2006 .01) Primary Examiner — Qiuwen Mi
A61K 35 /644 ( 2015 .01 ) (74 ) Attorney, Agent, or Firm — Amin Talati Upadhye
A61K 9 / 10 ( 2006 .01 ) LLP; George M . Carrera, Jr.; Brent A . Batzer
A61K 8 /97 ( 2017.01)
A61K 9 / 06 ( 2006 .01 ) (57) ABSTRACT
A610 19 / 08 ( 2006 .01) A two stage reaction platform is provided to produce a
A610 19 / 00 ( 2006 .01) pollen - based extract material, comprising the steps of a first
A61K 8 /92 ( 2006 .01 ) stage including: opening and / or germinating pollen grains,
A61K 47/ 06 (2006 .01) reacting the treated pollen grains with one or more beehive
A61K 47/08 ( 2006 .01 ) components , selected from beeswax, honey, or enzyme
A61K 47/ 10 (2017.01) containing material, and stirring to form a jelly ; and a second
A61K 47 / 14 ( 2017 .01 ) stage including heating the jelly in a closed container to
A61K 47 /20 ( 2006 .01) product an extract. The extract may be a fermented extract.
A61K 47 /26 (2006 .01) An aqueous skin cream includes the pollen -based extract
A61K 47/ 44 ( 2017 .01) and one or more of glycerine , natural oils, emollients ,
(52) U . S . CI. preservatives, vitamins, fragrance , emulsifiers , or waxes .
??? ... . A61K 36 / 18 ( 2013 .01 ) ; A61K 8 /927 The skin cream may be used for treatment of skin conditions
(2013 .01 ) ; A61K 8 /975 ( 2013.01 ); A61K or inflammation . In another embodiment a method of treat
9 / 0014 ( 2013 .01); A61K 9 / 06 ( 2013 .01) ; A61K ing eczema or psoriasis is provided , comprising administer
9 / 10 (2013.01) ; A61K 35 /644 ( 2013 .01 ); A61K ing to the individual in need of such treatment a therapeu
38 / 168 ( 2013 .01) ; A610 19 /00 ( 2013 .01) ; tically effective amount of an aqueous skin cream .
A61Q 19 / 08 (2013.01 ); A61K 47 /06 ( 2013 .01) ;
A61K 47/ 08 (2013 .01); A61K 47 / 10 (2013 .01 ); 6 Claims, 14 Drawing Sheets
US 9,877 ,991 B2
Page 2
( 56 ) References Cited
U . S . PATENT DOCUMENTS
2010 /0272790 A1 10 / 2010 Morariu
2012/0156673 A1 6 / 2012 De Lucca et al.
FOREIGN PATENT DOCUMENTS
EP 0136479 A2 4 / 1985
EP 0319062 B1 2 /1992
EP 654262 A1 5 / 1995
GB 2485483 A 5 /2012
WO 1999059523 A2 11/1999
WO 2002032442 Al 4 /2002
WO 2012092673 Al 7 / 2012
OTHER PUBLICATIONS
Weir , IE , et al. Oxidative Stress Is Generated Via the Mitochondrial
Respiratory Chain During Plant Cell Apoptosis . Cytometry. Aug.
2003 ; Part A 54A : 109 - 117 .
* cited by examiner
U . S . Patent Jan . 30 , 2018 Sheet 1 of 14 US 9 ,877 ,991 B2
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F erted
Roller
NZLABS sample Number ???
Date Supled 08104 / 2914
Testnelcoa Um
Fatty and profile 404093139. 989 .33 .963 . 22
04:0 Buric acid 100
C50 vaaric acid g1005
Cao Caxatc acid ) F1001
C70 Cranic acid ; 3:100g 2 .5
CS:0 (Cayic acid ) 000 KO .S
09 :0 Pelargoric 300, g 100g
O DO Camic acid ) OOKS 9 .5
2010 (uktecdc aco ) 9:100 05
002 : 0 Lauicace GLOBE ?
CB0 (TKonlic acid ) :100 0 .5
C14:0 M istic acci D/2009
014 feis Myristotec 3010 ???? ? 0 .5
C150 Pentadscylic acid ) OORS
6451cis gr009
??? ????: 9 : 4009 292
C16 : 1tans Paloniewdic and 9 :006 0 .5
Colcis (Paraticidad 38009
0170 (Margxic acid g 212 5
CV : tcis NIU 0 .5
C160 Semic act) grung 1.8
C16 : 1tans Elastic Sex }
081cis (Osica 91000 2
& tars Lincelaidis acid ) 1000 00
090 Nomeculce g ' 1908
018 :2 cia Lindaica ) 9: 3000
C200 ( kraciuidic acid) 2009 0 .5
016 :3 (GLA g 2009
0183 cia Ladlenic acid ??
C20: 1 cis (@ cosendic and 9:2004 40.5
C250 Mercioosacace DA1005 40.5
C220 Bebanie ucid 3: 0GS 07
020:2 cis 2009
C20 :3 vis PORT
C221 cis 0: 2009 .5
FIG . 1
U . S . Patent Jan . 30, 2018 Sheet 2 of 14 US 9 ,877,991 B2
36 otace
304 05
023 :0 Troen od
0222 03: 05
020 : 5 EPA
024 ,0 UKUR XXX 05
024 . is 02
022.5 CS @ pa om * .5
02268 094 08
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FIG . 1 Cont' d .
U . S . Patent Jan . 30 , 2018 Sheet 3 of 14 US 9 ,877 , 991 B2
Cuséwer Saxle D SARAite Fermented

Patent Foller
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NZLABS Sample Number 2355533 2378445
Date Sampied 2493/2011 2408241
{ sten Unt
Fatty mitwolie 404CX300
04: Brico ?
06:24 Valerici 100 2 :5 ?
Cocami 3 .5 »
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02 Fermi og 4
0 : 0 :0 CE BIODA ?
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034 01 Mywalczcie -0 .5
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05: a 10g *
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0210 Menecmacacir ? 30 . 5
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0273 vis 1649 < 3
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FIG . 2
U . S . Patent Jan . 30, 2018 Sheet 4 of 14 US 9,877,991 B2
TARerer Uni u
4890
C280 ( Hicosocia ) 60030 ki
0222413 sesió
023 :3 EPA Mos I
0240lmerke 345
0210s Bilo * $
2 - osipa 2003 05
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en un alt 2008 9839 03
2088 Fatty * sotila ( 1119 nyecer x 297 210 Mety

Xe 07529 & 0 376
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1 . 9333800 3Xweet 39 .
FIG . 2 Cont'd .
Protection of Coconut (1 :16 ) water extract to HL

60
on
ODRelative
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cellonlycellonly+H202 Extract
only
cellonly+Etoposide Extract+H2O2
Extracttreated+Etoposidetreated
FIG . 3A
U . S . Patent Jan . 30, 2018 Sheet 6 of 14 US 9,877,991 B2
protection ofCoconut ethanol extract
ODonm
@6rone
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1lea0tlnivmae tives
e
cells only H2O2
H202 Extract only Extract treated with
H202
FIG . 3B
atent Jan . 30 , 2018 Sheet 7 of 14 US 9 ,877,991 B2
» Extract Free Control

Protection from Tam water
Bethanol
longö
-
OD@6re0latnivme
Cells only H202 Etoposide
FIG . 4
U . S . Patent Jan .30, 2018 Sheet 8 of 14 US 9, 877, 991 B2
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U . S . Patent Jan . 30 , 2018 Sheet 9 of 14 US 9 ,877,991 B2
htens . MA279. 9 DES nODi RC1 01 17534 . d

X105 )
0.8 +
8 .68 min ; m / z 515 . 1200
( dicaffeoyl quinic acid )
S
10 .28 min ; m /z 529 . 1352

(dicaffeoyi tartaric acid
A
,
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ta ta
0 .2
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ntens . NA279 .4 DEG NODI RC2_ 01_ 17535 .0
X105
0.8 DE6 ' (24 hrs )
8 .41 min ; m / 2 515 . 1179
(dicaffeoyiquinic acid )
9 .79 min ; m /z 529 .1330
(dicaffeoyl tartaric acid
tetramethyl ether )
0.2
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Intens . MA279 .6 DEO noDi_ RC3 0117536 . d.
X105 )
0 .8 9 .03 min ; m /2 515 . 1189
DE9' (48 hrs ) (dicaffeoyl quinic acid )
10 .66 min ; m / z 529 . 1354
dicaffeoyl tartaric acid
tetramethyl ether )
0 .0 med
nters .
. . typen
DDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDD
* 105
Yu DE11' (72 hrs )
m /2515 . 1198
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m /z 529 , 1346
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mini
FIG . 6
000 W
FIG . 7A FIG . 7B
FIG . 8A FIG . 8B
FIG . 9A FIG . 9B
Average Severity Scoring ofAtopic Dermatitis

(SCORAD Index )
(Monitored from Baseline to Day 30 )
p - value - 0 . 049
A -value 1 ,004
30 .00 , ???????
3 . 787 3 .611 3 .811
25.00 3 . 182
20 .00 2874
2 .874
MSceoarens &
&
3.535
5 .00
0 .00
Baseline Day ? Baseline Day 15 Baseline Day 30
Pair ) Pair Pair 3
n 18 n 19 n = 19
Time points
FIG . 10
atent Jan . 30 , 2018 Sheet 14 of 14 US 9 ,877,991 B2
Average Dermatologic life Quality Index

(Monitored from Baseline to Day 30 )
Buvalue 0 .031
10 .00 1 .452
9.00 1.452
con W
1 . 138
1. 101
0 .956 1 . 155
5.00
SMceoarens 4 ,00
3 .00
2 .00
1 .00
0 .00
Baseline Day 7 Baseline Day 15 Easelin ??? 33
Par 1 air 2 Pair 3
20 18 0720
Timepoints
FIG . 11
US 9 ,877,991 B2
REACTION PLATFORM AND METHOD FOR creams are being used to treat dry and scaly skin (Loden , M .,
MAKING POLLEN BASED MATERIALS IN et al., “ A double -blind study comparing the effect of glyc
COMBINATION WITH BEESWAX AND USES erine and urea on dry , eczematous skin in atopic patients,"
THEREOF Acta Derm . Venereol. (2002 ) 82 :45 -47 ). Effectiveness of
5 such creams indicated a positive effect on the quality of life
This application is a U .S . National Stage application of the patients who tried the products (Eberlein , B ., et al.,
under 35 U .S .C . § 371 of International Application No. “ Adjuvant treatment of atopic eczema: assessment of an
PCT/IB2014 /002786 , filed on Aug. 12 , 2014 , which claims emollient containing N -palmitoylethanolamine ( ATOPA
the benefit of earlier filed U . S . Provisional Application No. study),” J. Eur. Acad . Dermatol. Venereol. (2008 ) 22 : 73 - 82 ).
61/865,011 , filed on Aug . 12 , 2013 , each ofwhich are hereby 10 Ifa
If a way could be found to use plant-based materials to
incorporated by reference herein . affect or control immune or PCD signaling pathways in
FIELD OF THE INVENTION mammalian cells , this would constitute a valuable contribu
tion to the nutraceutical and medical arts .
Further, if a way could be found to use plant-based
A process for making pollen -based products is described 15 materials
in connection with a reaction platform . Cosmetic and nutra including pollen -based extracts for treating
ceutical formulations containing pollen -based materials , inflammatory or skin conditions, this would constitute a
including polypeptides , amino acids, fatty acid triglycerides valuable contribution to the nutraceutical, cosmetic , and
and flavanoids in combination with beeswax suitable for medical arts .
administration to an individual are described . 20
SUMMARY OF THE INVENTION
BACKGROUND
A pollen -based extract includes germinated pollen , bee
Beehives in the natural state comprise interesting chem - hive components, and optionally enzyme- containing mate
istry including release of valuable bioactive components of 25 rial including honeydew or plant powders .
pollen that is transported into the hive, by the action of An aqueous skin cream includes the pollen -based extract
certain enzymes . However, use of the raw materials or and one or more of glycerine , natural oils , emollients ,
mixtures of the beehive presents problems including sepa - preservatives, vitamins, fragrance, emulsifiers , or waxes.
ration of components, contamination by microbial flora , The skin cream may be used for treatment of skin conditions
and / or exposure to pesticides used to eradicate pests such as 30 or inflammation .
mites . Thus, a method is needed to economize yet streamline A method of treating eczema or psoriasis is provided ,
production using the desired beehive components in syner- comprising administering to the individual in need of such
gistic combinations. treatment a therapeutically effective amount of an aqueous
The present inventor (Weir , formerly known as O 'Brien ) skin cream . The skin cream may be applied topically to the
has shown that plants possess internal genetic mechanisms 35 skin .
to control the process and progression of apoptosis , also A two stage reaction platform is provided to produce a
known as programmed cell death (PCD ). In one instance, pollen - based extract material, comprising the steps of a first
chromatin condensation , which is a hallmark of PCD in stage including: opening and/or germinating pollen grains ,
mammalian cells , may be reversible in plant cells during the reacting the treated pollen grains with one or more beehive
early stages of apoptosis ( O 'Brien , et al., The Plant Journal 40 components , selected from beeswax , honey , or enzyme
( 1998 ) 13 (6 ): 803 -814 ). containing material, and stirring to form a jelly ; and a second
Pollen has a hard shell known as the sporopollenin which stage including heating the jelly in a closed container to
is very resistant to chemical degradation . Also on the surface product an extract. The extract may be a fermented extract.
of the pollen are proteins which cause known allergies . In It is understood that the extract may be further fermentable
one method a reaction is used to " explode” or “ crack ” the 45 by the addition of one or more components .
pollen grains under pressure followed by a protease to
hydrolyse and deactivate the allergy producing proteins . BRIEF DESCRIPTION OF THE DRAWINGS
In another possible two -step reaction, pollen may be
manipulated to begin germination and thus release the FIG . 1 depicts a fatty acid analysis of the opened and
bioactive contents of the pollen more gently and naturally . 50 fermented pollen prepared according to an embodiment of
Therefore , if a way could be found to first open or the present invention .
germinate pollen grains to release beneficial components in FIG . 2 depicts a fatty acid analysis of the opened and
a biotic manner, followed by addition of other beehive fermented pollen (both upper layer from reaction and sedi
components, this would mimick a natural process, yet pro - ment) prepared according to an embodiment of the present
vide a novel way to obtain nutraceutical or cosmetic prod - 55 invention .
ucts . FIG . 3A depicts treatment of HL -60 cells in MTT assay
Atopic dermatitis, commonly known as eczema, is a with an extract based on coconut water having very potent
complex skin disease that is characterized by pruritus, cell protection activity as an unfermented " water " extract .
disrupted epidermal barrier function , and immunoglobulin FIG . 3B depicts treatment of HL -60 cells in MTT assay
sensitization to various food and environmental allergens 60 with an extract based on coconut water having very potent
(Sohn , A ., et al., “ Eczema,” Mt. Sinai J. Med . (2011) cell protection activity as a fermented 2 -Stage " ethanol”
78 :730 -739 ). Inflammation of the skin appears like ery - extract and promoting cell proliferation .
thema, which may include scaling and crusts (Krafchik , B . FIG . 4 depicts treatment of HL -60 cells in MTT assay
R ., “ Eczema,” Paediatr . Child Health (2000 ) 5 : 101 - 105 ). It with an extract based on coconutwater including tamarillo
usually occurs due to the interaction of the genes with the 65 powder having very potent cell protection activity as a
environment. Patients with eczema develop a higher risk for fermented 2 - Stage " ethanol” extract and promoting cell
skin infections. Skin care products including moisturizing proliferation .
US 9 ,877 ,991 B2
FIG . 5 depicts gel electrophoresis ( SDS -PAGE) analysis Pollen has a hard shell known as the sporopollenin which
of the opened and fermented pollen prepared according to an is very resistant to chemical degradation . Also on the surface
embodiment of the present invention . Lane markings are as of the pollen are proteins which cause known allergies. In
follows: DE2-DE9 (12 .5 ul loading), protein Markers, and one method a reaction is used to “ explode” or “ crack ” the
DE2 -DE7 ( 2 .5 ul) loading , as described below . 5 pollen grains under pressure (a commonly use abiotic
FIG . 6 depicts RP -UHPLC and negative ion electrospray approach ) followed by use of a protease to hydrolyse and
ionization (ESI) analysis of the opened and fermented pollen deactivate the allergy producing proteins. Optionally, a
prepared according to an embodiment of the present inven - peptidase may then be used to create peptides from the soup
tion ( Example 2A ), showing products of the stage 2 fermen - or mixture created
tation reaction up to 72 hrs . 10
Instead of trying to explode the pollen grain (which did
FIG . 7A shows a photograph of a hand of a human patient partially work ), an attempt was made to manipulate or treat
having severe eczema before treatment the pollen grains to begin germination and thus naturally
FIG . 7B shows a photograph of the eczema patient of FIG . release their contents thus optimizing the bioactivity and
7A after 4 weeks of topical treatment using a fermented providing a far more targeted product. This may be consid
pollen -based cream of Example 6B . 15 ered a “biotic” approach to open the pollen . If the pollen
FIG . 8A shows a photograph of an arm of a human patient grains are exploded then the compounds released are more
having severe eczema before treatment. in response to stress (which is good for some medical
FIG . 8B shows a photograph of the eczema patientof FIG . conditions), but if they are gently released through germi
8A after 4 weeks of topical treatment using a fermented nation then they promote cell renewal and repair , as dis
pollen -based cream of Example 6B . 20 cussed previously . By using the biotic germination
FIG . 9A shows a photograph of a leg of a human patient approach , the viable pollen grains began germinating whilst
having psoriatic lesions before treatment. the damaged or dead pollen grains remained and could be
FIG . 9B shows a photograph of the psoriasis patient of filtered out. Thus this approach was also an improvement
FIG . 9A after 4 weeks of topical treatment using a fermented through removal of the dead pollen grains .
pollen -based cream of Example 6D . 25 In one embodiment, it has been discovered that a two step
FIG . 10 depicts a bar graph showing Severity Scoring of process can be used to produce a pollen - based extract
Atopic Dermatitis (SCORAD ) index for a clinical treatment material, including opening and /or germinating pollen
group (n = 20 ) receiving a skin cream (Example 6B ) contain - grains using a biotic method , and reacting the treated pollen
ing fermented pollen - based extract prepared according to an grains with one or more beehive components, such as , for
embodiment of the present invention . 30 example , beeswax , propolis , etc .
FIG . 11 depicts a bar graph showing Dermatology Life Development of fermentation method and opening of
Quality Index (DLQI) for a clinical treatment group (n = 20 ) pollen grains.
receiving a skin cream ( Example 6B ) containing fermented Initially, small amounts of dry pollen (25 g ) were mixed
pollen -based extract prepared according to an embodiment with white sugar (5 g) and active yeast (5 g) in water (500
of the present invention. 35 mL ), inside of a sealed stainless steel bottle . At ambient
temperature this mixture produced fermentation after about
DETAILED DESCRIPTION 72 hours, with production of alcohol and attendant alcoholic
odor.
A detailed description of one ormore embodiments of the Follow on experiments performed with raw honey
invention is provided below along with accompanying tables 40 (Manuka , Rewa Rewa, clover ) and honeydew from beech
and figures that illustrate the principles of the invention . As trees in Southern New Zealand (Nothofagus spp .) beech
such this detailed description illustrates the invention by forests (isolated from the New Zealand beech scale insect
way of example and not by way of limitation . The descrip Ultracoelostoma assimile (Maskell)). Within New Zealand
tion will clearly enable one skilled in the art to make and use this insect occurs mainly in South Island and is most
the invention , and describes several embodiments , adapta - 45 common on black beech ( N . solandri solandri) and moun
tions, variations and alternatives and uses of the invention , tain beech ( N . s . cliffortioides ), but generally any beech will
including what we presently believe is the best mode for suffice (Gaze, et al., N . Z . J. Ecol. (1983 ) 6 :33- 37 ). In these
carrying out the invention . It is to be clearly understood that experiments the fermentation reaction occurred within about
routine variations and adaptations can be made to the 24 hr at higher temperatures (37° C ., 42° C ., or 65° C .),
invention as described , and such variations and adaptations 50 while it took 48 hr at 25° C . It was shown that about 66 %
squarely fall within the spirit and scope of the invention of the pollen grains open . Also , the use of honeydew did not
In other words , the invention is described in connection produce an alcoholic odor, but instead a very golden yellow
with such embodiments , but the invention is not limited to oily soup.
any embodiment. The scope of the invention is limited only Analysis of fatty acid (FA ) content was performed after
by the claims and the invention encompasses numerous 55 fermentation and pollen grain opening , as shown in FIGS. 1
alternatives, modifications and equivalents . Numerous spe and 2 (NZLabs, Auckland , New Zealand). See also , Pre
cific details are set forth in the following description in order parative examples A and B below for exemplary filtration of
to provide a thorough understanding of the invention . These sediment after fermentation .
details are provided for the purpose of example and the The results of the FA analyses indicates clearly that in the
invention may be practiced according to the claims with or 60 opened pollen grains the fatty acid content was preserved
without some or all of these specific details. and protected. The key thing that this experiment demon
A safe and effective pollen -based skin cream has been strated was that the fatty acids were released from the pollen ,
provided containing beeswax and optionally enzymes , while in non - exploded pollen there were no fatty acids in the
which can be administered in a therapeutically effective soup that was analyzed .
amount to an individual for treatment of eczemaor psoriasis. 65 Development of Reaction Platform .
The pollen -based skin cream may be applied topically to the Once a way had been found to efficiently open pollen
skin . grains, reaction of the pollen grain contents and other
US 9 ,877,991 B2
components from the beehive (such as beeswax , propolis, Canterbury, New Zealand . Propolis can be prepared for use
etc.) was performed . The process was developed via several as a water extraction by treating 25 g propolis / 50 mL water
preparative examples as described below . in a dark sealed glass vessel, with gentle rotation for 5 days
It was found that honeydew (from Southern Beech For at 28° C .
ests ) which contains insect enzymes worked better than raw 5 Glycerol (i.e ., glycerine ) is available from Sigma Aldrich
honey. Honeydew is a substance that is defecated from scale (St. Louis , Mo. ).
insects on beech trees as they eat the sap . This honeydew is Water may be filtered , sterilized rain water, distilled
a honey substance but contains enzymes from the scale water, deionized water, RO water, and the like .
insects and their " gut microflora , including fructophilic
lactic acid bacteria.” This finding led to the conclusion that 10 Preparative Example a (Including Pineapple
honeydew contains enzymes from the scale insect and from Powder )
the various bacteria , which of course was also in certain raw
honeys that were tested. Without being bound by theory, it The following ingredients were blended : raw pollen ( 50
is thought that the honeydew enzymes would be similar to g ), raw beeswax ( 25 g ), pineapple powder ( 12 .5 g ), raw
the bee saliva and thus also aid in the second stage of the 15 honey ( 12 .5 g ), glycerol ( 12 .5 g ), and water (1000 mL ).
process . After blending the reaction mixture in a stainless steel
Description of ingredients used in the representative two container washeated to 60° C . and stirred for 15 min . At this
step process in various useful forms are as follows. point the steel container was anaerobically sealed and pres
Raw pollen : contains fatty acids , polypeptides , EPA , surized to 120 psi. The container was stored at 60° C . for 24
DHA , long-chain alkanes , hormones, vitamins, phytohor - 20 hours. During this period pressure builds up in the container,
mones , cellulose , lignin , and flavanoids. and the mixture became bubbly and exothermic. Within 16
Raw beeswax : contains pheromones, phytic acid , chitin , to 24 hours the reaction naturally stopped. The solution was
and fatty acids. then filtered to remove sediments . This reaction product
Raw honey : contains naturally occurring bacteria , such as extract was heated , poured into a cream formula ( e . g ., Ex. 3 ),
lactic acid bacteria . 25 and whipped using an egg beatermanually until smooth (i.e .
Honeydew : contains potassium , sugars , insect enzymes having a viscous even consistency ).
and fructophilic lactic acid bacteria . This reaction resulted in a reaction product solution that
Pineapple powder : contains bromelain , a homologous still possessed protease activity , peptides and triglycerides.
serine protease , and peptidase enzymes ; contains potassium . In this manner the reaction product was formulated at 5 wt
Alternative plant powders containing flavanoids and /or 30 % in a commercially available aqueous cream (see , e.g .,
enzymes : tamarillo (antioxidant and enzymes ), blackcurrant Experimental Aqueous Cream Formulation , Example 3 ).
(antioxidant/cellular repair /brain function ), kawa kawa This formulated reaction product was trialled on subjects
(bladder and digestive health /antioxidant/ anti- inflamma- with various skin disorders . This reaction productworked on
tory/ detoxifying/acne), propolis (acne ), pine bark (female age spots , crusty eczema, psoriasis, herpes, corpus mollus
hormone regulation and sunscreen ), acai, pomengranate , 35 cum and acne. However, the reaction product caused irrita
cherry (sleep aid/melatonin ),kiwifruit, paw paw , and feijoa . tion when used on inflammed eczema or lupus.
Freeze dried plant powders can be prepared as follows. A
pulp of the desired plant is prepared , such as pineapple, Preparative Example B (Including Pineapple
tamarillo , kawa kawa, etc . The resulting pulp is frozen down Powder and Tamarillo Powder )
to - 20° C . over 48 hours and then freeze dried . Alternatively , 40
useful freeze -dried plant powders are available from Alaron The following ingredients were blended : raw pollen (50
GMP Manufacturers (Alaron Products , Nelson , New Zea - g ), raw beeswax (25 g ), pineapple powder (6 .25 g ), tamarillo
land ). Alternatively, spray dried or even fresh plant sources powder (6 .25 g ), mussel oil (25 g ), honey ( 12 .5 g ), glycerol
can be used in the reaction , for example pineapple stem and (12.5 g ), and water (1000 mL ). After blending the reaction
fruit was originally used prior to access of freeze dried 45 mixture in a stainless steel container was heated to 60° C .
powders. and stirred for 15 min . At this point the steel container was
Other useful plant components may include , but are not anaerobically sealed and pressurized to 120 psi. The con
limited to : apple flesh or skin ; avocado pulp or skin ; beech tainer was stored at 37° C . for 24 hours. During this period
bark or leaves ; blueberries , blackberries ; acai berries, gin - pressure builds up in the container, and the mixture became
ger ; grape skin ; hemp oil ; rimu bark or leaves ; turmeric ; 50 bubbly and exothermic . Within 16 to 24 hours the reaction
onion ; orange ; kiwifruit - flesh , skin , or seeds ; kauri bark or naturally stopped . The solution was then filtered to remove
leaves ; kohe kohe; mango ; manuka oil or leaves; noni; sediments .
olives — waste , skin or oil; grape seed oil, argan oil , jojoba In this experiment the temperature was lowered to protect
oil, and pohutakawa. the plant powders . This process resulted in peptides, esteri
Coconutmilk , i.e ., Coconutwater : contains hormones and 55 fied fatty acids and esterified flavanoids . This lower tem
nutrients . Coconut water /milk is obtained by buying fresh perature also protected the amino acids and vitamins from
coconuts and draining the fresh milk just prior to use . the pollen from heat denaturation . It is further contemplated
Commercially available coconut milk can be used but can that other oils may be used in place of mussel oil, including ,
not be pasteurized . but not limited to a combination of mussel oil/ coconut oil
Coconut oil: contains polypeptides, fatty acids, EPA , 60 ( 1 : 1 wt/wt).
DHA , and in particular myristic acid . One useful brand is To the extent the above reactions are producing and/or
Home Essentials brand , 100 % . undergoing fermentation , the pollen - based material pro
Mussel oil: contains polypeptides, fatty acids, EPA , and duced is considered to be fermentable .
DHA . In an embodiment, the pollen -opening fermentation reac
Certain beehive components , for example honey, honey - 65 tions can be performed as described in a sealed steel
dew , pollen , beeswax , raw propolis are sourced from Scott container or other appropriate pressure vessel. In an alter
Apiaries , Trading as Hanmer Bees, Hanmer Springs, North native embodiment, the pollen - opening fermentation reac
US 9,877 ,991 B2
tions can be performed as described aerobically in appro at 37° C . resulting in the reaction more anaerobic as it forms
priate plastic or glass containers or reaction vessels . a solid wax layer over the other components. If this reaction
One problem to this point was that all other competitors , is left for 7 days the beeswax is digested by the enzymes and
and the process as described herein involved cracking open
the pollen grain to release the contents within (an abiotic 5 becomes part of the solution (i.e., self- emulsifying ).
approach ) . What was most desired was a biotic method to Example 2A ( Eczema/Psoriasis)
produce those bioactive components in an enhanced state of
bioactivity , as in when a pollen grain begins to germinate . At
this point the contents are prepared during the osmosis
process to be bio -available to optimal germination . Ingredient Weight (g )
The plant cell germination process is equivalent to
embryogenesis in humans and thus optimal for cell renewal, Pollen 50 g
o
division , wound repair, etc . Beeswax 25 ??g

Coconut milk ( a.k .a . coconut water ) is known to contain Coconut Oil 25 g
all the nutrients required for embryogenesis and the plant Glycerol 12.5 g
u
Honeydew 12.5
hormones including indoleacetic acid , gibberillin , and aux - 15 Coconut Water 200 ml
ins , which stimulate cell division and growth . Coconutmilk
also contains potassium required for germination via osmo
sis. One study found that coconut milk (now known as ( Stage 1) Pollen was soaked in coconut water for 30
coconutwater) may be used to turn plant cells totipotent and minutes at 25° C . Concurrently the beeswax was melted in
to grow from a single cell into a whole new plant. This was 20 a stainless steel bowl, which is sitting in boiling water. Once
carried out with the species lisianthus in which no one has the beeswax was melted it was stirred vigorously for 5
previously been able to regenerate as it was recalcitrant to minutes, and the bowl placed into the incubator at 65° C .
normal hormone stimulants (O 'Brien , et al., Plant Cell resulting in the beeswax remaining liquid .
Tissue and Organ Culture ( 1993 ) 33: 31 - 37 ) . After 60 minutes soaking , glycerol was then stirred into
Instead of cracking open the pollen grain , a biotic method 25 the pollen mixture , followed by the honeydew . This mixture
is shown herein to trigger germinaton and thus activate it, is then stirred for 30 minutes at 25° C ., which completed part
that is produce enhanced bioactivity . Without being bound 1 . Next, the coconut oil was stirred into themelted beeswax ,
by theory, it is believed that if the pollen grain were which completed part 2
germinated , then enzymes in honeydew and /or from the
h The pollen
pollen itself can provide the protease /peptidase enzymatic 30 beeswax mixture (part 1 ) was then stirred into the
mixture ( part 2 ) and the whole mixture stirred
reactions that other components could provide (e .g ., Lacto vigorously by hand for about 15 minutes until a thick jelly
bacillus or pineapple powder ). formed (Stage 1 ends ). It is noted that stirring by hand was
Pollen grains will begin to germinate based on an osmotic laborious and that magnetic stirring on a hot plate would be
uptake of potassium . If used in the reaction platform ,
honey /honeydew and pineapple all contain enough potas - 35 use(Stage 2 ) This jelly mixture was then placed into a
sium to trigger this reaction , particularly at warmer tem
peratures. Coconut milk is also very high in potassium and stainless steel container with a screw sealable lid , and
thus the combination
thus the combination of
of the
the potassium
potassium with
with the
the phyto
phyto- incubated at 37° C . for a minimum of 72 hours, which
hormones result in triggering germination . produced the isolated 2 - stage extract (Stage 2 ends).
By using the coconut water it incorporates into the 40 In a preferred embodiment, plant powders (or other useful
product theminerals and growth hormones contained within plant parts) such as tamarillo , kawa kawa, and the like, may
the coconut water. These components work in synergy with be added after the jelly is formed , then the whole mixture
the pollen to promote cell renewal and cellular repair, in subjected to heating under sealed conditions and/ or fermen
contrast to the stressor effects of cracking or rupturing tation . In an alternative embodiment, after the jelly is
pollen . 45 formed , if any other components such as, for example ,
The methods described above may be further understood pineapple powder, fruit powder, etc . are required , these are
in connection with the following Examples . added as the mixture cools . That is , tamarillo , kawa kawa,
Example 1 (Reaction Platform ) etc . can be added after the end of Stage 2 .
50
In a further embodiment, other components such as, for
example , pineapple powder, tamarillo , kawa kawa , fruit
The following ingredients were blended : raw pollen (50 powder
g ), coconut milk (200 ml), and honeydew (12 .5 g ). These heated . , etc . may be added after the end of Stage 2 and
three ingredients were warmed to 30° C . and stirred for 30 It is understood that the stage 2 fermentation process can
minutes to stimulate germination . Next, the following ingre
dients were added : melted raw beeswax (25 g), glycerol 55 Inpe carried
55 be out in a range from about 24 hours to 168 hours.
one preferred embodiment, incubation is carried out at
(12 .5 g ), and water ( 1000 ml). Optional ingredients may
include: coconut oil, pineapple powder , or other plant pow 37° C . for about 24 hrs to 72 hrs. In a more preferred
ders (as listed above ). Depending on what components are embodiment, incubation is carried out at 37° C . for about 72
added and the end desired medical or nutraceutical treatment hrs .
the reaction is either carried out at 25° C ., 30° C ., 37° C ., 42° 60 In another alternative embodiment, increases in coconut
C ., or 65° C . The reaction platform can be carried out either water content of stage 1 can reduce manufacturing costs .
anaerobically or aerobically . In another alternative embodiment, the melted beeswax
( or part 2 component ) can be maintained at lower tempera
Example 2 ( Two Stage Reaction Platform ) tures ( e . g ., 42° C ., or 37° C .) in order to preserve other more
65 sensitive components (such as amino acids, peptides, or fatty
All reactions performed below are incubated anaerobi- acids). In this embodiment, the beeswax self-emulsifies into
cally for 72 hours at 37° C . Optionally, beeswax may be used the final product mixture.
US 9,877 ,991 B2
10
Example 2B (Age Spots / Actinic Keratosis) Example 2F (Core Formula _ Optional Tamarillo )
Ingredient Weight (g ) s Ingredient Weight (g)

Pollen in ?
Pollen 50 g
Beeswax Coconut Oil
12 . 5 oa
~
ucu Glycerol
Coconut Oil ~
Honeydew 12 .5 g
Glycerol 12 .5 ??g Coconut Water 200 ml
10
Honeydew 12 .5 g Tamarillo powder (optional) 12.5 g
Coconut Water 200 ml
Pineapple powder 12 .5 g
Example 2G (Cosmetic — Beauty /Female Hormonal)
15
Example 2C (Antimicrobial and Wound Healing )
Ingredient Weight ( g )
Pollen 50 g
Ingredient Weight (g ) 20
20 Coconut Oil 25 ??g
Glycerol 12. 5 g
Pollen 50 Honeydew 12. 5 g
Beeswax 25 ??g Coconut Water 180 ml
Coconut Oil 25 g Algae water 20 ml*
Glycerol 12 . 5 g Pine bark powder a 12.5 g
Honeydew 12 .5 g 25 *Micro algae is grown to an exponential phase, centrifuged and slurry added .
Coconut Water 200 ml
Propolis 5 ml* a Pine bark powder is prepared by hot water extraction of pine bark , then freeze drying.
* Added at the end of the reaction . Propolis is prepared as 25 g /50 ml water .
Chemical Analysis
30 Reaction samples made in accordance with the principles
Example 2D (Anti- Viral _ Herpes, Shingles, Corpus of the invention , namely Example 2A or 2B , were analyzed
Molluscum , Chicken Pox ) to assess how the chemical components in these mixtures
changed during incubation . Phytochemical analysis was
performed by liquid chromatograph -high resolution mass
35 spectrometry (LC -HRMS), and the protein /peptide compo
Ingredient Weight (g) sition was assessed by SDS acrylamide gel electrophoresis
(SDS -PAGE), using standard methods .
Pollen
After 0 hrs ( starting material) to 72 hrs of incubation in
Beeswax
Coconut Oil
Glycerol
Honeydew
N Uü
12 .5 g
12.5 g
09 the stage 2 fermentation reaction , the samples were thor
40 oughly mixed and a portion ( 1 .0 - 1 . 5 g ) extracted with
ethanol. Pineapple powder was added to starting material
Coconut Water 200 ml DE5 and DE7 ( Ex . 2A with or without beeswax , respec
Kawa Kawa powder 12 .5 g
Propolis 5 ml* tively ) after time 0 for incubation in accordance with
Example 2B . The resulting extracts were analyzed by LC
* Added at the end of the reaction. Propolis is prepared as 25 g/50 mlwater. 45 MS using reversed -phase ultra -high performance liquid
chromatography (RP -UHPLC ) and negative ion electro
In following examples 2E , 2F and 2G beeswax is omitted , spray ionization (ESI).
so the solution is more liquid ( less viscous) upon addition of Now referring to FIG . 5 , the samples tested were as
the oil, and the reaction takes another 24 -48 hours to follows:
complete . 50 DE2 : Extract sample 2A , without beeswax , 72 hrs
DE3: Extract sample 2A , 72 hrs
Example 2E (Cosmetic — Beauty / Anti-Aging ) DE4: Extract sample 2B , without beeswax , 24 hrs
DE5 : Extract sample 2A , 0 hrs (starting material — Stage
DE6 : Extract sample 2B , 24 hrs
Ingredient Weight (g ) DE7: Extract sample 2A , without beeswax , 0 hrs ( starting
material Stage 1)
Pollen
Coconut Oil
50 g
25 g
DE8: Extract sample 2B , without beeswax , 72 hrs
Glycerol 12.5 g
DES: Extract sample 2B , 72 hrs
Honeydew 12.5 g 60 A 12 . 5 % reduced SDS - PAGE gel was used . The markers
Coconut Water 180 ml were Biorad # 161-0373 , stain type : All Blue (Bio -Rad Lab .,
Algae water 20 ml* Hercules , Calif.).
* Micro algae is grown to an exponential phase, centrifuged and slurry added to reaction
Generally, FIG . 5 shows that the larger molecular weight
vessel just prior to fermentation at 30° C . proteins were degraded or digested into smaller molecular
65 weight protein components as part of the 2 - stage process .
Optionally, 12 .5 g blackcurrant powder is added to this Samples DE5 and DE7 were the (stage 1) starting mate
example as an antioxidant. rials for the 2 -stage process and showed that a significant
US 9 ,877,991 B2
11
amount of protein in the sample was of a large molecular
12
- continued
weight (> 250 kD ) so much so that much of it did not enter
the gel. Samples DE2 and DE3 showed a significant band Ingredient Wt/Wt ( % )
between 50 and 75 kD (which was also present in lesser Sodium Lauryl Sulfate 0 .9 %
amounts in the remaining samples ). Another band at about 5 Phenoxyethanol 1 .0 %
25 kD appeared in samples DE4 , DE6 and DES , and to a Purified Water 70 .0 %
lesser extent in DE9 .
Samples DE4 , DE6 and DE8 showed significant staining Aqueous cream without the extracts added was used as
at molecular weights less than 15 kD . Samples DE2 , DE3 the placebo , thus the prerequisite was that the addition of the
and DE9 showed protein / peptides to a lesser extent in this 10 extract had to be clinically significantly better than the
range . aqueous cream which is the current non - steroidal anti
Thus, the size profile of proteins in the incubated samples eczema treatment .
over time is overtly smaller compared to the starting mate Accordingly , the use of parabens significantly inhibited
efficacy of the extract in several human volunteers. Phe
rial (DE5 and DE7 ), either with or without pineapple powder 15 noxyethanol
enzymes , or with or without beeswax . did not fully inhibit but still reduced efficacy
extract in several human volunteers , which was hypoth
Turning now to FIG . 6 , the progression of the stage 2 esized as due to the bacteriocidal properties. Similar results
incubation was observed and analyzed for reaction products . to parabens were found with methyl parahydroxybenzoate ,
The samples tested were as follows: i.e . efficacy of the extract was significantly reduced .
DE5: Extract sample 2A , O hrs (starting material — Stage 20 Next, a batch of aqueous cream and was prepared using
1 ), as above Geogard 221, a bacteriostatic preservative as a replacement
for phenoxyethanol and / or parabens, containing dehy
DE6 ': Extract sample 2A , 24 hrs droacetic acid 8 % , benzyl alcohol 87 % (available from
DES': Extract sample 2A , 48 hrs Lonza , Allendale , N .J.). This formulation provided full
DE11': Extract sample 2A , 72 hrs efficacy of the extract when tested in several human volun
The progression of the stage 2 fermentation reaction teers . Thus , the type of preservative was found to be
showed a pattern of compounds that were determined to be important, arguably an additive having more " natural prod
dicaffeoyl quinic acid isomers , and, it is believed , dicaffeoyl uct ” similarity .
tartaric acid ester or ether compounds. As shown in FIG . 6 , Example 4 (Natural Base Cream Formulation )
after 72 hours , it was observed that multiple isomers of the 30
phenolic compound dicaffeoyl quinic acid had formed ,
which is an indicator or marker of the 2 - stage reaction . It is
believed thatmultiple dicaffeoyl tartaric acid ester or ether Ingredient Wt/Wt ( % )
isomers formed as well (e. g ., a tetramethyl ether of the Cetostearyl Alcohol 8.1 %
caffeoyl portions) . These samples have been shown to 35 Cetomacrogol* 15. 0 %
contain substantial amounts of the caffeoyl quinic acid Glycerine 2. 5 %
isomers , and, it is believed , dicaffeoyl tartaric acid ester or Crodamol (coconut oil ) 2 .0 %
ether compounds (i. e ., chicoric acid derivatives, including Beeswax
Geogard * *
2 .0 %
0 .5 %
methyl ethers or esters ). Purified Water 70 .0 %
Thus, while not intended to be bound by theory, peptides 40 * Cetomacrogol 1000 , a PEG polymer
are created in the 2 - stage method according to a process of * * Geogard 221
“ lipophilization ” or “ lipolyzation ” in which protein is
digested , phenolic compounds are isomerized ormethylated , Thus , a useful aqueous cream formulation was discovered
and fatty acids may be involved in esterification reactions to that is very stable and uses no bacteriocidal preservatives .
produce useful and beneficial new products . In another 45 This material is a beautiful soft cream that may be applied
embodiment, it is expected that hydroxylation and/or acety to human skin .
lation of flavonoids may occur in conjunction with fatty To the base cream of Example 4 was added the two - stage
acids . In another embodiment, it is expected that hydrolysis extract of Example 2A in an amount of 5 % by wt. based on
of tannins, or glycosylation of aglycones may occur during the total weight of the formulation . Useful amounts of the
the 2 - stage fermentation reaction . In yet another embodi- 50 two -stage extract can range from about 0 . 5 % by wt. to about
ment, it is expected that hydroxylation of fatty acids may 10 % by wt. based on the total weight of the formulation . A
occur during the 2 - stage fermentation reaction . In fact, the preferred range of the two - stage extract can range from
lipid composition of the 2 - stage reaction product produced about 0 .5 % by wt. to about 5 % by wt. based on the total
increased lipid content as observed by HPLC analysis. weight of the formulation .
An emollient cream was developed as follows.
55 In addition , essential oil or fragrance was added to the
base cream of Example 4 , in that tangerine oil extract was
added in an amount of 0 .4 % by wt. based on the total weight
Example 3 (Experimental Aqueous Cream of the formulation . Useful amounts of one or more essential
Formulation ) oils can range from about 0 .4 % by wt. to about 1 % by wt.
60 based on the total weight of the formulation .
Example 5 (Testing of Base Cream Including
Ingredient Wt/Wt (% ) Example 2A Extract)
Cetostearyl Alcohol 8.1 %
White soft paraffin 15 . 0 % 65 The base cream of Example 4 including 5 wt % Example
Liquid paraffin 6 .0 % 2A extract and 0 . 4 wt % tangerine oil extract was tested as
follows using human volunteers ( Table 1) .
US 9, 877,991 B2
13 14
TABLE 1 Olivem 1000 , available from B & T Srl (Milan , Italy ), is a
Subject
nonionic, non - ethoxylated self-emulsifying system derived
Skin / Inflammatory from olive oil for oil- in -water creams and lotions.
No. Sex Age Condition Result
Initial testing using Example 6A in human volunteers as
M 51 psoriasis on limbs redness and scaly skin 5 in Example 5 gave similar results with respect to eczema and
=WN cleared within 5 days psoriasis, etc .
F 6 corpus molluscum skin cleared within 4 days
F 48 lupus history of steroids ,
showed reduced flare Example 6B (Skin Cream Formulation )
ups and severity
4. F 35 lupus history of steroids,
showed reduced flare 10 In a further embodiment, a skin cream formulation was
ups and severity prepared .
F 22 lupus used instead of steroids ,
redness reduced , no need
for steroids
over 4 days itching,
Ingredient
Ingredient Wt ( % )
eczema 15
redness and infection Olivem 1000 ECO CERT (Cetearyl Olivate
cleared & Sorbitan Olivate )
agespots , solar over 2 weeks crusted and
VEN OWN
3
Shea Butter Organic
keratosis exfoliated off 2 - Stage Extract ( Example 2A ) "
o
F 15 eczema
eczema over 4 days itching, Safflower Oil Organic
redness and infection
cã?EouAWNva F 23 eczema
cleared
over 4 days itching,
cleared
20 Macadamia Nut Oil Organic
Deionized Water
Glycerine
Geogard 221
Vitamin E
10 M 45 eczema over 4 days itching, Tangerine Fragrance (tangerine essential oil)
cleared 20 lipolyzed fatty acid and peptides, as shown in FIGS. 1, 2 and 5 as described herein .
M 28 eczema over 4 days itching ,
redness and infection The final formulation is an off -white viscous cream , pH
cleared 4 .0 -5 .0 . Microbiological properties: aerobic plate count :
12 F 33 eczema over 4 days itching,
cleared
( 30° C .) 377 cfu /g ; yeast andmold : < 1 cfu / g . The cream may
F over 4 days itching, 30 be applied topically by hand to a human skin surface .
13 16 eczema In an embodiment, the 2 -stage extract may be used in a
cleared range of from about 1 % by weight to about 10 % by weight .
14 7
42 eczema over 4 days itching , In another embodiment, water may be used in a range of
redness and infection from about 70 % by weight to about 90 % by weight.
cleared 35 Increases in water content, and /or alternatively , increases in
15 M 58 psoriasis redness and scaly skin
reduced coconut water content of stage 1 ( as discussed above ) can
16 M 22 psoriasis redness and scaly skin reduce manufacturing costs .
17 F 17 psoriasis
reduced
redness and scaly skin
The cream formulation of Example 6B was tested in a
reduced clinical trial as follows.
M 51 itch
athletes foot/jock fungal infection clears 40
Example 6C (Clinical Study Eczema )
As shown in Table 1 , this formulation cleared the eczema nessTheofpurpose
Example
of this study was to determine the effective
6B in the treatment of mild to moderate
and psoriasis on the volunteers . Subjects 1 , 3 , 4 , 5 , 8, 9 and
10 spent a week with no cream and then trialled Example 6 45 eczema, including reducing the appearance of lesions,
cream (below ) prepared for the clinical trial with equivalent reducing the symptoms of itching and scaling, and reducing
results to Example 4 . redness .
Clinical Study Design
Example 6A (Skin Cream Formulation ) This study was designed as an open -label, adaptive
50 design pilot study.
A skin cream formulation was prepared as follows. Otherwise healthy subjects , 18 - 70 years of age , with mild
to moderate eczema were included in the study. A total of
n = 40 subjects were screened , and from this , n = 21 were
Ingredient Wt ( % ) randomized ; however, only n = 20 subjects completed the
55 study
S . Other standard inclusion / exclusion criteria were used .
Olivem 1000 ECO CERT (Cetearyl Olivate Adverse events were monitored . Initial IRB approval of the
& Sorbitan Olivate )
Shea Butter Organic protocol was granted by the MaGil IRB (Rockville , Md.).
UNA
Macadamia Nut Oil Organic All recruitment materials were approved by the IRB prior to
Myristyl Myristate use .
Safflower Oil Organic 60 The study duration was up to 5 weeks for each subject
Glycerine
Geogard * * comprising 5 visits , with the testing phase being 30 days
Vitamin E (approx . 4 weeks ).
2 - Stage Extract (Example 2A ) V1: Screening Visit - Day -7
Tangerine Fragrance (tangerine oil extract)
Deionized Water V2: Day0Baseline
6 V3 : Day 7
* *Geogard 221 V4: Day 15
V5: Day 30 — End of Study
US 9,877 ,991 B2
15 16
Subjects were required to undergo a 7 -day washoutof any Redness of the Eczema Skin Lesions. Analyses revealed
and all treatments for eczema. significant decreases in the Average VAS Symptom Score
Dermatologic Assessments . Dermatologic examinations for Itching, Scaling , and Redness from Week 1 to Weeks 2 ,
by a qualified practitioner were performed . This assessment 3 , and 4 (p - values0 .05 , for all time points ).
includes the Severity Scoring of Atopic Dermatitis (SCO - 5 In accordance with Table 1A , Itching ( VAS) Symptom
RAD ) an evaluation and grading of lesion quality . Subjects Score was reduced - 30 . 27 % from baseline from week 1 to
completed the Dermatologic Life Quality Index (DLQI), a week week 4.
In accordance with Table 1A , Scaling (VAS) Symptom
self-assessment on each subject 's own skin condition .
Photographs. Subjects were photographed to examine and WScore was reduced - 30 .53 % from baseline from week 1 to
evaluate skin lesion (s ), which were identified by anatomical In accordance with Table 1A , Redness (VAS) Symptom
location and size .
Dispensing Procedures . The following were dispensed at week Score was reduced - 33 . 95 % from baseline from week 1 to
this visit: 4.
Severity of Eczema was found to have statistically sig
Subjects were given a one -week supply of study product 1515 nificant changes from Baseline to Day 30 (p -values0 .05 ).
(Example 6B cream ) and instruct to apply it twice out a day. Overall, moderate eczema was reported to decrease in
Subjects were given a daily dosing diary .
Instruction : Patients were instructed to apply the cream severity and 19 .44 % lesions had disappeared by Day 30 . It
topically to the affected area( s ) twice per day. That is, were alsothatreduced
is noted the majority of the moderate to severe lesions
(based on a count of lesions , and also
patients with eczema were instructed to apply the cream a ,20 assessment of type: mild , moderate , or severe ). See Table
topically to a given lesion twice per day . 1A .
Dosages : Based on the clinical trial presented herein , It was also observed that the overall average size (lengthx
average Example 6B cream use per day, per patient was 2 .46 width ) of the eczema skin lesions decreased substantially
g . Average Example 6B cream use per lesion was 1 . 52 g . upon treatmentwith the topical cream . For example, average
Data Analysis. Statistical Software for Social Sciences 25 length
(SPSS version 19 ) was used to run all descriptive and averagewas reduced - 23 .8 % ( from 6 .3 cm to 4 .8 cm ), while
width was reduced -22.8 % ( from 3.9 cm to 3 .0 cm ).
inferential analyses for all endpoints . SCORAD is a clinical tool used to assess the extent and
The primary objective was to assess the reduction in the severity of eczema (Scoring Atopic Dermatitis ). Dermatolo
appearance of lesions by examining skin lesion photographs gists may use this tool before and after treatment to deter
(FIGS. 7A -7B and 8A - 8B ). The second objective was to 30 mine whether the treatment has been effective .
assess the reduction in the symptoms of itching , scaling , and The Severity Scoring of Atopic Dermatitis (SCORAD )
redness via different dermatologic assessments, as shown in index was used as the standardized ratings scale for eczema .
Table 1A . This composite scoring index was developed by the Euro
pean Task Force on Atopic Dermatitis in 1993 . It has
TABLE 1A 35 undergone testing for validity and reliability and has shown
Signif sensitivity change in trials of topical steroids and UV - A
Endpoint Visit/Day Change icance P -value therapy . It combines an assessment of disease extent using
Severity of Baseline vs. Visit 5 Decreased Yes 0 . 001 the rule of nines with 6 clinical features of disease intensity
Eczema (Day 30 ) overall (assessed at a single representative site ), plus a visual
lesions 40 analogue score for itch and sleep loss. The index has shown
VAS Week 1 vs. Week 2 Decreased from Yes 0 . 001 agreement with global assessments of disease severity as
SYMPTOM Baseline well as with various circulatory factors thought to reflect
SCORE
(ITCHING )
Week 1 vs . Week 3 Decreased from
Baseline
Yes 0 .011 disease activity in atopic dermatitis .
Week 1 vs. Week 4 Decreased from 0 . 031 In accordance with Table 1A , within -subject analyses
Baseline 45 showed that subjects in the treatment group had statistically
VAS Week 1 vs. Week 2 Decreased from Yes 0 .0001 significant decreases in the average SCORAD index score
SYMPTOM Baseline from baseline to Day 7 (p - value = 0 . 049 ) and Day 30
SCORE Week 1 vs . Week 3 Decreased from Yes 0. 003 ( p - value = 0 . 004 ). Moreover , consistent decreases from Base
(SCALING ) Baseline
Week 1 vs. Week 4 Decreased from Yes 0 .031 line to all time points were noted and the largest decrease
Baseline 50 was found at Day 30 ( - 49. 11 % ). FIG . 10 shows the SCO
VAS
SYMPTOM
Week 1 vs. Week 2 Decreased from
Baseline
Yes 0.005 RAD results .
SCORE Week 1 vs. Week 3 Decreased from Yes 0 .001 Subjects completed the Dermatologic Life Quality Index
(REDNESS ) Baseline (DLQI), a self-assessment on each subject' s own skin con
Week 1 vs. Week 4 Decreased from Yes 0 .043 dition . Dermatology Life Quality Index (DLQI) consists of
Baseline
SCORAD Baseline vs. Visit 3 Decreased from Yes 0.049 55
D 10 questions concerning patients ' perception of the impact
INDEX (Day 7) Baseline of skin diseases on different aspects of their health related
Baseline vs. Visit 5 Decreased from Yes 0 .004 quality of life over the last week .
(Day 30 ) Baseline In accordance with Table 1A , the Average Dermatology
Dermatologic Baseline vs . Visit 5 Decreased from Yes 0 .031 Life Quality Index (DLQI) scores of the subjects were
Life Quality (Day 30 ) Baseline
Index 60 shown to have constant decreases from baseline to all time
(DLQI) points, with the largest decrease at Day 30 (- 48 .03 % ).
VITALS
(Weight)
Baseline vs. Visit 5 Increased from
( Day 30 ) Baseline
Yes 0 .032 Statistically significant changes were observed from base
line to Day 30 (p -values0 .05 ). FIG . 11 shows the DLQI
results .
Visual analogue scale (VAS) Symptom Score for Itching, 65 The vital scores of the subjects were also assessed from
Scaling , and Redness . Subjects were also required to Day 0 to Day 30 . It was observed that the subjects had no
undergo Dermatologic Assessment for Itching , Scaling and statistically significant changes in their Body Temperature,
US 9 ,877,991 B2
17
Systolic Blood Pressure , Diastolic Blood Pressure , Pulse ( 3 - (4 ,5 -dimethylthiazol- 2 -yl) -2 , 5 -diphenyltetrazolium bro
Rate , and Respiratory Rate from baseline to any time point mide ) to formazan in the mitochondria of living cells. If the
despite exhibiting either an increasing or decreasing trend . cells are stressed or dying they cannot carry out this reaction
On the other hand , significant but minor increase in Weight and thus OD (optical density ) is low . If the cells are
was observed from baseline to Day 15 (p 0 .05 ). In accor - 5 functioning well in the presence of the extract then cell
dance with Table 1A , on Day 15 weight increased + 0.81 % proliferation occurs. Therefore OD directly relates to the
(about 1.2 lbs), and by Day 30 , weight increased + 0 .97 % number of viable ( living ) cells.
(about 1. 5 lbs ) respectively, compared to baseline. This In FIGS. 3A , 3B and 4 as shown , the higher the OD the
result could be valuable from a nutritional standpoint for more living cells present. None of the extracts show cyto
patients suffering from various conditions or diseases as 10 toxicity as the bar is the same height as the cell only
described herein . treatment.
There were no adverse events or serious adverse events The traditional MTT assay was adapted to measure cell
reported for this study. protection by an extract. This provides a measure of whether
In conclusion , the results of the clinical study indicated
that product Example 6B was effective in treating mild to 15 the extract protects cells from stress . HL -60 cells were
tested . As exemplified in FIG . 3A , the premise of the assay
moderate atopic dermatitis, also known as eczema, as dem is as follows:
onstrated by the reduction in the size of skin lesions along 1. cells only — only has cells growing for 48 hours ;
with other hallmarks of the disease such as itching , scaling 2 . cells plus hydrogen peroxide. The cells are grown for
and redness .
It is expected that the skin cream will successfully treat 20 24 hours and then hydrogen peroxide a pro -oxidant is added
symptoms associated with eczema, psoriasis, and related for a further 24 hours . Hydrogen peroxide is toxic to cells
inflammatory conditions when applied topically . The cream and thus inhibits growth even killing cells;
is generally applied topically to the skin in a manner 3 . cells plus etoposide . The cells are grown for 24 hours
substantially covering the affected surface area . The cream and then etoposide a chemotherapy drug is added for a
may be formulated as a cosmetic , pharmaceutical, or nutra - 25 further 24 hours . Etoposide is very toxic to cells and kills by
ceutical composition , including a pharmaceutically or nutra damaging DNA and preventing cell division and thus cell
proliferation . Thus etoposide results in inhibiting cellular
ceutically acceptable carrier, respectively.
Useful therapeutic dosages of the skin cream (Example growth and ultimately cellular death ;
6B ) can range , but are not limited to , from about 1 g to about 4 . extract only is the cells incubated with the extract for
5 g in a human individual. Another narrower suitable dose 30 48 hours . If the extract is toxic to the cells then the OD
range is from about 1 g to about 2 g , for example , 1- 2 would be lower than the cell only treatment;
g /lesion. Another useful therapeutic dosage for application 5 . extract plus hydrogen peroxide the extract is added for
to human skin is from about 0 .5 g /lesion to about 1. 5 24 hours to the cells and then the hydrogen peroxide is
g /lesion . added . If the extract can protect the mitochondria against the
The skin cream (Examples 6A -6D ) can be provided in 35 oxidative stress (i.e . free radicals ) from the hydrogen per
daily dosages of from about 1 g to about 5 g , in a human oxide then the cells will continue to grow ; and
patient, for example . Another narrower suitable dosage 6 . extract plus etoposide. The extract is added for 24 hours
range is from about 1 g to about 2 g daily. Another narrower to the cells and then the etoposide is added . If the extract can
suitable dosage range is from about 1 g to about 3 g daily . protect the DNA against the etoposide chemotherapy then
40 the cells will continue to grow .
Example 6D (Psoriasis ) Potent extracts such as tamarillo , apple skin , and kawa
kawa may be used to assess bioactivity . For example ,
A test cream of was prepared by substituting the 2 -Stage tamarillo extracts were prepared and either reacted as
Extract (Example 2B ) for Extract 2A in Example 6C . described above by adding tamarillo powder just before
A psoriasis patient having scaly outbreaks on a leg ( FIG . 45 sealing the container (prior to Stage 2 ) , or not reacting but
9A ) was treated with the psioriasis test cream . Topical adding tamarillo powder after the reaction was completed
application of the cream (as shown above in Example 6C ) (either after Stage 2 , or entirely omitting Stage 2 ). By
over 4 weeks provided a noticeable improvement by reduc reacting with the other components it appears that the
ing encrustation and scaling. (FIG . 9B ). bioactivity is either enhanced or even changed which is
It is further contemplated that the 2 - Stage reaction plat - 50 either because the lipids in the reaction mixture are syner
form as described herein can be used to produce oral gistically enhancing bioactivity by acting as a lipid mem
formulations of pollen -based reaction products in combina - brane carrier, and/or by esterifying the flavonoids . In the
tion with the components of beehives . Such products may following examples, "water " samples mean that the tama
include a 2 -stage reaction mixture which has been freeze rillo extract or coconut water extract without tamarillo (as in
dried or lyophilized , then appropriately formulated for oral 55 Ex. 2F) was not reacted or fermented in Stage 2 , while
administration . " ethanol” samples mean that the tamarillo extract or coconut
water extract without tamarillo (as in Ex . 2F ) was reacted
Example 7 (Cell Protection Assay ) and fermented in Stage 2 .
Thus the MTT assay was performed as a standard method
The MTT assay has been adapted to measure whether an 60 using Example 2F without tamarillo (at 1: 16 dilution ) as a
extract could protect the cells from apoptosis (programmed "water " extract, meaning that in this case Stage 2 fermen
cell death ) either by : tation was not performed . This extract demonstrated potent
1. Oxidative stress using hydrogen peroxide , or efficacy in that it is not cytotoxic and protected the cells from
2 . DNA damage using etoposide ( chemotherapy drug ). the DNA damaging and free radical stress . It was shown in
The MTT assay is traditionally used to measure the 65 FIG . 3A that the extract based on coconut water (without
cytotoxicity of a compound / extract. This is done by mea - tamarillo ) has very potent cell protection activity as a
suring cell proliferation of the cells by the reduction ofMTT "water " extract, and also in FIG . 3B that the extract based on
US 9 ,877,991 B2
20
coconut water (without tamarillo ) is even more potent as an Example 9 (Acne Cream Formulation )
“ ethanol” extract in promoting cell proliferation .
Next, the same assay protocol was performed using the In a further embodiment, an acne treatment cream for
tamarillo extract (Ex . 2F ) described above. The tamarillo mulation was prepared .
" water" extract on its own has shown partially reduced cell 5
growth , and has potent cell protection bioactivity against
hydrogen peroxide and etoposide . When incorporated into Ingredient Wt ( % )
the fermentation reaction to provide a tamarillo “ ethanol” Olivem 1000 ECO CERT (Cetearyl Olivate
extract, there was significant cell proliferation and cell & Sorbitan Olivate )
protection , as shown in FIG . 4 (i.e. protection provided from Aloe vera gel
“ Tam ” ). Thus the bioactivity of tamarillo has been syner
gistically changed by the reaction.
2 -Stage Extract (modified Example 2C ) 2
Hazelnut oil
Blackcurrant oil
Kiwifruit seed oil
NON
1. 5
The inventive extracts tested in this example demon
O
strated potent efficacy in that they are not cytotoxic and have 15 . Deionized Water
Bioactive honey (Manuka )
protected the cells from the DNA damaging and free radical Geogard 221
stress. It is noted thatboth hydrogen peroxide and etoposide Vitamin E
Vitamin C ?
induce apoptosis . That is how chemotherapy works — it Vitamin A ?
induces DNA damage and then the cell dies . The results Kiwifruit powder
from these experiments demonstrate that the inventive 20 Totara extract
Fragrance
extracts have potent cell protection activity, not only against
oxidative stress but also against DNA damaging chemo- ? no beeswax is included.
therapy. Therefore these results demonstrated that these
extracts protect against induction of apoptosis. Beeswax is omitted since it is not conducive to skin
25 treatment for acne .
Example 8 ( Immune Assays ) Optionally , salicylic acid up to 2.0 % of the total weightof
the composition may be added . In one embodiment, 0 . 5 % by
Flow cytometry was performed using standard methods to weight salicylic acid is added .
measure cytokine expression using dual labelled antibody Totara extract is obtained from tree bark of a native
markers for IL - 10 and TNF a (BioActives Research New 30 species.
Zealand Ltd ., Auckland, N . Z .). For this experiment the cells It is expected that the acne cream will successfully treat
were treated in the following way : symptoms associated with acne , and related inflammatory
1. cells only — only has cells growing for 48 hours ; conditions when applied topically . The cream is generally
2. (control) cells plus lipopolysaccharide (LPS ). The cells 35 applied topically to the skin in a manner substantially
are grown for 24 hours and then LPS a pro - inflammatory » covering the affected surface area . The cream may be
bacterial toxin is added for a further 24 hours ; formulated as a cosmetic , pharmaceutical, or nutraceutical
3 . extract only is the cells incubated with the extract for composition , including a pharmaceutically or nutraceuti
48 hours; and cally acceptable carrier, respectively .
4 . extract plus LPS. The extract is added for 24 hours to 10 The cosmetic ( or cosmeceutical), or nutraceutical com
the cells and then the LPS is added . positions of the present invention may be administered in
combination with a nutraceutically acceptable carrier. The
TABLE 2 active ingredients in such formulations may comprise from
LPS ( - )) LPS (- )) LPS ( + )/ LPS( + )/
1 % by weight to 99 % by weight, or alternatively, 0 . 1 % by
Extract TNF IL - 10 TNF IL - 10 45 weight to 99.9 % by weight. Alternatively , the active ingre
dients can range from about 5 % by weight to about 75 % by
Control 19 . 7
21. 3
13 . 3
23. 9
31. 7
19 . 1
9 .7
19 . 5
weight, or from about 10 % by weight to about 75 % by
Ex. 2F weight. “ Nutraceutically acceptable carrier” means any car
(Coconut " water" -
no tamarillo ) rier, diluent or excipient that is compatible with the other
Ex. 2F Tamarillo 22. 1 24 . 8 19 . 1 20 . 2 50 ingredients of the formulation and not deleterious to the user.
" water"
Ex. 2F Tamarillo 19..99
19 24 ..88 19..33
19 21.. 55
21 Useful excipients include microcrystalline cellulose , mag
" ethanol” nesium stearate , calcium stearate , any acceptable sugar ( e . g .,
mannitol, Xylitol), and for cosmetic use an oil-base is
preferred .
TNF is a proinflammatory cytokine. An increase in TNF 55 The topical pharmaceutical compositions of the present
indicates inflammation . LPS (lipopolysaccharide ) is a bac invention may be administered in combination with a phar
terial toxin which in this assay was used to induce inflam - maceutically acceptable carrier. The active ingredients in
mation ( TNF went from an average of 19 . 9 to 31.7 ) . such formulations may comprise from 1 % by weight to 99 %
IL - 10 is an anti-inflammatory cytokine and has been by weight, or alternatively , 0 . 1 % by weight to 99 . 9 % by
implicated in reducing allergic responses such as eczema . 60 weight . “ Pharmaceutically acceptable carrier" means any
By increasing IL - 10 , TNF is decreased along with other carrier, diluent or excipient that is compatible with the other
inflammatory cytokines . LPS also by triggering an increase ingredients of the formulation and not deleterious to the user.
in TNF results in a cascade that reduces IL - 10 . As shown in In accordance with certain embodiments , the topical phar
Table 2 , coconut and tamarillo extracts prepared in accor- maceutical compositions disclosed herein can be provided in
dance with the invention maintained , in the presence of LPS, 65 the form of an ointment, cream , lotion , gel or other trans
TNF expression at a normal level whilst increasing IL - 10 to dermal delivery systems as described in L . V . Allen , Jr., et
counter the pro - inflammatory attack . al., Ansel's Pharmaceutical Dosage Forms and Drug Deliv
US 9 ,877,991 B2
21 22
ery Systems, 9th Ed ., pp . 272 - 293 ( Philadelphia , Pa.: Lip In addition , shampoos or other types of cleansing prod
pincott Williams & Wilkins, 2011 ) which is incorporated ucts are contemplated . Also , topical sprays are contemplated
herein by reference . including sprays useful for one or more applications to the
Ointments, as used herein , refer to semi-solid preparations skin , back , neck , arms, legs , and torso , for example .
including an ointment base having one or more active 5 For oral administration , fermented pollen - based extract,
ingredients incorporated or fused (i.e ., melted together with or a solid formulation thereof such as lyophilized powder,
other components of the formulation and cooled with con - may be combined with one or more solid inactive ingredi
stant stirring to form a congealed preparation ) therein . The ents for the preparation of tablets , capsules , pills , powders ,
ointment base may be in the form of: an oleaginous or granules or other suitable dosage forms. For example , the
hydrocarbon base ( e . g ., petrolatum or a petrolatum /wax 10 active agent may be combined with at least one excipient
combination ); an absorption base which permits the incor - such as fillers, binders, humectants , disintegrating agents ,
poration of aqueous solution resulting in the formation of a solution retarders , absorption accelerators , wetting agents ,
water - in -oil emulsion ( e . g ., hydrophilic petrolatum ) or absorbents , or lubricating agents . Other useful excipients
which is a water - in - oil emulsion that permits the incorpo - include magnesium stearate , calcium stearate , mannitol,
ration of additional quantities of aqueous solutions ( e . g ., 15 xylitol, sweeteners , starch , carboxymethylcellulose , micro
lanolin ); a water -removable base which are oil- in -water crystalline cellulose , silica , gelatin , silicon dioxide , and the
emulsions that may be diluted with water or aqueous solu like.
tions ( e. g ., hydrophilic ointment, USP ) ; or a water - soluble Routes of Administration
base that do not contain oleaginous components (e .g ., poly The fermented pollen -based extract may be administered
ethylene glycol (PEG ) formulations which combine PEGs 20 by any route , including but not limited to oral, sublingual,
having an average molecular below 600 with a PEG having buccal, ocular, pulmonary , rectal, and parenteral adminis
an average molecular weight above 1,000 ); and the like . tration , or as an oral or nasal spray ( e.g . inhalation of
Creams, as used herein , refer to semisolid preparations nebulized vapors, droplets , or solid particles). Parenteral
containing one or more active , fermented pollen - based administration includes , for example, intravenous , intramus
material, or medicinal agent dissolved or dispersed in either 25 cular, intraarterial, intraperitoneal, intranasal , intravaginal,
a water - in -oil emulsion or an oil- in -water emulsion or in intravesical ( e . g ., to the bladder ), intradermal, transdermal,
another type of water-washable base . Generally, creams are topical, topical spray , or subcutaneous administration .
differentiated from ointments by the ease with which they The treatment may be carried out for as long a period as
are applied/ spread onto a surface such as the skin and the necessary , either in a single , uninterrupted session , or in
ease with which they are removed from a treated surface . 30 discrete sessions . The treating physician will know how to
Lotions, as used herein , refer to suspensions of solid increase , decrease , or interrupt treatment based on patient
materials in an aqueous vehicle . Generally , lotions have a response . According to one embodiment, treatment is carried
non - greasy character and increased spreadability over large out for from about four to about five weeks. The treatment
areas of the skin than ointments, creams, and gels. schedule may be repeated as required .
Gels, as used herein , refer to semisolid systems including 35 Furthermore , it is believed that this technology and pro
a dispersion of small and/ or large molecules in an aqueous cess may be applied to other reaction platforms. The afore
liquid vehicle which is rendered jellylike by the addition of mentioned examples describe a technology platform in
a gelling agent. Suitable gelling agents include , but are not which different startingmaterials can be added depending on
limited to , synthetic macromolecules (e .g ., carbomer poly the desired end product. By altering the settings, e .g . aero
mers ), cellulose derivatives ( e . g ., carboxymethylcellulose 40 bic / anaerobic , temperature and pressure different end prod
and/ or hydroxypropyl methylcellulose ), and natural gums uct compounds are obtained . The end products resulting
( e .g ., tragacanth gum , carrageenan , and the like ). Gel prepa - from the reaction platform are analogous to those produced
rationsmay be in the form of a single -phase gel in which the in nature . These compounds mimic those in nature which
active or medicinal ingredients are uniformly dispersed control immune and cell death signaling pathways and can
throughout the liquid vehicle without visible boundaries or 45 be used to treat a range of immune and apoptosis related
a two -phase gel wherein flocculants or small distinct par diseases .
ticles of the active or medicinal ingredient are dispersed In an alternative embodiment, a technology platform may
within the liquid vehicle . comprise a bioreactor , that uses naturally occurring chemical
Transdermal preparations may be formed from an oint - reactions to produce modified or new bioactive compounds
ment, cream , or gel that has been combined with a penetra - 50 that regulate mammalian cell signaling . These chemical
tion enhancer and are designed to deliver an active or reactions can be applied to any plant, microbial, marine or
medicinal ingredient systemically . Penetration enhancers insect derived extract to create bioactive components that
include, for example , dimethyl sulfoxide , ethanol, propylene interact directly with the human signaling pathways to
glycol, glycerine , PEG , urea , dimethyl acetamide , sodium regulate immune and cell death responses .
lauryl sulfate , poloxamers, Spans, Tweens, lecithin , and /or 55 While in the foregoing specification this invention has
terpenes amongst others . been described in relation to certain embodiments thereof,
Other suitable semi-solid forms for use as cosmetic and /or and many details have been put forth for the purpose of
topical pharmaceutical compositions include pastes (prepa illustration , it will be apparent to those skilled in the art that
rations containing a larger proportion of solid material the invention is susceptible to additional embodiments and
rendering them stiffer than ointments ) and glycerogelatins 60 that certain of the details described herein can be varied
(plastic masses containing gelatin , glycerine , water, and an considerably without departing from the basic principles of
active or medicinal ingredient). the invention .
In other embodiments the topical and/or cosmetic com The use of the terms“ a," " an ,” “ the," and similar referents
positions can be prepared in accordance with dosage forms in the context of describing the presently claimed invention
as described in Sample Preparation of Pharmaceutical Dos - 65 (especially in the context of the claims) are to be construed
age Forms, B . Nickerson , Ed . (New York : Springer, 2011) to cover both the singular and the plural, unless otherwise
herein incorporated by reference . indicated herein or clearly contradicted by context. Recita
US 9 ,877,991 B2
23 24
tion of ranges of values herein are merely intended to serve (c ) melting beeswax and maintaining liquid beeswax at
as a shorthand method of referring individually to each about 65° C .;
separate value falling within the range , unless otherwise (d ) treating the melted beeswax with an equal weight of
indicated herein , and each separate value is incorporated into coconut oil to provide a beeswax mixture;
the specification as if it were individually recited herein . All 5
methods described herein can be performed in any suitable ( e ) adding the germinated pollen mixture to the beeswax
order unless otherwise indicated herein or otherwise clearly mixture ;
contradicted by context. The use of any and all examples , or (f) stirring the combined mixtures to form a jelly ; and
exemplary language ( e.g ., " such as” ) provided herein , is (g ) incubating the jelly in a sealed vessel for about 72
intended merely to better illuminate the invention and does 10 hours at 37° C . to produce the fermented pollen -based
not pose a limitation on the scope of the invention unless composition .
otherwise claimed . No language in the specification should 2 . The process of claim 1, wherein the soaked pollen is
be construed as indicating any non - claimed element as germinated
essential to the practice of the invention .
A11 references cited herein are incorporated by reference 15 3 . The process of claim 1, wherein the pollen is about
in their entirety. The present invention may be embodied in twice the weight of the beeswax , and wherein the glycerol
other specific forms without departing from the spirit or and honey dew are about half the weight of the beeswax .
essential attributes thereof and , accordingly , reference 4 . The process of claim 1, wherein an enzyme-containing
should be made to the appended claims, rather than to the component is added prior to or after step (g ).
foregoing specification , as indicating the scope of the inven - 20 5 . The process of claim 4 , wherein the enzyme-containing
tion . component is selected from the group consisting of pine
I claim : apple powder , tamarillo powder, kawa kawa powder , kiwi
1. A process for making a pollen -based fermentable fruit powder , honey, additional honeydew , and mixtures
composition comprising the steps of: thereof .
( a ) soaking dry pollen grains in coconut water at ambient 25
temperature to provide soaked pollen ; 6 . The process of claim 1 or 4 , further comprising step (h )
(b ) treating the soaked pollen with glycerol and honeydew addingpropolis
a substance to stop fermentation selected from
, pine bark powder , or mixtures thereof.
at ambient temperature to provide a germinated pollen
mixture ; * * * * *

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US009877991B2

(12) United States Patent ( 10 ) Patent No.: US 9 ,877, 991 B2

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