|HAO WALA NAMAN UTAMA UNATAMANI NA WA AINA TATTI
US010004679B2(12) United States Patent( 10) Patent No.: US 10 , 004,679 B2

Prasad(54) COMPOSITIONS OF AMORPHOPHALLUS

KONJAC AND METHODS FOR THEIR USE(45 ) Date of Patent:

References Cited
U . S . PATENT DOCUMENTS(56 )IN SKIN CARE

5,578,641 A * 11/1996 Jackson ................. A61K 8 /606
424 /401. ... A23G 3 /346
6 ,162,906 A * 12/2000 Ohashi(71 ) Applicant: Kodimule Shyam Prasad , Bangalore( IN )(72 ) Inventor: Kodimule Shyam Prasad , Bangalore( IN )

426 /573

2010 /0255076 A1* 10 /2010 Heber ...................... A61K 8/ 73
424 /450( 73 ) Assignee : Vidya Herbs, Inc ., Fullerton, CA (US )( * ) Notice: Subject to any disclaimer,the term of thispatent is extended or adjusted under 35

U . S .C . 154 (b ) by 0 days . days .
(21 ) Appl. No.: 14 /975 ,863

FOREIGN PATENT DOCUMENTSWO

WO2013/101650* 7 /2013 ............. C08B 15 /02

OTHER PUBLICATIONS
Hajime Kimata , Improvement of Atopic Dermatitis and Reduction

Dec . 21, 2015of Skin Allergic responses by Oral Intake of Konjac Ceramide , 23
Ped . Dermatol. 386 (2006 ).*Prior Publication Data
US 2016 /0184218 A1 Jun . 30 , 2016

erties of Konjac Glucomannan Extracted from Konjac Flour by a
Simple Centrifugation Process , 44 Food Sci. Tech . 2059 ( 2011).*(22 ) Filed :(65 )Jun . 26 , 2018

Related U .S . Application Data
(60 ) Provisional application No. 62 /097 ,579, filed on Dec .
29, 2014* cited by examinerPrimary Examiner — Sean M Basquill(74 ) Attorney, Agent, or Firm — Law Office of Timothy
M . Brown(51) Int . CI.
A61K 8 /97(2017.01)

A610 19 / 08
A610 19 /02
A61K 36 /888
A61K 8 /68( 2006 .01 )( 2006 .01 )( 2006 .01 )(2006 .01)

A610 19 /00(2006 .01 )(52 ) U .S . Ci.

Orawan Tatirat & Sanguansri Charoenrein , Physicochemical Prop

ABSTRACT(57)
Compositions derived from an extract of Amorphophallus
konjac and methods for their use in the treatment of skinconditions . The compositions of the invention find use in

A61K 8 / 97 (2013.01); A61K 8 /68

maintaining the health of the skin and treating a variety ofskin conditions, including wrinkles and fine lines , loss of

19 /00 (2013 .01 ); A61Q 19/02 ( 2013 .01 );any particular theory ormechanism , the compositions of theinvention treat skin conditions through the inhibition of( 2013 .01); A61K 36 /888 ( 2013 .01); A610

A610 19 /08 ( 2013 .01)

(58 ) Field of Classification Search
CPC .......... A61K 8 / 97 ; A61K 36 /888 ; A61K 8 /68 ;A61Q 19/08; A61Q 19/02 , A61Q 19 /00
See application file for complete search history .

skin tone, and hyperpigmentation . Without being limited toenzymes, including but not limited to, collagenase , elastaseand tyrosinase .

20 Claims, 7 Drawing SheetsU . S . Patent

Jun . 26, 2018

Sheet 1 of 7

FIG . 1

(PRIOR ART)

*

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FIG . 2A

US 10 ,004 ,679 B2

U . S . PatentJun. 26 , 2018

US 10 ,004 ,679 B2

Sheet 2 of 7

FIG . 2B

FIG . 3
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Sheet 3 of 7

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FIG . 5A

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Sheet 5 of 7

FIG . 7

( PRIOR ART)

FIG . 8A

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Sheet 6 of 7

US 10,004 ,679 B2

FIG . 8B

FIG . 9

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U . S . PatentJun. 26 , 2018

Sheet 7 of 7

FIG . 10BER

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US 10 ,004 ,679 B2

US 10 ,004 ,679 B2
A . konjac is a rich source of glucosylceramides which isa glycoside of ceramide, a major spingo (glycol) lipid in

COMPOSITIONS OF AMORPHOPHALLUS
KONJAC AND METHODS FOR THEIR USE
IN SKIN CARE

plants. Glucosylceramides are essential structural compo
nents of mammalian cell membranes and are mostly found
FIELD OF THE INVENTION
5 at the cell surface , which serve to maintain mammalian skinbarrier function through their role as intracellular lipids.
The invention generally relates to compositions derived
It is an object of the invention to provide a method for skinfrom herbal extracts.More particularly, the invention relates care in a subject comprising administering to the subject a
to compositions derived from Amorphophallus konjac (koncomposition derived from konjac in an amount effective tojac) and methods for their use in skin care .

BACKGROUND

10 maintain the skin architecture .
It is a further object of the invention to provide a com

position derived from a herbal extract for skin care, wherein

the extract comprises by weight, about 5 % spingolipids
The human skin consists of epidermis , corium and tela , containing glucosylceramide .
subcutanea . The epidermis is classified into four layers ,It is a further object of the invention to provide a method
namely stratum corneum , stratum granulosum , stratum
for treating a skin condition in a subject comprising admin

spinosum and stratum basale. The quantity of ceramides is istering to the subject a composition comprising an amountlower in the stratum corneum of atopic dermatitis, dry skin of a composition derived from an extract of konjac sufficientand the skin of aged individuals . The ceramide content of the 20 to inhibit an enzyme selected from tyrosinase, elastase ,skin declines with increasing age (e .g . over 70 years old ) collagenase and combinations thereof.
with dry appearance in xerotic skin . In addition , there is a
A further object of the invention is to provide a method formarked reduction in the amount of total ceramide in both treating a skin condition in a subject comprising adminis
lesional and non - lesional skin as compared to that of healthy tering to the subject composition comprising an effective
individuals of the same age .
25 amount of an extract of konjac .
More than just a cosmetic problem , skin aging exposes
A further object of the invention is to provide a compo
individuals to significant health risks from infectiousmicro - sition for skin hydration comprising 3 % konjac extract andorganisms, UV radiation , defective permeability to topical 5 % ceramide -basedrugs, and increased susceptibility to contact dermatitis
from environmental chemicals . The skin barrier is essential 30
for protecting skin against physical stimuli, thermal chal

BRIEF DESCRIPTION OF THE DRAWINGS

lenge and ultraviolet radiation, chemical substances and

FIG . 1 shows 3D structure of tyrosinase .
FIG . 2A shows the interaction of a composition derived

microbial attack and preventing water loss . The skin needs

to be at its functional best in order to properly function (i.e.

from konjac with tyrosinase .

environment) . In vitro studies demonstrate that the moisture

FIG . 3 shows in vitro anti - tyrosinase activity of a com

regulate body temperature , synthesize optimal amounts of 35
vitamin D , and provide critical sensory input from the naseFIG. . 2B shows the interaction of kojic acid with tyrosi

content of the stratum corneum is decreased with age. In

derived from konjac .
maturing individuals, the stratum corneum is also suscep position
FIG.
4
3D structure of elastase .
tible to inflammation and infection . Aging skin also suffers 40 FIG . 5Ashowsshows
the interaction of elastase with a compofrom reduced structural integrity due to lower lipid content,
mostly a lower proportion of ceramides. The decline of the sition derived from konjac.

ceramide content in aging human skin is possibly due to a
FIG . 5B shows the interaction of elastase with EGCG .
FIG . 6 shows anti -elastase activity of a composition
decline in enzyme activity that helps too deliver ceramides inusable form to the skin . When aging skin 's ability to make 45 derived from konjac.

FIG . 7 shows 3D structure of collagenase.

osad cer
ceramides diminishes, as it inevitably does, increasedcer

FIG . 8A shows the interaction of collagenase with aamide intake becomes essential.

Synthetic and animal ceramide have been used as mate -composition derived from konjac .

rials in cosmetic manufacture . However, it was discovered
FIG . 8B shows the interaction of collagenase with EGCG .
that Creutzfeldt- Jakob disease may be contracted from eat- 50 FIG . 9 shows in vitro anti- collagenase activity of aing or using contaminated animal products , especially thatcomposition derived from konjac.

FIG . 10 shows the moisturizing effect of a compositionof cattle .

What is needed in the art therefore is a safe and effective

derived from konjac.

treatment for improving ceramide content in the skin .

55

SUMMARY OF THE INVENTION

The inventors of the present disclosure, therefore envis -age a cost- effective and safe skin care treatment derived

DEFINITIONSAs used herein , the phrase “ Amorphophallus konjac material,” or “konjac material,” refers to any portion of the

Amorphophallus konjac plant, including, but not limited tofrom herbal extracts including Amorphophallus konjac (“ A . 60 tubers , seeds, leaves, stems, fruit, fruit skin , flowers , roots ,konjac ” or “ konjac” ). Konjac is a plant of the genus Amor phophallus . It is native to warm subtropical to tropicalbark , pollen , and combinations thereof.
As used herein , “ treating ” or “ treat” includes: (i) prevent

eastern Asia , from Japan and China south to Indonesia . It is ing a pathologic condition from occurring (e.g . prophylaxis );a perennial plant growing from a large corm up to 25 cm in( ii ) inhibiting the pathologic condition or arresting its devel
diameter. The single leaf is up to 1.3 m across , bipinnate , and 65 opment; ( iii) relieving the pathologic condition , and/ or ( iv )divided into numerous leaflets. The flowers are produced on
diminishing symptoms associated with the pathologic cona spathe enclosed by a dark purple spadix up to 55 cm long. dition .

US 10 ,004 ,679 B2
As used herein , " extract” refers to a substance obtained byextracting a raw material, using a solvent system or physicalprocess.
As used herein , the phrase " skin condition ” refers to aa single or multiple dosage form . It is important to admin

ister the composition in the minimum amount that can

exhibit the maximum effect without causing side effects , inview of all the above - described factors , this amount can bepathological condition affecting the skin . Some non - limiting 5 easily determined by a person skilled in the art .

examples of skin conditions include , but are not limited to ,The preferred administration dose of the pharmaceuticalwrinkles, fine lines, sagging skin , dryness, xerosis, itching ,composition of the present invention may be 0 . 1 mg/kg/day

dermatitis , asteatotic eczema, atopic dermatitis , elastolysis,dermatitis , hand dermatitis , dyshidrotic eczema, stasis der -to 1, 000 mg/kg/day .
Another aspect of the invention concerns methods formatitis, seborrheic dermatitis, psoriasis, pityriasis, vitiligo , 10 making the subject compositions. The compositions canhyperpigmentation, liver spots , acne, lacerations, eczema, comprise an extract of Amorphophallus konjac. The com
lymphoma of the skin , actinic keratosis, scleroderma, rosa

cea , an allergic skin disorder, radiation and/ or chemotherapy

induced mucositis, or a combination thereof.

positions of the invention can comprise an extract of Amor
vents . The compositions can comprise an extract ofphophallus konjac obtained from polar or non -polar sol

As used herein , “ therapeutically effective amount," or 15 Amorphophallus konjac that is obtained by ethanol extrac" effective amount,” refers to an amount of a substance ( or tion . The compositions can comprise an extract of Amor

compound ) that is sufficient to treat the condition that is phophallus konjac that is obtained by aqueous extraction .
intended to be addressed by the administration of the sub
The compositions can comprise an extract of Amorphophallus konjac that is obtained by supercritical fluid extraction .
stance or compound to a subject.
The term “ subject” as used herein typically refers to a 20 As used herein , “ supercritical fluid extraction ” or “ SFE”

human . In certain embodiments , a subject is a non - humanrefers to the process of separating one or more components

tory, domestic , pet, sport , and stock animals, e .g .,mice , cats ,as the extracting solvent. The compositions can be preparedmammal. Exemplary non -human mammals include labora -(extractant) from another (matrix ) using supercritical fluids

by concentrating a solution obtained from an Amorphophal
dogs, horses, and cows.
The term “ about” or “ approximately ” as used herein 25 lus konjac material such as by extrusion .
ususually means within 20 % ,more preferably within 10 % , andThe compositions of the invention can comprise konjac

most preferably still within 5 % of a given value or range .

extract in combination with a pharmaceutically acceptable

Alternatively , especially in biological systems, the term

carrier. Suitable carriers for combining with konjac extract“ about” means within about a log (i.e ., an order of magni

are disclosed in Remington 's Pharmaceutical Sciences, 19th

DETAILED SPECIFICATION
The invention generally relates to compositions derived

their entirety for all purposes . The pharmaceutical carriertude) preferably within a factor of two of a given value . 30 Edition , Mack Publishing Co., Easton, Pa . 1995 , the entire
contents of which are incorporated herein by reference in

can be an artificial pharmaceutical carrier. The konjac

extract may be combined with the pharmaceutical carrier atfrom herbal extracts and methods for their use in skin care . 35 any concentration that permits the konjac extract to treat a
More particularly, the invention relates to compositions
skin condition as described herein . The concentration of thederived from herbal extracts of Amorphophallus konjac konjac extract can range from 10 ug/mL to about 1,000
extract and methods for their use in the treatment of skin ug /mL, including all intervening concentrations at about 1
conditions .
ug/mL increments. In one non - limiting embodiment, theThe compositions of the invention can comprise an 40 concentration of konjac extract is 100 ug/mL .
extract derived from Amorphophallus konjac . Such extracts
can be derived from any portion of Amorphophallus konjac ,including the tubers , seeds , leaves , stems, fruit, flowers ,pollen , and combinations thereof. The compositions of the

The compositions of the present invention may be admin
istered by various routes including but not limiting to
topically, orally, buccal, intraaeterially, intravenously, intra

peritoneally , sub - lingually , rectally, and by inhalation . The

invention can comprise an extract of Amorphophallus kon - 45 compositions may be in a dosage form that includes but isjac from tubers . The compositions of the invention cancomprise an extract of Amorphophallus konjac from leaves .

The compositions of the invention can comprise Amor-

phophallus konjac extract from tubers . In a preferred

not limited to powders , pills , tablets , pellets, capsules, thinfilms, solutions, sprays , syrups, linctuses, lozenges, pastilles,

chewing gums, pastes, vaporizers , suspensions, emulsions,ointments , creams, lotions, liniments, gels, drops, topicalembodiment, the extracts described herein are free of the 50 patches (e. g . adhesive topical patches or transdermal

solvent( s ) that is used to obtain the extract from the Amor phophallus konjac material. The composition of the invention can comprise a tincture of Amorphophallus konjac. The

patches ), buccal patches, beads, gummies, gels , sols , injec
t ions and the like. The compositions of the invention cancomprise an extract that is dried to form one or more of the

Amorphophallus konjac material used to make the compo
foregoing dosage administration forms.
sitions described herein can be fresh , dried , rehydrated , 55 In some aspects of the invention, the compositions herein
powdered , or a combination thereof.
The composition of the present invention may be admin -istered in a therapeutically effective amount. The effective

dosage level may be determined depending on a subject's

include one or more additional constituents to facilitate

topical application . The compositions can comprise one ormore flow regulating agent( s ), including but not limited to ,silica (e.g ., colloidal silica ), silica gel, magnesium trisilicate ,type, severity of the disease, the subject ' s age and sex , 60 or cornstarch . A flow regulating agent may be included toactivity of the composition , administration time, adminis increase flow of the formulation , depending on the applica

tration route , excretion rate, duration of treatment, and otherfactors known in the medical field . The composition of thepresent invention may be administered alone or in combi-

tion of use . A powder formulation of the compositions may

include one or more such flow regulating agent(s ). The
compositionsmay further include one or more filler (s ), suchnation with other therapeutic agents , and may be adminis - 65 as titanium dioxide, spray dried lactose , or starch , and may

tered sequentially or simultaneously with conventional

therapeutic agents. The composition can be administered in

optionally further include one or more excipient(s ) such as

magnesium stearate to facilitate compression . The compoUS 10 , 004 ,679 B2
sitions for topical administration can comprise a formulationand combinations thereof. The term “ reduce” as used hereinthat is an anhydrous absorption base composition . For
example , a liquid or semi- liquid anhydrous absorption baserefers to any measurable decrease in a parameter that is
produced as a result of administering a composition of theof the composition may contain one or more humectant(s )invention , relative to the amount of the parameter that is( one or more substance( s ) that seals moisture in skin , e . g ., 5 observed in the absence of such administration . The term

act as moisturizers ), for example , coconut oil , almond oil," increase ” as used herein refers to any measurable increase

example , lecithin , and one or more consistency regulator ( s )or thickener ( s ), for example , beeswax .

a composition of the invention , relative to the amount of theparameter that is observed in the absence of such adminis

lanolin ; one or more surfactant(s ) or emulsifier (s ), forin a parameter that is produced as a result of administering

Oral formulations of the composition can be combined 10 tration .

with ordinary foods to enhance the value of the palatability.
of the composition . For example , the compositions can be

The compositions of the invention may be administered tomodulate one or more enzymes in the treatment of a skinmixed with drinks, food supplements , snacks, bars , andcondition or maintenance of the health of the skin . Somevirtually any other food , nutritional product or drink that cannon -limiting examples of enzymes that are modulated by thebe supplemented . Thus, the invention specifically includes 15 compositions of the invention include , but are not limited to ,food substances of specific types combined with the com collagenase , tyrosinase , elastase , and the enzymes involved

positions of the invention in specified forms and quantities .

in ceramide production, regulation and transport. In some

The compositions of the invention may be combined withembodiments , the compositions of the invention are used to

one or more substances to increase the nutritive value of the inhibit such enzymes in the treatment of a skin condition . As
composition , such substances including, but not limited to , 20 used herein , the term " inhibit” refers to any measurable
vitamins, minerals, amino acids, proteins, and combinations decrease in the activity of an enzyme that is brought about

thereof.
Compositions intended for oral use may be preparedaccording to any known method, and such compositions

by contacting the enzyme, or an enzyme precursor, with anagent.
The compositions of the invention can be administered to

may contain one or more agents selected from the group 25 a subject to inhibit tyrosinase as a means for lightening theconsisting of sweetening agents, flavoring agents, coloringskin tone of the subject. Tyrosinase is a multifunctional,

positions. Tablets may contain konjac extract in admixturenase is initiated with the hydroxylation of L -tyrosine toagents , and preserving agents in order to provide commer
cially viable, pharmaceutically elegant and palatable com -glycosilated , copper containing oxidase and found exclu
sively in melanocytes. The biosynthetic pathway of tyrosi

with non -toxic pharmaceutically acceptable excipients 30 3 -4 - dihydroxyphenylalanine ( L -DOPA ) and the oxidation ofwhich are suitable for the manufacture of tablets. These
L -DOPA to o -dopaquinone . This o -dopaquinone is a highly

excipients may be, for example, inert diluents , such asphate or sodium phosphate ; granulating and disintegrating
calcium carbonate , sodium carbonate , lactose , calcium phos -reactive compound and can polymerize spontaneously toform the pigment melanin ; this presents a serious aesthetic
problem in humans as it results in the darkening of the skinagents, for example , corn starch or alginic acid ; binding 35 (Briganti et al. 2003 ). Without being limited to any particu
agents , for example , starch , gelatin or acacia ; and lubricating lar mechanism or theory , the compositions of the invention

agents , for example, magnesium stearate , stearic acid or talc . can be administered to a subject to inhibit tyrosinase and
The tablets may be uncoated or they may be coated byprovide a depigmenting (e . g . skin lightening) effect in the
known techniques to delay disintegration and absorption in
subject.
the gastrointestinal tract and thereby provide a sustained 40 Hyper -pigmented skin is observed in various dermato
action over a longer period. For example , a time delay

logical conditions namely melasoma, solar lentigines andmaterial such as glycerylmonostearate or glyceryl distearate

ephilides (Maeda & Fukuda , 1991) . The above mentioned

may be employed . They also may be coated for controlled

clinical conditions are due to frequent exposure to UV rays ,delivery . For example , a " delayed release ” dosage form

certain drugs and chemicals resulting in skin darkening.

releases a product or substance at a time other than promptly 45 Depigmenting agents commonly are prescribed to treat suchafter administration . Examples of delayed -release systems

conditions. The most common skin lightening and depig

include repeat-action tablets and capsules , and entericmentation agents available commercially are kojic acid ,coated tablets where timed release is achieved by a barrier

arbutin , catechins , hydroquinone and azelaic acid (Maeda &variety of therapeutic and preventive applications . In someembodiments of the invention , the composition is adminis
tered to a subject for restoring skin balance . In otherembodiments of the invention , the compositions find use innosis, for example . Thus , the compositions of the invention
can be used to treat hyperpigmented skin in a subject ,including the treatment of melasoma, solar lentigines andephilides .

the invention may be used in the treatment of hyperpigmen -a subject to inhibit elastase as a means for treating wrinkles ,fine lines, sagging skin , dermatitis , psoriasis , and improving

the chymotrypsin family of proteases, is responsible primar

skin texture . In someaspects , the inventive compositions are

ily for the breakdown of elastin which is an importantcoating
Fukuda , 1996 ) . Some adverse effects of these synthetic
The compositions of the present invention find use in a 50 compounds are irreversible cutaneous damage and ochro

treating skin conditions . For example , the compositions of 55

tation , loss of skin turgor, loss of skin tonicity, wrinkling ,The compositions of the invention can be administered tofine lines, and loss of skin elasticity . Elastase , a member ofused in lightening the color of the skin (i. e. treating hyper - 60 protein found within the extracellular matrix (ECM ). Elaspigmentation ), reducing, slowing or preventing the wrinkle
formation such as caused by sun damage and aging, increas ing skin turgor, increasing skin tone, and combinations
thereof. In embodiments of the invention , the compositions

tin , due to its unique elastic recoil properties, is vital forgiving elasticity to arteries , lungs, ligaments and skin (Kimet al. 2004 ; Baylac & Racine, 2004 ; Melzig et al. 2001;
Siedle et al. 2002). Elastases can cleave elastin as well as

including but not limited to , reducing wrinkles , skin brightcollagen , fibronectin and other ECM proteins (Melzig et al.

are administered as a means for managing skin balance , 65 having a broad substrate portfolio including ability to cleave

ening (e .g . lightening of skin color ), moisturizing the skin ,2001; Siedle et al. 2002 ). As with the metalloproteinases ,US 10 ,004 ,679 B2
under normal conditions elastase activity is necessary after

continuous circulation of extract with a transfer pump. Afterwounding to degrade foreign proteins within the ECM
completion of the extraction , the extract was filtered throughduring phagocytosis by neutrophils to enable tissue repair a 5 micron SS candle filter and clear extract was collected in(Melzig et al. 2001 ; Siedle et al. 2002). In terms of anti
a receiver tank . The bed was re -extracted by adding 6 bed
aging, finding inhibitors of elastase enzymes can be useful 5 volumes of 95- 98 % ethyl alcohol 3 more times at room
to prevent loss of skin elasticity and thus skin 's aging .
temperature for about 7 - 8 hrs and filtered through 5 micron
The compositions of the invention can be administered to SS candle filter. All the extracts were collected in a receiver

a subject to inhibit collagenases and/ or metalloproteinases astank and combined extract was concentrated in a reactor
a means for treating wrinkles , fine lines , and loss of skinunder vacuum at 80 - 85° C . until the extract moss TDS
elasticity . Matrix metalloproteinases (MMPs ) are part of a 10 reached
60 - 70 w /w % . The extract moss was dried in agroup of transmembrane zinc containing endopeptidases vacuum tray
drier at 80 - 85° C . under vacuum until dry . The
which include collagenases and gelatinases . Collagenases yield of the konjac
composition was about 5 . 8 : 0 . 2 w / w % .
are metalloproteinases capable of cleaving other moleculesThe
resultingcomposition
was used in the following
found within the cell for example collagenase - 2 (MMP- 8 ) s examples.

can cleave aggrecan , elastin , fibronectin , gelatine and lami- 15
nin as well as collagen (Raffetto & Khalil , 2008 ). Collage

EXAMPLE 2
Anti - Tyrosinase Activity of Konjac Compositionnase cleaves the X - gly bond of collagen and also synthetic

peptides that contain the sequence - Pro - X -Gly - Pro where X
is almost any amino acid provided that the amino terminus
is blocked (Van Wart & Steinbrink , 1981) . Collagenase from 20

the bacteria clostridium histolyticum (ChC ) also degrades
ECM . This bacterial collagenase hydrolyses triple -helicalcollagen in both physiological conditions and in vitro con
ditions using synthetic peptides as substrates (Kim et al.

In Silico Docking Studies of Koniac Composition with

Enzyme Tyrosinase

AutoDock tools were utilized to generate grids, calculate
dock scores and evaluate the conformers of the konjac
composition bound in the active site of elastase as targets for2004 ; Van Wart & Steinbrink, 1981) .

In aspects of the invention , the compositions are admin

anti -wrinkle activity . Automated docking is a graphical user

istered in the maintenance of thehealth of the skin . Thus, thebinding scores ; which is implemented by the Lamarckian

tain skin hydration , function , turgor, tone , and /or color.

genetic algorithm method. The ligand molecules i.e ., thecompositions of the invention may be administered to maininterface . AutoDock 4 . 2 was employed to get docking andkonjac composition and epigallocatechin gallate (EGCG )The compositions of the invention can be administered to 30su were designed and the structure
struc was analyzed using ACD /a subject for increasing the moisture content of the skin . Chemsketch . The PRODRG server was used to minimize
Without being limited to any particular theory or mecha energy of drug compounds and 3D coordinates were prenism , administering the compositions of the invention to a pared. The protein structure file (PDB ID : 1WX2) (FIG . 1)
subject improve the appearance of the skin by fighting was takenfrom PDB and was edited by removing the hetero
dryness, balancing skin tone and improving skin texture . 35 atoms using
Python molecule viewer . The grid map was
Compositions of the invention can comprise konjac extract
centered at particular residues of the protein and was gen

and ceramide. Such compositions can be combined with any erated with AutoGrid . As per genetic algorithm all theof the other constituents described in the present disclosure . torsions were allowed to rotate during docking . The
The compositions can comprise konjac extract in any ratio Lamarckian genetic algorithm and the pseudo -Solis andthat permits the composition to provide any of the thera - 40 Wets
methods were applied for minimization , using default
Wets methods

peutic effects described herein . The compositions can com
parameters (Rodriguez and Infante , 2011 ).
prise konjac extract and ceramide (e. g. a purified ceramide
The results of molecular docking studies were highlyor a ceramide rich material) in a ratio of about 1 : 1 , 1 :2 , 1 : 3 ,appreciable and showed good interaction of konjac compo
1 :4 , 1 : 5 , 1:6 , 1:7 , 1 :8 , 1: 9 ; 1: 10 , or any ratio intervening sition with the enzymes' active pocket amino acid residues .
those specifically described here . In one non - limiting 45 The molecule was bound with tyrosinase having a minimal
embodiment, the ratio of konjac extract to ceramide is 1 :1.7 . binding energy of - 6 .64 kJmol-1 ( Table 1). The interaction
The present disclosure is further described in the light of was more firm as evident by the formation of 4 hydrogenthe following non - limiting examples which are set forth for bonds with tyrosinase as compared to the standard kojic acidillustration purpose only and not to be construed for limiting ( 3H -bonds) ( FIG . 2 ). The inhibition of tyrosinase which is
50 involved in melanin production clearly indicated the poten
the scope of the disclosure .
tial of the konjac composition in exhibiting the skin whit
ening property more convincingly and we further carried out
EXAMPLE 1
in vitro enzyme assay to confirm its activity .

Preparation of Konjac Composition
55
100 kg of Amorphophallus konjac (konjac ) powder was

TABLE 1
Molecular docking of konjac composition with tyrosinase
Thermodynamic parameterstransferred into a cleaned vertical 1 .0 KL extractor. The
bottom of the extractor comprised a perforated plate on
which filtration cloth was fixed . The bottom of the extractor

Binding

was connected to a transfer pump input and output of the 60

transfer pump was connected to a T bend. One end wasconnected to the extractor top for circulation of extraction

mass while extraction period and the other end of T bend
were connected to a receiver tank .

Konjaccompo

sitionKojic
The above mentioned mass was extracted with eight bed 65 acidvolumes of 95 - 98 % v /v ethyl alcohol (aqueous). Extraction

was continued at room temperature for about 7 - 8 hrs with

LigandInhibi-Intereffi -

tion

molecularenergybonds actions
Lys5,

energyciencyconstant- 6.64- 0.3665 .29- 8 .99- 5 .71- 0 .66

13 .68- 7.53

H - Inter

Ser80
3

Glu14 ,

Thr82 ,Lys5

US 10 , 004,679 B2
10In Vitro Anti - Tyrosinase Activity of Konjac Composition
Mushroom tyrosinase (Prod . No. 17755 ) and L -DOPA

TABLE 3
Molecular docking of konjac composition with elastase

were purchased from Sigma Adrich . Tyrosinase activity was

Thermodynamic parameters
LigandInhibiInter

determined by spectrophotometry 4w75 with minor modi

fications 20 uL of mushroom tyrosinase ( 1000 U /mL ), 20 uL
sample solution (20 % ) containing 20 uL of konjac compo
sition , were mixed (called sample solution with enzyme).
We also prepared positive controls of Img/mL kojic acidsolutions (with water), with enzyme. Twenty 20 uL of 0 . 85

of 0 . 1 M phosphate buffer (pH 6 .8 ) and 100 uL of the test

Mole
cule

Konjaccompo
sitionEGCG

mM L -DOPA solution as the substrate was added into every
sample and blank . These assay mixtures were incubated at

Binding
EnergyEffi

ciency(kJmol-1) (kJmol- 1)

tion

molecular(UM )(kJmol-1) bonds actions

energyH - Inter

Constant- 7. 16-0 .3930. 54-9. 44

3 Cys182
Val163,,- 9.84- 0 .361.17- 13.42

6 Met180 ,Tyr165,Blank
20

The assay employed was based on methods from the

20 literature . This assay was performed in 0 .2 mM Tris -HCL
buffer (pH 8 . 0 ) . Porcine pancreatic elastase (PEKonjac extract- 32 .67

60

64 . 46
77 .66
88 .3280

91 .3730 .69100

93 .9050 .4940

Asn178In Vitro Anti-Elastase Activity of Konjac Composition

Anti-tyrosinase activity of konjac composition% inhibition of tyrosinase activity

Kojic acid

Leu130 ,15

TABLE 2Concentration in ug/ml

Ile129,

Asp164 ,25° C . for 10 min . The amount of dopachrome produced in

the reaction mixture was measured at 475 nm .

Lue1306 .93
25 .74EXAMPLE 3

Evaluation of Anti-Wrinkle Activity of Konjac
Composition

E .C .3 .4 .21. 36 ) was dissolved to make a 3.33 mg/mL stock

solution in sterile water. The substrate N -succinyl-Ala -AlaAla -p -nitroanilide (AAAPVN ) was dissolved in buffer at 1 .6
25 mM . The test extracts were incubated with the enzyme for
15 minutes before adding substrate to begin the reaction .
The final reaction mixture (250 uL total volume) contained

buffer, 0 .8 mM AAAPVN , 1 mg/mL PE and 25 mg test
extract. EGCG ( 1 mg/mL ) was used as a positive control.
30 Negative controls were performed using water. Absorbancevalues between 381 and 402 nm (following pre - screen
scans ) weremeasured immediately following addition of thesubstrate and then continuous for 20 minutes . FIG . 6
35

In Silico Studies of Konjac Composition with Elastase
AutoDock tools was utilized to generate grids, calculate
dock score and evaluate the conformers of the konjac
composition bound in the active site of Elastase as targets foranti -wrinkle activity . Automated docking is a graphical userinterface . AutoDock 4. 2 was employed to get docking andbinding scores ; which is implemented by the Lamarckian 45
genetic algorithm method . The ligand molecules i. e ., theTABLE 4Anti-elastase activity of konjac composition% inhibition of Elastase activity

Concentration in ug/ml

Konjac
EGCG

extract

43. 989 .801
20 .471
24 . 194
26 .427
31 .266

Blank

20

40

54. 3660

53. 32

80
100

60. 3762 . 45

konjac composition and epigallocatechin gallate ( EGCG )were designed and the structure was analyzed using ACD /Chemsketch . The PRODRG server was used to minimize

EXAMPLE 4

pared . The protein structure file (PDB ID : 1GVK ) (FIG . 4 )was taken from PDB and was edited by removing the hetero
atoms using Python molecule viewer . The grid map was

Anti -Collagenase Activity of Konjac Composition
Molecular Docking Studies of Konjac Composition with

Wets methods were applied for minimization, using default

for anti-wrinkle activity. Automated docking is a graphicalenergy of drug compounds and 3D coordinates were pre - 50

centered at particular residues of the protein and was gen - Collagenase
erated with AutoGrid . As per genetic algorithm all the 55 AutoDock tools was utilized to generate grids , calculate
torsions were allowed to rotate during docking. The dock scores and evaluate the conformers of the konjac
Lamarckian genetic algorithm and the pseudo - Solis andcomposition bound in the active site of collagenase as targets

user interface . AutoDock 4 . 2 was employed to get docking
parameters (Rodriguez and Infante, 2011) .
60 and binding scores ; which is implemented by Lamarckian
The konjac composition exhibited pronounced elastase genetic algorithm method . The ligand molecules i.e., theinhibition activity as evident from the thermodynamic konjac composition and epigallocatechin gallate (EGCG )parameters studied . The interaction ofmolecule with active were designed and the structure was analyzed using ACDI
pocket residues required less binding energy comparable to Chemsketch . The PRODRG server was used to minimize
standard EGCG . The konjac composition formed three 65 energy of drug compounds and 3D coordinates were prehydrogen bonds in the active pocket of the protein . The pared . The protein structure file (PDB ID : 1CGL ) (FIG . 7 )was taken from PDB and was edited by removing the hetero
results are depicted in Table 3 (FIG . 5 ).

US 10 ,004 ,679 B2

12
atoms using Python molecule viewer. The grid map was

distilled water in the ratio 3 % , ceramide-base 5 % andcentered at particular residues of the protein and was gen -distilled water 92 % .

Wets methods were applied for minimization , using default

was calculated as follows:

erated with AutoGrid . As per genetic algorithm all theCondition : Temperature 35° C ., RH 40 %torsions were allowed to rotate during docking. The
1 g of test sample was weighed and added to vessel ( 3Lamarckian genetic algorithm and the pseudo - Solis and 55 cm ). Samples were weighed 8 hours later.Moisturizing ratio
parameters (Rodriguez & Infante , 2011).
The konjac composition binds very efficiently within theactive pocket of collagenase (FIG . 8 ). The result obtained iscomparable to orientation of standard EGCG ( Table 5 ).

Mole

Moisturising Ratio (% )= { (Weight at 0 hour -Weight10

at Time(t))/Original Weight}x100
The konjac composition demonstrated superior moistur

TABLE 5izing effect with maximum moisturizing ratio of 80 .45 % as

Molecular dockingof konjac extract with collagenase
Thermodynamic parameterscompared to control which is 31.02 % . ( FIG . 10 ). Our study
shows that the konjac composition can be exploited as
15 moisturizer to make the skin look younger and brighter.

LigandInhibi-Inter

Binding

Effi

tion

molecular

EnergyREFERENCES

ciencyConstantenergycule(kJmol-?) (kJmol- 1)
Konjac-5.89-0 .37(UM )48 .23(kJmol- l) bonds actions
1. Boisnic S, Branchet M ? . Interet clinique d 'un ingre- 9.17 5 Arg214 , 20 dient alimentaire avisee hydratante :LipowheatTM . Etude ran

EGCG -8.41 -0.25

689 .45- 11. 99

EGCG

- 0 .25- 8 .41

H - Inter

Gln247, 20 domisee en double aveugle versus placebo . J Med Esth etAsp 245

Chir Derm 2007 , 34 (136 ): 239-42 .

Asp 2

2 . Menon G K , Feingold KR, Moser A H , Brown BE,
Elias PM . De novo sterologenesis in the skin . I I. Regulation

Ser243,

Ile232,Gln24725 by cutaneous barrier requirements . J Lipid Res 1985, 26 (4 ):418 - 27 .
In Vitro Anti -Collagenase Activity of Konjac Composi
3. Briganti S , Camera E , Picardo M . Chemical andtion
instrumental approaches to treat hyperpigmentation . Pig

The assay employed was based on spectrophotometric ment Cell Res 2003, 16 : 101- 110 .
methods reported in the literature . The assay was performed 20 4 . Maeda K . Fukuda M . Arbutin : mechanism of its
in 50 mM Tricine buffer (pH 7 .5 with 400 mM NaCl and 10 3 depigmenting action in culture . J pharmacol Exp Ther 1996 ,mM CaC12 ). Collagenase from Clostridium histolyticum
276 : 765 - 769 . human melanocyte(ChC -EC . 3 .4 .23.3 ) was dissolved in buffer for use at an
5 . Kim Y , Uyama H , Kobayashi S . Inhibition effects ofinitial concentration of 0 . 8 units/mL according to the sup+ )-catechin -aldehyde polycondensates on proteinases causplier 's activity data . The synthetic substrate N -[3 -(2 - furyl735) (ingproteolytic degradation of extracellular matrix . Biochem

acryloyl]-Leu -Gly -Pro - Ala (FALGPA ) was dissolved in Tri - 35 Biophys Res Commun 2004 , 320 : 256 - 261.
cine buffer to 2 mM . The konjac composition was incubated
6 . Raffetto JD , Khalil R A .Matrix metalloproteinases andwith the enzyme in buffer for 15 minutes before adding

their inhibitors in vascular remodeling and vascular disease .

substrate to start the reaction . The final reaction mixture

Biochem Pharmacol 2008, 75 : 346 - 359.

( 150 ul total volume) contained Tricine buffer , 0 .8 mm
FALGPA . 0 . 1 units ChC and 20 - 100 ug test extracts Nega 40 l. Van Wart H E , Steinbrink D R . A continuous spectro
assay for Clostridium histolyticum collagenase .
tive controls were performed with water. Absorbance at 335 photometric
Anal
Biochem
1981, 113 :356 - 365 .
nm was measured immediately after adding substrate and8.
BaylacS,
Racine P. Inhibition of human leukocytethen continuous for 20 minutes. EGCG , 250 UM (0 . 114
elastase by natural fragrant extracts of aromatic plants . Int J

mg/mL) was used as a positive control. FIG . 9

45 Aromather 2004 , 14 : 179 - 182 .

9. Melzig M F, Loser B , Ciesielski S . Inhibition of

TABLE 6

neutrophil elastase activity by phenolic compounds from

In vitro anti- collagenase activity of konjac composition% inhibition of Collagenase activity

KonjacConcentration in ug/ml

EGCG

extract
19 .7860
80

8 . 3016 .9325 . 83
32 . 70

100

41.52

34. 20
41.07

Blank
20

40

21.01
26 .37EXAMPLE 5

plants . Pharmazie 2001, 56 : 967 - 970 .

10 . Siedle B , Cisielski S , Murillo R , Loser B , Castro V ,50 Klaas C A , Hucke O , Labahn A , Melzig 3999M F , Merfort
I. Sesquiterpene lactones as inhibitors of human neutrophil
elastase. Bioorg Med Chem 2002, 10 : 2855 - 2861.
11 . Narisa Kamkaen , Narong Mulsri, Charoen Treesak .
Screening of some tropical vegetables for Anti- Tyrosinase
55 Activity. Thai Pharm Health sci J 2007 , 2 ( 1): 15 - 19
12 . Tamsyn Thring S A , Pauline Hili , Declan P N .
Anti-collagenase , anti -elastase and anti -oxidant activities ofextracts from 21 plants . BMC Compl Alt Med 2009, 9 : 27
60

The Moisturizing Effects of Konjac Composition
The moisturizing effect ofkonjac extract was examined in65
vitro .
Sample preparation : The konjac composition sample
solution was prepared by mixing with ceramide -base and

The invention claimed is :1. A method of treating a skin condition in a subject inneed thereof, the method comprising :administering to the subject a composition comprising an
Amorphophallus konjac tuber extract that is substan

tially free of glucomannan ;contacting Amorphophallus konjac tuber with alcohol

wherein the extract is made by a process comprisingUS 10 ,004 ,679 B2
13

to produce a mixture comprising a glucomannan coagu
late , and removing the glucomannan coagulate from the

mixture thereby producing an extract that is substan

tially free of glucomannan ;14removing the glucomannan coagulate from the mixture

thereby producing an extract that is substantially free of
glucomannan ; andwherein administering the composition treats the skinwherein the extract is capable of inhibiting tyrosinase , 5
condition in the subject.
elastase , collagenase or combinations thereof; and11.
Themethod of claim 10 , wherein the skin condition iswherein administering the composition treats the skinselected from the group consisting of wrinkling, loss of skincondition in the subject.
turgor, loss of skin tone, dryness, hyperpigmentation , or

2 . The method of claim 1 , wherein the skin condition isthereof.
selected from the group consisting of wrinkling , loss of skin 10 combinations
12.
The
method10 , wherein the composition isturgor, loss of skin tone , dryness, hyperpigmentation , or administered orally oforclaim
topically.
combinations thereof.
13.
The
method
of
claim
10
,
whereinconcentration of
3. The method of claim 2 , wherein the skin condition is the extract in the composition is about the100
ug/mL.
hyperpigmentation and administering the composition
14 . The method of claim 10 , wherein the composition
inhibits tyrosinase in the subject .
the extract and ceramide in a ratio of about 1 : 1 .7 .
4 . The method of claim 2 , wherein the skin condition is comprises
15 . The method of claim 10 , wherein the form of theselected from the group consisting ofwrinkling , loss of skin compositionis selected from the group consisting of atone , and loss of skin torgor and administering the compo
powder, pill , tablet, pellet , capsule , thin film , solution , spray,
sition inhibits elastase in the subject.

5 . The method of claim 2 . wherein the skin condition is 20 syrup , linctus, lozenge, pastille , chewing gum , paste , vapor,suspension , emulsion , ointment, cream , lotion , liniment, gel,selected from the group consisting of wrinkling, loss of skindrop , topical patch , buccal patch , bead , gummy, gel, sol andtone, and loss of skin torgor and administering the compo injection .
sition inhibits collagenase in the subject.
16 . A composition for treating a skin condition , the
6 . The method of claim 1, wherein the composition is os composition
comprising konjac extract and ceramide in a
25 wompadministered orally or topically.
ratio
of
about
1 : 1 . 7 , wherein the extract is substantially free
7. The method of claim 1, wherein the concentration of of glucomannan
and is made by a process comprising
the extract in the composition is about 100 ug/mL .
contacting
Amorphophallus
konjac tuber with alcohol to
8 . The method of claim 1, wherein the composition
comprises the extract and ceramide in a ratio of about 1 : 1. 7 . produce a mixture comprising a glucomannan coagulate ,orm ofthe glucomannan coagulate from the mixture
9 . The method of claim 1 , wherein the formof thethe 3030 and removing
producing an extract that is substantially free ofcomposition is selected from the group consisting of a thereby.
powder, pill, tablet, pellet, capsule, thin film , solution , spray, glucomannan
17 . The composition of claim 16 , further comprising asyrup , linctus , lozenge , pastille , chewing gum , paste , vapor ,suspension, emulsion , ointment, cream , lotion , liniment, gel, material selected from the group consisting of colloidal
drop , topical patch , buccal patch. bead, gummy, gel, sol or 35 silica, silica gel, magnesium trisilicate , or combinations
thereof.
injection .
18 . The method of claim 1 , wherein the glucomannan
10 . A method of treating a skin condition in a subject in coagulate
is removed by filtration and the extract is a filtrate .
need thereof, the method comprising :19 . The method of claim 10 , wherein the glucomannan
administering to the subject a composition comprising an coagulate
is removed by filtration and the extract is a filtrate .
40
extract of Amorphophallus konjac tuber ;20. The composition of claim 16 , wherein the glucoman
wherein the extract is substantially free of glucomannan
and is made by a process comprising contacting Amor nan coagulate is removed by filtration and the extract is aphophallus konjac tuber with alcohol to produce a filtrate .
* * * * *mixture comprising a glucomannan coagulate, and