# Patent

## Title
Short peptides and a method of use as an antioxidant

## Abstract

A short chain polypeptide having antioxidant activity is provided. The short chain polypeptide has 2 to 4 amino acids bonded to form a polypeptide chain having an amine-terminal end and a carboxyl terminal end; wherein a net charge of the short chain polypeptide is positive, the amino terminal amino acid is an amino acid selected from the group consisting of arginine, lysine and histidine, the polypeptide chain following from the amino terminus comprises a hydrophobic or neutral amino acid, and the polypeptide chain is free of an amino acid having a negatively charged side-chain group. A method to prepare a composition having antioxidant activity and a composition having antioxidant activity are also provided.

## Background
BACKGROUND OF THE INVENTION
Antioxidants are compounds which can delay or inhibit the oxidation of organic molecules by inhibition of the initiation and/or propagation of oxidizing chain reactions, generally free radical reactions. Species associated with free radical oxidation processes include peroxyl radicals (ROO.), superoxide radicals (O2.−) and hydroxyl radicals (.OH). Many natural and synthetic molecules have antioxidant properties and such character has been quantified, collected and published by the United States Department of Agriculture by listing of oxygen radical absorbance capacities (ORAC). Generally, a wide range of spices, fruits, berries and legumes have been identified as having antioxidant properties. Natural antioxidants provide platforms for the quenching of free radicals.
Conventionally employed biologically safe antioxidants include Vitamin C, Carnosine and Resveratrol. Carnosine (β-alanyl-histidine) is a natural dipeptide that is innate to vertebrates and found to act as a pH buffer, ion-chelating agent and in lipid peroxidation in vitro. Such activity for Carnosine spurs interest in peptide structures, because peptides offer a wide variety of structural modification and molecular design possibilities upon which antioxidant molecules of designed properties may be prepared.
Proteins have also been shown to have antioxidative activities against free radical oxidation of lipids and/or fatty acids. Certain peptides having electron donor properties can react with free radicals to terminate the radical chain reaction, although the exact mechanism of action for such antioxidant peptides is not clearly known. Some aromatic amino acids and histidine have been reported to play a vital role in peptides having antioxidant properties.
In view of growing demand for antioxidants designed for attractive cost and structure activity performance that may be used in food, cosmetic and other applications, economical antioxidants based on natural product raw material building blocks are sought. Proteins or long chain polypeptides having interesting antioxidant properties are known; however, the cost of producing synthetic peptides are five to twenty times higher than the cost of conventional antioxidants.
Therefore, the present inventors have studied the antioxidant properties of short polypeptide molecules and have surprisingly learned specific structure activity relationships which have led to the discovery of short chain polypeptides having antioxidant activity comparable to or better than conventionally known antioxidants such as Vitamin C, Resveratrol and Carnosine which are also cost effective.
Therefore an object of the present invention is to provide novel short chain polypeptides that have high antioxidant activity and are structurally tailored for a specific utility. The short chain polypeptides must be biologically safe and ideally at least economically competitive with the conventionally employed antioxidants described above.
A further object of the present invention is to provide a method to protect a composition from oxidation or to impart antioxidant properties to a composition.
An even further objective of the present invention is to provide a method to protect a keratinous material from free radical degradation.
SUMMARY OF THE INVENTION
These and other objects have been achieved by the present invention, the first embodiment of which includes a short chain polypeptide, comprising: 2 to 4 amino acids bonded to form a polypeptide chain having an amine-terminal end and a carboxyl terminal end; wherein a net charge of the polypeptide is positive, the amino terminal amino acid is an amino acid selected from the group consisting of arginine, lysine and histidine, the polypeptide chain following from the amino terminus comprises a hydrophobic or neutral amino acid, and the polypeptide chain is free of an amino acid having a negatively charged side-chain group.
In one embodiment of the present invention, the short chain polypeptide comprises four amino acids.
In another embodiment, the present invention includes a method to protect a composition from oxidation wherein the short chain polypeptide of the first embodiment is added to the composition.
In a further embodiment, the present invention includes a method to protect a keratinous material from free radical degradation by application of a composition containing the short chain polypeptide of the first embodiment to the keratinous material.
The foregoing paragraphs have been provided by way of general introduction, and are not intended to limit the scope of the following claims. The described embodiments, together with further advantages, will be best understood by reference to the following detailed description taken in conjunction with the appended claims.


DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
As used herein, the words “a” and “an” and the like carry the meaning of “one or more.” The phrases “selected from the group consisting of,” “chosen from,” and the like include mixtures of the specified materials. Terms such as “contain(s)” and the like are open terms meaning ‘including at least’ unless otherwise specifically noted. Where a numerical limit or range is stated, the endpoints are included. Also, all values and subranges within a numerical limit or range are specifically included as if explicitly written out.
Additional advantages and other features of the present invention will be set forth in part in the description that follows and in part will become apparent to those having ordinary skill in the art upon examination of the following or may be learned from the practice of the present invention. The advantages of the present invention may be realized and obtained as particularly pointed out in the appended claims. As will be realized, the present invention is capable of other and different embodiments, and its several details are capable of modifications in various obvious respects, all without departing from the present invention. In this regard, the description herein is to be understood as illustrative in nature, and not as restrictive.
In the following description peptide sequences are described in terms of one-letter abbreviations of the amino acids according to the following chart.
















Abbre-

Abbre-

Abbre-


Amino acid
viation
Amino Acid
viation
Amino Acid
viation







Alanine
A
Arginine
R
Asparagine
N


Aspartate
D
Cysteine
C
Glutamate
E


Glutamine
Q
Glycine
G
Histidine
H


Isoleucine
I
Leucine
L
Lysine
K


Methionine
M
Phenylalanine
F
Proline
P


Serine
S
Threonine
T
Tryptophan
W


Tyrosine
Y
Valine
V









The capital case letters represent naturally occurring amino acids in the L configuration. When a lower case letter is shown, the amino acid is in the D configuration.
According to conventional practice amino acid sequence description is provided by the one letter abbreviation sequence stated with the N-terminal end (N-terminus) amino acid first in the sequence and ending with the carboxyl terminal (carboxy terminus) amino acid. For example, the sequence RYHM (SEQ ID NO:4) is a polypeptide containing four amino acids linked via peptide bonds in the order from N-terminus to carboxy terminus: arginine-tyrosine-histidine-methionine. When the carboxyl terminus of the sequence is designated with —NH2, the end carboxyl group is in the form of an amide. When the N terminal group is derivatized, the nature of the derivative is designated by standard organic chemistry abbreviations. For example, “Ac” indicates an acetyl group.
Throughout the following description, terms such as “polypeptide,” “short chain polypeptide” and peptide molecule may be used interchangeably. According to the present invention, a short chain polypeptide contains 2 to 10 amino acids linked through a sequence of peptide bonds.
As understood by one of ordinary skill, the amino acids may be grouped according to the chemical structure of the side chain. Thus glycine, alanine, valine, leucine, isoleucine, methionine, tryptophan, phenylalanine and proline are described as nonpolar (hydrophobic) amino acids. Serine, threonine, cysteine, tyrosine, asparagine and glutamine are described as polar (hydrophilic), neutrally charged amino acids. Aspartic acid (aspartate) and glutamic acid (glutamate) are ionic with a negative charge and lysine, arginine and histidine are ionic with a positive charge (basic).
Standard abbreviations conventionally employed in organic chemistry may also be employed. As an example, “Ac” represents an acetyl group.
In the study of and search for molecules that have potent antioxidant activity and at the same time are biologically safe, economical to produce and may be tailored for physical properties including bioavailability, solubility and dispersibility, the inventors have recognized that polypeptides are an interesting class of molecules that have the potential to meet all the criteria for development of novel new antioxidants.
Thus a study was undertaken to understand the structural property and molecular composition parameters of polypeptide molecules that contribute to maximum antioxidant activity.
As a result of this study, the inventors have discovered the present invention, the first embodiment of which is a short chain polypeptide, comprising: 2 to 4 amino acids bonded to form a polypeptide chain having an amine-terminal end and a carboxyl terminal end; wherein a net charge of the polypeptide is positive, the amino terminal amino acid is an amino acid selected from the group consisting of arginine, lysine and histidine, the polypeptide chain following from the amino terminus comprises a hydrophobic or neutral amino acid, and the polypeptide chain is free of an amino acid having a negatively charged side-chain group.
Tables I to IX shown below are a compilation of polypeptides studied by the inventors to ascertain those elements of the molecular structure that contribute to or negate antioxidant activity. In the course of the study, polypeptides were prepared by conventional synthesis methods and screened in tubo for antioxidant activity for peroxyl radicals (ROO.), superoxide radicals (O2.−) and hydroxyl radicals (.OH).
In the screening standard testing for oxygen radical absorbance capacity (ORAC), hydroxyl radical absorbance capacity (HORAC), superoxide radical absorbance capacity (SORAC) and superoxide dismutase activity (SOD) were conducted with the test polypeptides. Testing was conducted according to USTM 190 (HORAC), USTM 191 (SORAC), USTM 192 (ORAC) and USTM 193 (SOAC) (SOD). Upon review of the results as indicated in the Tables, the inventors have discovered certain elements of the polypeptide structure as recited in claim 1 that are key contributors to determination of antioxidant activity.
Thus as shown in Table III, short chain polypeptides having neutral and/or hydrophobic amino acids at the N-terminal exhibited low or limited activity in HORAC and SORAC. Table IV shows that polypeptides having a negatively charged amino acid at the N-terminus have no activity according to both ORAC and HORAC, while Table V shows that short chain polypeptides having a positive charged (basic) amino acid at the N-terminus have high HORAC activity. In fact, the HORAC activity is 10,000 μmol GAE/g or higher and is significantly greater than the values shown in any of the previous Tables.
Table VI shows that short chain polypeptides of four or less amino acids having a net sum of positive charge across the side chain structures have the greatest overall antioxidant activity. However as indicated by Table VII, a polypeptide structure having high net positive charge, but not containing a neutral or hydrophobic amino acid had no ORAC activity. Thus as shown in Table VIII, inclusion of at least one hydrophobic and/or neutral amino acid in the amino acid sequence after the N-terminal amino acid leads to significantly higher antioxidant activity as demonstrated by the ORAC values.
Further, the inventors have discovered, that even though a given sequence may contain positively charged amino acids and neutral and/or hydrophobic amino acids, inclusion of a negatively charged (acidic) amino acid in the chain leads to a molecule having no ORAC or HORAC activity.
Thus, the inventors have discovered that the placement of the sequence and overall positive charge are crucial factors that determine the antioxidant activity of the short chain polypeptide. The first amino acid should be positively charged as a neutrally/hydrophobic and/negatively charged amino acid has limited to low antioxidant activity, especially for HORAC. Though an overall positive charge is a crucial factor contributing to antioxidant activity, a hydrophobic/neutrally charged amino acid within the chain subsequent to the N-terminal amino acid is necessary for increased activity as seen in ORAC activity. Further, the presence of negatively charged amino acids eliminates any antioxidant activity of the short chain polypeptide.
In one preferred aspect of the first embodiment, the short chain polypeptide of the present invention contains four amino acids. The inventors have taken all the information obtained from the study described above as indicated from Tables III to IX and designed the short chain polypeptides of formulas (I) to (IV) (SEQ ID NOS:1-4) as explicit embodiments of the present invention.







The structural formulas, mass weights, single letter abbreviation and net polypeptide charge for each of the novel short chain polypeptides are shown in Table I. ORAC, HORAC and SOD test results for each of the structures of formulas (I) to (IV) are shown in Table II. Also shown in Table II are antioxidant results for Vitamin C, Carnosine and Resveratrol. As indicated in Table II, the short chain polypeptides according to the present invention have ORAC activity comparable to those of the conventional antioxidants, while having significantly higher HORAC activity. Such increased HORAC activity is an unexpected and significant improvement obtained with the compounds of the present invention.
In another embodiment, the present invention also includes a method to prepare a composition having antioxidant properties. The method includes adding a short chain polypeptide according to the first embodiment to the composition. Thus the short chain polypeptide added comprises 2 to 4 amino acids bonded to form the polypeptide chain, wherein a net charge of the short chain polypeptide is positive, the amino terminal amino acid of the polypeptide chain is an amino acid selected from the group consisting of arginine, lysine and histidine, the polypeptide chain following from the amino terminus comprises a hydrophobic or neutral amino acid, and the polypeptide chain is free of an amino acid having a negatively charged side-chain group.
In explicit aspects of this embodiment, the short chain polypeptide may be one or more of compounds of formulas (I) to (IV).
The composition may be of any physical form including, for example, a solid, a paste, a cream, a gel or a liquid. The composition may be a solution, a dispersion, an emulsion or a suspension. The content of the short chain polypeptide according to the invention will depend upon the nature and intended use of the composition and may be easily formulated by one of ordinary skill in the respective technology of the composition.
In general the content of the short chain antioxidant polypeptide of the invention may be from 0.01 to 40 wt % of the total weight of the composition. However, the content is not limited and depending on a given utility, a content different from the range of 0.01 to 40 wt % may be employed.
In a further embodiment, the present invention includes a composition comprising a short chain polypeptide, according to the first embodiment to the composition. Thus the short chain polypeptide added comprises 2 to 4 amino acids bonded to form the polypeptide chain, wherein a net charge of the short chain polypeptide is positive, the amino terminal amino acid of the polypeptide chain is an amino acid selected from the group consisting of arginine, lysine and histidine, the polypeptide chain following from the amino terminus comprises a hydrophobic or neutral amino acid, and the polypeptide chain is free of an amino acid having a negatively charged side-chain group.
A short chain polypeptide of the invention may be advantageously formulated in a composition that may be in any galenical form normally available for the intended indication and mode of administration. The composition may comprise a physiologically or pharmaceutically acceptable medium.
According to one embodiment, a topical composition according to the invention may advantageously be formulated in any galenical form that is suitable for caring for the skin and its integuments, and may be in the form of ointments, creams, solutions, gels, emulsions, foams or aerosol compositions containing a propellant, milks, pomades, powders, impregnated pads, lotions or suspensions. A composition intended for topical administration may be an aqueous, aqueous-alcoholic or oily solution, a solution or a dispersion of the lotion or serum type, an emulsion of liquid or semiliquid consistency of the milk type, obtained by dispersing a fatty phase in an aqueous phase (O/W) or conversely (W/O), a suspension or an emulsion, of soft, semisolid or solid consistency, of the cream type or of the aqueous or anhydrous gel type, a multiple emulsion (W/O/W or O/W/O), a microemulsion, a nanoemulsion, a preparation of microcapsules, a preparation of microparticles, a vesicular dispersion of ionic and/or nonionic type, or a wax/aqueous phase dispersion.
In the case of a composition in accordance with the invention for oral administration, the use of an ingestible support, whose nature is adapted according to the type of composition under consideration, is preferred. Tablets, gel capsules or lozenges, suspensions, oral supplements in dry form and oral supplements in liquid form, milk, yoghurt, cheese, fermented milks, milk-based fermented products, ice creams, cereal-based products or fermented cereal-based products, milk-based powders, infant and baby formulae, food products of confectionery, chocolate or cereal type, and animal feed in particular for pets, are thus especially suitable as food supports.
The term “oral composition” means, for example, nutritional, nutraceutical, cosmeceutical or pharmaceutical compositions comprising at least one compound according to the invention. The formulation of the oral compositions according to the invention may be performed via any common process known to those skilled in the art for producing drinkable solutions, coated tablets, gel capsules, gels, emulsions, tablets to be swallowed or chewed, capsules, especially soft or hard capsules, granules to be dissolved, syrups, solid or liquid foods and hydrogels allowing controlled release, food bars, powders, in compacted or non-compacted form, liquid solutions or suspensions, confectioneries, fermented milk, fermented cheeses, chewing gums, toothpastes or spray solutions.
A short chain polypeptide of the invention may moreover be formulated with the usual excipients and components for such oral compositions or food supplements, i.e. especially fatty and/or aqueous components, humectants, thickeners, preserving agents, texture agents, taste agents and/or coating agents, and/or antioxidants. The formulating agents and excipients for oral compositions, and especially for food supplements, are known in this field and will not be the subject of a detailed description herein.
A composition according to the invention may also comprise any formulating agent or any cosmetically or dermatologically acceptable additional active agent. The amounts of these various active agents are those conventionally used in the field under consideration, and are especially determined so as not to affect the desired properties for a compound of the invention or for a composition of the invention.
In another embodiment, the short chain polypeptides according to the present invention may be included in nutritional compositions as an antioxidant or stabilizer of the composition that may also function as a solubilizer or as a dispersant. In other aspects of this embodiment, the short chain polypeptides according to the invention may be combined with other antioxidants to stabilize a composition to a broad spectrum of oxidative degradation mechanisms. The composition may contain nutrient fats, oils and/or proteins and may be aqueous or oil based solutions or emulsions or dry powders. In addition to the conventional antioxidants described above, other antioxidants known to one of skill in the art may be employed in combination with the short chain polypeptides of the present invention. Examples of adjuvant antioxidants may include, but are not limited to butylated hydroxy toluene, α- or β-carotene, citric acid or a derivative thereof, p-aminobenzoic acid, tocopherols and vitamins e, k and q10. One of ordinary skill may formulate an effective anti-oxidant combination for a nutritional composition employing conventional laboratory test methods.
In another embodiment, the present invention provides a method for attenuating effects of free radicals on a keratinous material by application of the composition described above to the keratinous material. The keratinous material may be human skin or hair and the composition may be in the form of, for example, a sunscreen, a skin cream, a shampoo or a hair conditioner.
The composition may be of any physical form including, for example, a solid, a paste, a cream, a gel or a liquid. The composition may be a solution, a dispersion, an emulsion or a suspension. The content of the short chain polypeptide according to the invention will depend upon the nature and intended use of the composition and may be easily formulated by one of ordinary skill in the respective technology of the composition.
In general the content of the short chain antioxidant polypeptide of the invention may be from 0.01 to 40 wt % of the total weight of the composition. However, the content is not limited and depending on a given utility, a content different from the range of 0.01 to 40 wt % may be employed.
In explicit aspects of this embodiment, the short chain polypeptide may be one or more of compounds of formulas (I) to (IV).
In another embodiment, the present invention provides a method to protect a keratinous material from free radical degradation by application of the composition described above to the keratinous material. The keratinous material may be human skin or hair and the composition may be in the form of, for example, a sunscreen, a skin cream, a shampoo or a hair conditioner.
The above description is presented to enable a person skilled in the art to make and use the invention, and is provided in the context of a particular application and its requirements. Various modifications to the preferred embodiments will be readily apparent to those skilled in the art, and the generic principles defined herein may be applied to other embodiments and applications without departing from the spirit and scope of the invention. Thus, this invention is not intended to be limited to the embodiments shown, but is to be accorded the widest scope consistent with the principles and features disclosed herein. In this regard, certain embodiments within the invention may not show every benefit of the invention, considered broadly.
EXAMPLES
Method of Peptide Synthesis
N-α-Fmoc-L-amino acids, Fmoc-amide resin and reagents used for peptide synthesis were obtained from Iris Biotech. Solvents were analytical grade products from ThermoFisher. The three peptides were chemically synthesized by the solid phase method using an automated peptide synthesizer (Applied A433). Peptide chains were assembled stepwise on 0.75 meq of Fmoc-amide resin using 1 mmol of Fmoc L-amino acids.
The following reagents were used: Fmoc-amino acids (lmmol), activator (0.5 M HOBT/HBTU in dimethylformamide), base (2 M diisopropylethylamine in N-methyl-pyrrolidone) and deprotecting mixture (20% piperidine in N-methyl-pyrrolidone).
After peptide chain assembly, peptidyl-resins were treated 2 h at room temperature with a mixture of TFA/water/phenol/thioanisole/ethanedithiol (92.5/2/1/2.5/2). The peptide mixtures were then filtered, and the filtrates were precipitated by adding cold diethylether. The crude peptides were pelleted by centrifugation (3,000 g; 10 min), and the supernatants were discarded.
Peptides were purified by C18 reversed-phase (RP) High Performance/Pressure Liquid Chromatography (HPLC) using an Onyx Jupiter column (250×10 mm, 50. Elution of the peptides was performed with a linear gradient of 0 to 40% acetonitrile in 0.1% TFA (run duration of 150 min). The collected fractions were analyzed for their peptide content by analytical C18 RP-HPLC (Onyx monolithic column, 100×4.6 mm). The target peptides were characterized by matrix-assisted laser desorption ionization-time-of-flight (MALDI-TOF) mass spectrometry.
The purity of each peptide sample tested was >98%.
Peptides according to the present invention are listed in Table I. The Table shows the sequence of the peptide in one-letter abbreviation, the structural formula, the molecular weight and the net charge of the tetrapeptide.











TABLE I





Name
Sequence
Structure
Mass (g/mol)
Net Charge



















CP2
RRHM
formula (I)
598.31
+2.5



(SEQ ID NO: 1)





AO1
HYHY
formula (II)
618.25
+1



(SEQ ID NO: 2)





AO2
RYHW
formula (III)
680.31
+1.5



(SEQ ID NO: 3)





AO2.1
RYHM
formula (IV)
605.27
+1.5



(SEQ ID NO: 4)









The peptides were tested in tubo for their antioxidant properties and tested against reference/known antioxidants. The evaluation of the antioxidant properties were conducted by determination of the ORAC (USTM 192), HORAC (USTM 190) and SOD (USTM 193) test methods. The results are listed in Table II for the peptides of the invention and for the conventionally known antioxidants











TABLE II







ORAC/
HORAC/
SOD/




peroxyl,
hydroxyl,
superoxide,



Sequence
μmol GAE/g
μmol GAE/g
Active50 (U/mg)



















Name






CP2
RRHM
1204
321448




(SEQ ID NO: 1)





AO1
HYHY
4748
33489
6 (Active25)



(SEQ ID NO: 2)





AO2
RYHW
9328
41882
4 (Active25)



(SEQ ID NO: 3)





AO2.1
RYHM
5390
132591




(SEQ ID NO: 4)





Supplier






DSM

4456
0
752


Symrise

354
2099
 0


Symrise

25462
14190
626 (Active25) 









Table III shows the results obtained for short chain polypeptides having a neutral and/or hydrophobic amino acid at the N-terminal.












TABLE III





Name
Sequence
ORAC
HORAC
SORAC



















RED9
fkwR-NH2
2202
 752
No capacity


RED25
WKYK-NH2
4168
2476
No capacity



(SEQ ID NO: 5)





RED31
wRwR-NH2
4652
—
No capacity


RED6
WRYR-NH2
4488
1548
No capacity



(SEQ ID NO: 6)





RED18
WrYr-NH2
4758
2982
No capacity


RED19
WRYr-NH2
4350
1457
No capacity


RED20
WrYR-NH2
4448
1666
No capacity


RED21
WRyR-NH2
4816
2472
7(IC25)


RED22
wRyR-NH2
5383
1686
No capacity


RED24
WRYR-NH2
4077
2225
No capacity


RED23
WRYR
4091
2089
No capacity



(SEQ ID NO: 6)





RED29
wRyRw-NH2
5512
—
No capacity


RED28
wRyRy-NH2
5216
—
3(IC25)


RED34
YkYkYk
355
1053
—









Table IV shows the results obtained for short chain polypeptides having a negatively charged amino acid at the N-terminal. These sequences have no activity in both ORAC and HORAC.










TABLE IV





Tradename
Sequence
ORAC
HORAC







AC-SYN1
Ac-EEVKRK-NH2
No capacity
No capacity



(SEQ ID: 7)




 


AC-SYN2
Ac-EELMSDIKK-NH2
No capacity
No capacity



(SEQ ID: 8)




 


AC-SNP3
Ac-EEMLERQRR-NH2
No capacity
No capacity



(SEQ ID: 9)




 


AC-SNP4
Ac-DESLESTRRM-
No capacity
No capacity



NH2





(SEQ ID: 10)




 


AC-SNP5
Ac-EEMQRRKKK-NH2
No capacity
No capacity



(SEQ ID: 11)









Table V shows the results obtained for short chain polypeptides having a positively charged amino acid at the N-terminal.












TABLE V






Tradename
Sequence
ORAC
HORAC
SORAC





















CP2
RRHM
1204
321448
6(IC25)




(SEQ ID NO: 1)






AO2.1
RYHM
5390
131574
—




(SEQ ID NO: 4)






AO2
RYHW
9328
41882
4(IC25)




(SEQ ID NO: 3)






CP2.6
RRH
0
41073
—



AO1
HYHY
4748
33489
6(IC25)




(SEQ ID NO: 2)






CP2.5
HRHM
952
10192
—




(SEQ ID NO: 12)









Table VI shows the results obtained for polypeptides having four or less amino acids.












TABLE VI










Charge


Tradename
Sequence
ORAC
HORAC
SORAC
(pH 7)




















CP2
RRHM
1204
321448
6(IC25)
  2.5+



(SEQ ID NO: 1)






AO2.1
RYHM
5390
131574

  1.5+



(SEQ ID NO: 4)






AO2
RYHW
9328
41882
4(IC25)
  1.5+



(SEQ ID NO: 3)






CP2.6
RRH
0
41073

  2.5+


AO1
HYHY
4748
33489
6(IC25)
1+



(SEQ ID NO: 2)






CP2.5
HRHM
952
10192

2+



(SEQ ID NO: 12)






IMM2
YHFR

3036
5(IC25)
1+



(SEQ ID NO: 13)






RED18
WrYr-NH2
4758
2982
No capacity
2+


RED25
WKYK-NH2
4168
2476
No capacity
2+



(SEQ ID NO: 5)






RED21
WRyR-NH2
4816
2472
7(IC25)
2+


RED24
wRYR-NH2
4077
2225
No capacity
2+


RED23
WRYR
4091
2089
No capacity
2+



(SEQ ID NO: 6)






RED22
wRyR-NH2
5383
1686
No capacity
2+


RED20
WrYR-NH2
4448
1666
No capacity
2+


RED6
WRYR-NH2
4488
1548
No capacity
2+



(SEQ ID NO: 6)






RED19
WRYr-NH2
4350
1457
No capacity
2+









Table VII shows the results obtained for a short chain polypeptide having a positive charge (2.5+) is important to contributing to HORAC activity, the lack of a hydrophobic/neutrally charged amino acid has given this sequence no capacity in ORAC activity.











TABLE VII





Tradename
Sequence
ORAC
HORAC
SORAC







CP2.6
RRH
No capacity
41073
—









Table VIII below indicates that the presence of at least one neutral and/or hydrophobic amino acid in the chain after the N-terminal amino acid contributes to the ORAC activity.











TABLE VIII





Tradename
Sequence
ORAC
HORAC
SORAC







AO2
RYHW
9328
41882 
4(IC25)



(SEQ ID NO: 3)





S1
HWPY
5955
—
13(IC25) 



(SEQ ID NO: 14)





S2
YWPW
5917
—
No capacity



(SEQ ID NO: 15)





RED29
wRyRw-NH2
5512
—
No capacity


AO2.1
RYHM
5390
131574 




(SEQ ID NO: 4)





RED22
wRyR-NH2
5383
1686
No capacity


RED27
Ac-wRyR-NH2
5342
—
No capacity


RED28
wRyRy-NH2
5216
—
3(IC25)


RED21
WRyR-NH2
4816
2472
7(IC25)


RED18
WrYr-NH2
4758
2982
No capacity


AO1
HYHY
4748
33489 
6(IC25)



(SEQ ID NO: 2)





RED31
wRwR-NH2
4652
—
No capacity


RED6
WRYR-NH2
4488
1548
No capacity



(SEQ ID NO: 6)





RED26
RwRyR-NH2
4461
—
No capacity


RED20
WrYR-NH2
4448
1666
No capacity


RED19
WRYr-NH2
4350
1457
No capacity


RED25
WKYK-NH2
4168
2476
No capacity



(SEQ ID NO: 5)





RED23
WRYR
4091
2089
No capacity



(SEQ ID NO: 6)





RED24
wRYR-NH2
4077
2225
No capacity


RED32
RyR-NH2
3385
—
No capacity


IMM1
HWRF
2422
—
5(IC25)



(SEQ ID NO: 16)





RED9
fkwR-NH2
2202
 752
No capacity


RED7
kfRw-NH2
2120
1124
No capacity


RED11
CWRYR-NH2
2007
 625
No capacity



(SEQ ID NO: 17)





RED2
KRKYWW-NH2
1837
1063
No capacity



(SEQ ID NO: 18)









Table IX shows results obtained for polypeptides containing a negatively charged amino acid. These sequences have no activity in both ORAC and HORAC. It shows that despite the presence of positively charge amino acids and neutral/hydrophobic amino acids, these peptide sequences have no antioxidant activity.










TABLE IX





Tradename
Sequence
ORAC
HORAC







AC-SYN1
Ac-EEVKRK-NH2
No capacity
No capacity



(SEQ ID: 7)




 


AC-SYN2
Ac-EELMSDIKK-NH2
No capacity
No capacity



(SEQ ID: 8)




 


AC-SNP1
Ac-RRMLQLVEE-NH2
No capacity
No capacity



(SEQ ID: 19)




 


AC-SNP3
Ac-EEMLERQRR-NH2
No capacity
No capacity



(SEQ ID: 9)




 


AC-SNP4
Ac-DESLESTRRM-NH2 
No capacity
No capacity



(SEQ ID: 10)




 


AC-SNP5
Ac-EEMQRRKKK-NH2
No capacity
No capacity



(SEQ ID: 11)




 


AC-SNP6
Ac-Valérate-
No capacity
No capacity



EEMQRRKKK-NH2





(SEQ ID: 11)




 


AC-SNP7
Ac-KKKEEMQRR-NH2
No capacity
No capacity



(SEQ ID: 20)









The above description is presented to enable a person skilled in the art to make and use the invention, and is provided in the context of a particular application and its requirements. Various modifications to the preferred embodiments will be readily apparent to those skilled in the art, and the generic principles defined herein may be applied to other embodiments and applications without departing from the spirit and scope of the invention. Thus, this invention is not intended to be limited to the embodiments shown, but is to be accorded the widest scope consistent with the principles and features disclosed herein. In this regard, certain embodiments within the invention may not show every benefit of the invention, considered broadly.

## Claims

1. A short chain polypeptide, comprising:
4 amino acids bonded to form a polypeptide chain having an amine-terminal end and carboxyl terminal end;
wherein the carboxy-terminal end is amidated;
wherein the net charge of the polypeptide is positive, the amino terminal amino acid is an amino acid selected from the group consisting of arginine, lysine and histidine, and the polypeptide chain following from the amino terminus comprises a hydrophobic or neutral amino acid, and the polypeptide chain is free of an amino acid having a negatively charged side-chain group;

and wherein the polypeptide is selected from the group consisting of formula (I), formula (II) and formula (III):

2. A method to prepare a composition having antioxidant properties, comprising:
adding the short chain polypeptide of claim 1 to the composition.

3. A composition, comprising:
the short chain polypeptide of claim 1 in a physiologically or pharmaceutically acceptable medium.

4. The composition of claim 3, further comprising an antioxidant that is not a short chain polypeptide.

5. The composition of claim 3, further comprising a nutritional ingredient selected from the group consisting of a fat, an oil and a protein.

6. The composition of claim 3, wherein a content of the short chain polypeptide is from 0.01 to 40 wt % based on a total weight of the composition.

7. A method for attenuating effects of free radicals on a keratinous material, comprising applying the composition of claim 3, to the keratinous material of a subject in need thereof.