L. Rigano and c. AndoLfatto

Laboratori L. Rigano, Milan, Italy

F. RastreLLi

Kalichem Italia, Botticino Sera, Italy

KEY WORDS: antiaging, sodium DNA, cell renewal, skinmoisturization, skin elasticity

ABSTRACT: A particular extract of DNA from the gonadic tissueof male sturgeons is shown to have cell renewaleffects with possible antiaging beneJits for skinmoisture, thickness, elasticity and a reduction in skinwrinkledness.

S odium DNA is an ingredient withactivity at the cellular level. This fact

has led to its incorporation in numeroushigh-end antiaging skin care products. Anexplanation of that activity and results ofseveral tests of one sodium DNA materialare presentedin this article.

Moreover) as the years pass) biologicaland environmental damage accumulatesand wiq!not be repaired naturally.The most important changes concerncollagen and elastin fibers) the basicconstituents of the connective tissue. Theamount of collagen) which is synthesizedby the fibroblasts) tends to decrease asthe activity of the fibroblasts is modi-fied;this adds to the effects of the sunradiatlon and the fal1 in estrogen during

menopause.

Action is necessary

on different levels

to slow down

the skin aging process.

During menopause the elastin fibersatrophyas the dermis thins and their pro-duction is altered; thus, the dermis loosesits viscoelastic properties. In addition,the epidermis becomes less efficient inperforming its barrier function becauseof a loss of keratinocytes and a reductionin the thickness of the hydrolipid fi1mthickness on its surface.

The skin tends to look dull, becomesdry and dehydrated, less firm and

not homogeneous, grows thin andslackens. This is caused by a numberof factors. On the one hand, the inputof biological nutriènts becomes poordue to reduced skin vascularizationand slow cell renewalj on the otherhand, the progressive degeneration ofthe dermal connective tissue and thestructural changes in the epidermisplay their role. Environmental stressmust also be considered. Skin cells areexposed daily to UV rays, infrared (IR),osmotic stress and poor moisturization;therefore, they become an easy target forthousands of free radicals. Furthermore,psychological stress weakens the body'sdefense system, making it more vulner-able totheir attacks.

In conclusion, it seems clear thataction is necessary on different levels toslow down the skin aging process.

Extracted DNA to StimuLate

CeLl Repair

More than 30 years ago in Russia,experiments were performed with theaim of developing an effective treat-ment for diseases related to ionizingradiation. Among the biologica1ly activematerials tested was deoxyribonucleicacid (DNA) extracted from the gonadictissue of male sturgeons. The extractwas carefully purified, depolymerizedand neutralized with sodium hydroxideaccording documented procedures.1.2The extract later received the INCIname Sodium DNA.

Positive feedback on sodium DNAwas obtained in 1986, when it wasemployed to treat diseases related to theChernobyl nuclear disaster, howeverthe results were never precisely quanti-fied. Indeed, in animal studies carriedout some years later,3 the same com-

The Many LeveLs of Aging

www.CosmeticsandToiletries.com Cosmetics & Toiletries'" magazine I 57Vol. 121, No. 11/November 2006

The slow, inevitable skin agingprocess is characterized by a progressivedegeneration of the skin tissue as well asby a variety of attendant visible changesin the skin surface. The skin acquiresa new appearance as wrinkles formand become increasingly conspicuous,the epidermallayer thins, and the skindecreases in firmness and elasticity.These changes show the passage fromyouth to adulthood to old age.

Such visible effects can be seen wellbefore the age of 30 in humans andresult from major changes in skin cellsand the structures supporting the tissue.They are caused by a variety of genetic,metabolic, hormonal, biological andenvironmental factors.

The first and most notable sign ofaging is a decrease in the skin's waterretention capability and the resultantdecrease in dermis elasticity. This is vis-ible when facial muscles are contractedand face wrinkles become deeper.

pound was shown to protect and repairy-radiation-induced lesions. In the fol-lowing years, many clinical tests provedits efficacy at treating different types ofskin lesions and illnesses.4,5 Scientistslooked at its nucleotides, the basic unitsof nucleic acids. They found that nucleo-tide segments of DNA with a molecularmass from 250 to 500 kD were able tocontrol the formation of wrinkles. More-over, intradermal administration ofDNAfragments in aesthetic surgery patientsaccelerated wound healing.6,7 This pavedthe way to research on sodium DNA asan antiaging active in cosmetics.

0.00+.+ 4> ..

00.0.'~ 0 +00 +

.Oò..~0 O y+

0..Oo. .

~ + +Ò Ò v0 + 0#0 + i9 O

,b OÒoO+O

Figure 1. Pinocytosis as a method of transport of exogenous molecules in the celi

bases, which are key molecules for thevitality of all cells. Sodium DNA passesthrough the cell membrane by pinocy-tosis (Figure 1), an endocytosis methodof transport facilitated by sodium ions,which are combined with the poly-deoxyribonucleotides. Therefore, the cel1spresumably use the acquired amount ofsodium DNA both as a structural baseto synthesize the nucleic acids and theircofactors and to metabolize their ownDNA. These processes occur very easilyin cells that are under metabolic condi-tions or extreme stress. This is the caseof altered keratinocytes or fibroblasts,both of which are typical in aged skin.Tha.~ to the cell integration process,

sodium DNA acts to stimulate celI repairactivity, regenerate epithelial and granu-lation tissues and reduce the symptomsof inflammation, thus accelerating thehealing of skin microlesions.8-11

Sodium DNA acts

to stimulate

cell repair activity.

In vitro Tests

In vitro tests were used to assess theregenerating and photo-protective prop-erty of sodium DNA toward two ceUtypes-keratinocytes and fibroblasts.The particular form of sodium DNAused in these tests was a highly purifiedcommercial producta with molecularThe most likely explanation of this

ingredient's antiaging effect is based onthe fact that some segments of DNAact as donors of vurine and vvrimidine

.Kalinat Anti- Wrinkle (INCI: Sodium DNA), KalichemItalia LC, Botticino Sera, Italy. Kalinat Anti. Wrinkle is aregistered trademark of Kalichem Italia.

Statement or Ownership, Management and Circulation

(Act or August 12, 1970; Section 3685, Title 39, United States Code)1. Publication lil/e: C08metics & Toi1etries2. Publicalion number: 0361-43873. Filing date: September 13.20064. Issue frequency:Month1y5. Numberofissuespublishedannually: 126. Annual subscription price. $98.007. Complete mailing address of known office ofpublication: 362 South Schmale Road. Carol Stream. IL 60188-2787 Te/ephone: 630-653-21558. Complete mailing address ofheadquarters or generai business offices ofpublisher: Same as above9. Full names ond complele mailing addresses ofpublisher. editorond managing editor~ Publisher~ Matt Gron1und. 362 South Schmale Road, Carol Stream. IL60188-2787

Editor: Rache1 Chapman. 362 South Schmale Road, Caro1 Stream, IL60J88-2787 Managing Editor: Lois Hince, 362 South Schmale Road, Carol Stream.1L60J88-278710. Owner: Allured Publishing Corporalion, 362 South Schmale Road, Carol Stream. IL60188-2787; Stan1ey E. Allured. Nancy Al1ured, Janet Ludwig1J. Known bondholders. mol1gages and olher security holders owning or holding I pert:entor more oftotal amountofbonds. mortgages or other securities' None.12.

tax purposes: Not applicable13. Publi",tion title:C08metics &Toi]etries magazine14. I.,.,ue date jor cirt:ulation data helaw: September 200615. Extent ond nolure ofcirculation Average number of copies each Actual number of single issues

issue during preoeding 12 months published nearest to fi1ingdateA. Totalnumberofcopies(netpressrun): 3.861 5,027B. Paid andlor requesled circulation:

I. PaidlRequested Oulside County

MaiISubscription., 1,514 1.6182. Paid In-County Subscriptions 0 03. Sales through dealers and carriers, street vendors.

counter sa/es ond other non-USPS Paid Distribution 1.478 1.477C. Total poid ondIor requested circulation: 2.992 3.095D. Free distribution by mail: 0 0E. Free distribution oul.'ide Ihe mail (carrier., or olher means): 338 515F. Tolalfreedistribution: 338 515G. Total djstribution: 3.330 3.610H. Copies no1 distributed 530 1.4171. Total{SumofI5G.I5H(I)ond.l5H(2)J: 3.860 5.027

Percent poid andlor requesled cjrculation~ 89.85% 85.73%16. Publicalion of Ihi., Slalement of Ownership.- Will be printed jn the November 2006 i.,sue of Ihis publication.17. Sjgnolure and tit/e of editor. publisher, business manager or owner: Janet Ludwig. Owner

tion requested on theform may be subjecl to crjminal sanctions (includingfines ond imprisonment) andlor civil sanclions (including mu/tip/e damages ond cjvil penalties).

58 I Cosmetics & Toiletriese magazine www.CosmeticsandToiletries.com Vol. 121, No. 11/November 2006

Figure 2. Growth of keratinocytes 72 h after exposure at di.fferent concentrations ofONA-Na

weight in the range 250-500 kD.This product will be called DNA-Na inthe following discussion.

Keratinocytes and fibroblasts wereobtained from two healthy donors bybiopsy. Both specimens were grownin culture and incubated on platescontaining DNA-Na at differentconcentrations.

The skin's cell renewal rate decreasesnaturally as the years go by. This is thecause of skin aging. As a measure ofthe regenerating power, the growthrate of the cells treated with DNA-Nawas determined at 24, 48 and 72 hafter incubation and compared to theuntreated cells used as controls.

To assess theproperty of protectingthe cells exposed to radiation, the vital-ity of the cells treated with DNA-Nawas tested 24 h after exposure to a UVsource and compared to the untreatedcells used as) controls. Test resultsshowed that DNA-Na stimulated cellproliferation and proved effective inprotecting them.

In detail, it stimulated the growthof keratinocytes. Moreover, increasedcellular growth was recorded 72 h afterexposure at 1% concentration of DNA-Na (Figure 2). DNA-Na also improved thevitality of fibroblasts, whose growth wasincreased 24 h after exposure (Figure 3).Furthermore, phototoxicity tests sug-gested that DNA-Na had no harmfulcytotoxic effects and carried out a protec-tive function from the damage inducedby UV rays toward fibroblasts.12.13

Figure 3. Growth percentage of fibroblasts 24 h after exposure at differentconcentrations of DNA-Na In vivo Tests

To assess the efficacy of an emu1sion(Formula l) containing DNA-Na aimed

at increasing moisturization, elasticityand thickness and reducing wrinkledness,a test was performed after prolonged useand its results were compared to resultsfrom a placebo emulsion.14 This double-b1ind study enrolled 20 Caucasian femalevolunteers aged 30--60, with an averageage of 49 years. These volunteers appliedthe test products on the two periocularzones, twice daily for eight weeks.

Numerous instrumental measure-ments of skin properties were takenbefore the first application and on theday following the final application..Moisturization level (in arbitrary cor-

neometric units) was measuredl5 by a

Vol. 121, No. 11/November 200660 I Cosmetics & Toiletriese magazine www.CosmeticsandToiletries.com

r.nntrnl TrA~tArl

Figure 4. Wrinkles in the skin site before (control area) and after (treated area) theapplication of ONA-Na

comeometer>. It is rdated to the modi-fication of conductance as measured bythe probe applied to the skin surface.

.Elasticity was measuredl6 by acutometer' .Its probe exerts a cyclingsuction on the skin surface and theconsequent skin deformation ismeasured by electrical means.

.Skin wrinkledness was measuredl7 onskin imprints that are prepared witha quick-hardening resind and imageanalysise of skin replicas.

.Skin thickness measurementsl8 usedscanning softwaref and an ultrasoundscanner at high resolution, with highfrequency (320 MHz) ultrasoundemission. This frequency al1ows skinscanning to a depth of 3 cm, withan axial resolution of 50 !lm and alateral resolution of 350 1.1In.18

During the eight-week productapplication period, the test subjects werenot a1lowed to use emulsions otherthanthe ones being evaluated and were askedto abstain from prolonged exposure toUV rays.

The resulting instrumental datarevealed that the emulsion containingthe active principle had induced anincrease in the mean basal values of skinmoisturization from 60.9 corneometricunits before treatment to 63.7 corneo-metric units after treatment, whereasthe placebo values were essentiallyunchanged. The data also revealed asignificant (P<0.05) decrease of 4% inthe mean basaI values of maximum

wrinkledness (Figure 4). However, thebest results were recorded for biological

elasticity and skin thickness. In the sitetreated with the active emulsion, the

mean basal values of elasticity and skinthickness showed a highly significant

(P<0.01) increase of 25.6% and 8.7%,

respectively.Formulas 2-3 are given as guidance

for an optimized use of sodium DNA.

Conclusions

Epidermal keratinocytes and skin

fibroblasts ofhuman origin w ere tested

for cell vitality and phototoxicity. Within

.Model CM 825 Comeometer, Cou"'ge & Khazaka, Germany, Cutometer 575, Courage & Khazaka

d Si!flo-F/exico Ltd., UK, Quantilines, Monaderm

f Dermascan C Version 3, Cortex Technology, Denmark.

Dermascan C is a registered tnldemark of Cortex Technology.

www.CosmeticsandToiletries.com Cosmetics & Toiletries.' magazine I 61Vol. 121, No. 11/November 2006

the bounds of in vitro models, the testresults suggested that nucleotide seg-ments of sodium DNA in a proprietarymaterial called DNA-Na in this articlehad a protective and regenerating effecton the skin. In vivo tests of a formula-tion containing DNA-Na showed asignificant effect on increasing the basalvalues of moisturization, elasticity andskin thickness as well as in reducing skinwrinkledness.

A. Water (aqua) 71.00% wt/wt

Betaine 1.00

Panthenol 0.20

Allantoin 0.10

Disodium EDTA 0.05

Glycerin 4.00

B. PVP/VA copolymer 0.20

(. Ammonium acryloyl-dimethyl taurate/VP copolymer 1.40

D. Olivoyl hydrolyzed wheat protein (and) cetearyl alcohol (and)

glyceryl oleate (and) glyceryl stearate (and) potassium hydroxide 2.00

Butylene glycol dicaprylate/dicaprate 6.00

Phenoxyethanol 0.80

Salicylic acid 0.20

Tocopheryl acetate 0.30

E. Potassium azeloyl diglycinate 1.00

Alcohol (and) water (aqua) (and) Plantago lanceolata (and)

Berberis aquifolium 2.00

Arctium lappa (and) propylene glycol 1.00

Hedera helix (and) propylene glycol 1.00

Urtica dioica (and) propylene glycol 1.00

Water (aqua) 4.00

Sodium DNA 0.15

Diazolidinyl urea 0.30

Fragrance (parfum) 0.30";I;r~ ? nn

The best resuLts

w ere recarded

far biaLagicaL eLasticity

and skin thickness.

Even if it is well-known that nucleo-tides, nucleosides, purine bases andpyrimidine bases enhance cell pro-liferation in vitro, the mechanismsinvolved in these actions are stilIcontroversial. These compounds arereported both to synergize with growthfactors and to act directly on purinergicreceptor A2 inducing per se a prolif-erative response. Moreover, they couldstimulate the skin's photoprotectivemechanisms and an immediate actionagainst free radicals.19 Indeed, whencultured fibroblasts were incubatedwith radioactive amino acids in thepresence of oligonucleotides, the incor-

2.00

IL 1.00

1.00

,"'.u, ~I ~L_.'- :.. -_V, 1.00

i 4.00

0.15

I 0.30

I ."--"-.i , 0.30

J,..'-- l..QQ

J 100.00

Procedure: Prepare A in the main mixer under vatuum. Add B; after complete

dispersion, add C, then mix and homogenize until complete swelling. Heat to

70°C. Melt D at 70-75°C; then add D to the main mixer. Homogenize ABCD for 10

min. Cool to 40°C while mixing; then add E in order. Cool to RT under vacuum.

Characteristics: White-ivory creamy emulsion; pH 5.3; viscosity (Brookfield RVT):

23000 mPa.s 5 rpm, 25°C.

Comment: Gel-cream easy to spread. The synergy between sodium DNA and the

epithelizing, soothing, anti-inflammatory activity of the other actives protects and I

moisturizes, thus making the product especially suitable for dry skin.

62 I Cosmedcs & Toiletries~ magazine www.CosmedcsandToiletries.com Vol. 121, No. 11/November 2006

poration of the tracer into secretedproteins increased significantly.9

In conclusion, regardless of theprotection and repair mechanisminvolved, cell proliferation after contactwith DNA- Na takes place, and the testedactive principle proved in vivo to have amultipurpose activity in improving theappearance of age signs. This activityis related not only to improved watercoordination, but also to increasedcohesiveness of superficial skin layers,as demonstrated by the improvementof skin thickness and elasticity.

A. Water (aqua) 51.00% wt/wt

Panthenol 0.20

Allantoin 0.20

Disodium EDTA 0.10

B. Xanthan gum 0.25

C. Carbomer 0.60

D. Olivoyl hydrolyzed wheat protein (and) cetearyl alcohol (and)

glyceryloleate (and) glyceryl stearate (and) potassium hydroxide 4.00

PPG-15 stearyl ether 1.00

Phenoxyethanol 0.60

Shorea stenoptera butter 2.00

C12-15 alkyl benzoate 6.00

Cetyl alcohol 0.60

Tocopherylacetate 0.50

BHT 0.01

E. Water (aqua) 14.44

Sodium DNA 0.50

Quaternium-15 0.10

Water (aqua) (and) phospholipids (and) superoxide dismutase 1.50

Water (aqua) (and) Fagus sylvatica 2.00

GLycerin (and) water (aqua) (and) Buddleja davidjj(and) Thymus vulgaris 3.00

Glycerin (and) water (aqua) (and) Plantago lanceolata 1.00

Water (aqua) 4.00

Sodium hyaluronate 0.10

Water (aqua) 2.00

citric acid 0.20

Sodium hydroxide 3.80

FraQrance (.Darfum) Q,J..Q

Reproduction of alI or part of this article is strictly

prohibited.

To get a copy of this artide or others from asearchable database, visit the C& T magazine ArtideArchives at www.CosmeticsandToiletries.com/articles.

(and) Thymus vulgaris 3.00

Glycerin (and) water (aqua) (and) Plantago lanceolata 1.00

Water (aqua) 4.00

Sodium hyaluronate 0.10

Water (aqua) 2.00

citric acid 0.20

Sodium hydroxide 3.80

Fragrance (parfum) Q,.JQ100.00

Procedure: Prepare A in the main mixer under vacuum. Add B; mix with turbine

under vacuum; after complete dispersion of AB, add c. Mix under vacuum until

complete swelling. Heat to 70°C. Melt D at 70°C then add to the main mixer.

Homogenize 10 min. Cool to 40°C while mixing, then add E in order. Cool to room

temperature while mixing with blades under vacuum.

Charaderistics: Ivory creamy emulsion; pH 6.8, viscosity (Brookfield RVT): 68,000

mPa.s at 5 rpm, 25°CComment: With emollient and rich texture, this emulsion is effective in controlling the

age signs at different skin levels. The principles include sodium DNA with antiaging

effects, in synergy with actives of vegetable origin, with antioxidant molecules and

soothing, moisturizing, protective actives having filming and moisturizing properties.

relli, p Di lorio and F Caciagli, Trophic effects

of purines in neurons and glial cells Prog

NeurobioI59(6}663-690 (1999)12. F Marzatico, Study concerning the in-vitro

evaluation of keratinocytes and fibroblasts

proliferation in presence and in absence of a

substance that works as cell-growth stimulator,

University of Pavia, Department of Physiologi-

cal-Pharmacological-Cellular & Molecular

Sciences. Prot. N' M320503V (2003)

13. F Marzatico. In-vitro phototoxicity study on

modified 3T3 NRU to evaluate the vitality of

fibroblasts irradiated with a UVA source in

presence and in absence of a cell-protecting

substance. University of Pavia, Department

of Physiological-Pharmacological-Cellular &

Molecular Sciences. Kalichem Study (2003)

14. Instrumental evaluation of the efficacy of

an anti-wrinkle cosmetic ingredient; Study

176/05/01-02. ISPE, Milano

15. E Berardesca, EEMCO guidance for the

assessment of stratum corneum hydra-

tion: Electrical methods, Skin Res Technol

3 126-132 (1997)16. L Rodriguez, EEMCO guidance for the

assessment of tensile functional properties

of the skin-Part 2, Skin Pharmacol Appl Skin

Physio/14(1) 52-67 (2001)17. JL Leveque, EEMCO guidance for the

assessment of skin topography, Eur Acad

Dermatol Venereo/14(1) 52-67 (1999)18. JM Kirsch, J Hanson and G Tikjob, The

determination of the skin thickness using

non-conventional diagnostic ultrasound

equipment, Clin Experimerital Dermatol

9280-285 (1984)

19. IM Hadshiew, MS Eller, I Moli and

BA Gilchrest, Photoprotective mechanisms o(j

human skin. Modulation by oligonucleotides,Hautarzt53(3) 167-173 (2002) C&T

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64 I Cosmedcs & Toiletriese magazine www.CosmedcsandToiletries.com Vol. 121, No. 11/November 2006