Journal of Health Science, 55(3) 447–455 (2009) 447

Ferrous Ferric ChlorideStimulates the Proliferation ofHuman Skin Keratinocytes,Melanocytes, and Fibroblastsin Culture

Tomohisa Hirobe∗

Radiation Effect Mechanism Research Group, National In-stitute of Radiological Sciences, 4–9–1 Anagawa, Inage-ku,Chiba 263–8555, Japan and Graduate School of Science,Chiba University, 1–33 Yayoi-cho, Inage-ku, Chiba 263–8522,Japan

(Received January 27, 2009; Accepted February 24, 2009;Published online February 25, 2009)

Ferrous ferric chloride (FFC R©) is a distinct formof aqueous iron made up of a complex of ferrouschloride and ferric chloride that participates in bothoxidation and reduction reactions. Although FFCis known to stimulate cellular function, its precisemechanism of action is largely unknown. My pre-vious study showed that FFC stimulated the pro-liferation of cultured epidermal keratinocytes andmelanocytes derived from newborn mice, suggestingthat FFC stimulates their function of mice. However,it is not known whether FFC stimulates the prolifer-ation of keratinocytes and melanocytes derived fromhuman skin. Moreover, it is unknown whether FFCstimulates the proliferation of human skin fibroblasts.Pairogen R© (Akatsuka Co., Tsu, Japan) is a drinkcomprising FFC, vinegars, and vitamins, PairogenGold R© (Akatsuka) contains herbal medicines in ad-dition to FFC, vinegars, and vitamins, while Non-calorie Pairogen R© (Akatsuka) contains low molecu-lar weight collagen and hyaluronic acid in additionto FFC, vinegars and vitamins. To clarify whetherFFC in addition to the natural factors included inthese drinks stimulates the proliferation of humanskin cells, FFC and these different drinks were addedto culture media and tested for their proliferation-stimulating activity on skin cells. FFC, Pairogen, andPairogen Gold equally doubled the growth rate ofcultured human keratinocytes, melanocytes, and fi-broblasts. Non-calorie Pairogen tripled the growth

∗To whom correspondence should be addressed: Radiation Ef-fect Mechanism Research Group, National Institute of Radio-logical Sciences, 4–9–1 Anagawa, Inage-ku, Chiba 263–8555,Japan. Tel.: +81-43-206-3253; Fax: +81-43-206-4638; E-mail:thirobe@nirs.go.jp

rate of human fibroblasts, but Non-calorie Pairogenonly doubled the growth rate of human keratinocytesand melanocytes. FFC plus low molecular weight col-lagen tripled the growth rate of human fibroblasts,whereas FFC plus low molecular weight hyaluronicacid only doubled the growth rate of human fibrob-lasts. Low molecular weight collagen, with or withouthyaluronic acid failed to stimulate the proliferationof fibroblasts. These results suggest that FFC stim-ulates the proliferation of human skin keratinocytesand melanocytes. Moreover, FFC markedly stimu-lates the proliferation of human skin fibroblasts syn-ergistically with low molecular weight collagen.

Key words —— keratinocyte, melanocyte, fibroblast,ferrous ferric chloride, proliferation, collagen

INTRODUCTION

Ferrous ferric chloride (FFC R©, Akatsuka Co.,Tsu, Japan) is a distinct form of aqueous iron madeup of a complex of ferrous chloride and ferric chlo-ride that participates in both oxidation and reduc-tion reactions.1) However, the mechanism of actionby which Fe(II) and Fe(III) contained in FFC af-fects living organisms is not well understood. FFCelicits a specific biological effect such as stimulat-ing plant growth, especially root growth.2) In an-imals, FFC also increases red blood cell numbercount and the hemoglobin content in rats.3) My pre-vious study showed that FFC stimulated the prolif-eration of cultured keratinocytes, melanoblasts, andmelanocytes derived from newborn mice.4) More-over, FFC stimulated the differentiation of murinekeratinocytes and melanocytes.4, 5) The proliferationof keratinocytes, melanoblasts, and melanocyteswas stimulated to the same extent (a two-fold in-crease), suggesting that the proliferation of kerati-noocytes and melanocytes in the mouse epidermismay be equally affected by FFC.4) Thus, FFC isthought to activate skin function by promoting cellrenewal through the stimulation of the prolifera-tion and differentiation of both keratinocytes andmelanocytes.4, 5) However, it is unknown whetherFFC stimulates the proliferation of human epider-mal keratinocytes and melanocytes. Also, whetherFFC stimulates the proliferation of human dermalfibroblast, which is the primary constituent of skindermis, remains to be clarified.

Pairogen R© (Akatsuka) is a refreshing drink thatmainly comprises FFC water along with other puri-

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fied ingredients (not artificially synthesized). Thesecomponents include rice vinegar, Japanese apricotvinegar, apple vinegar, persimmon vinegar, vita-mins B2, B6, and C, glucose, fructose, citric acid,and malic acid. Pairogen Gold R© (Akatsuka) is an-other refreshing drink that comprises mainly FFCwater with other purified ingredients (not artifi-cially synthesized). In this case the other com-ponents include apple vinegar, vitamins B1, B2,B6, and C, glucose, fructose, citric acid, malicacid, polydextrose and extracts of Korean gin-seng (Panax ginseng), Chinese wolfberry (Lyciumchinense), and Siberian ginseng (Eleutherococcussenticosus). Moreover, the drink referred to as Non-calorie Pairogen R© (Akatsuka) also contains FFCwater as the main component and other purified in-gredients (not artificially synthesized), such as ricevinegar, Japanese apricot vinegar, apple vinegar,persimmon vinegar, lemon vinegar, vitamins B2,B6, B12, and C, citric acid, malic acid, as well aslow molecular weight collagen and hyaluronic acid.

Pairogen Gold is known to be more effectivefor maintaining the normal condition of internal or-gans, such as the intestines than Pairogen. More-over, my previous study indicated that PairogenGold was more effective in inducing the differen-tiation of cultured mouse epidermal melanocytes innewborn mouse primary skin culture compared withPairogen.5) FFC was shown to stimulate the differ-entiation of cultured mouse epidermal melanocytesto a greater extent in the presence of extracts ofChinese wolfberry and Siberian ginseng included inPairogen Gold.5) Non-calorie Pairogen seems to bemore effective in maintaining a healthy skin thanPairogen. Because the FFC content in Pairogenand Non-calorie Pairogen does not differ, this ob-servation may be the result of the combined effectsof FFC and the natural factors included in Non-calorie Pairogen. It seems that the low molecularweight collagen or hyaluronic acid that is includedin Non-calorie Pairogen but not Pairogen, seems tobe responsible for this effect. In this study, FFC,Pairogen, Pairogen Gold, and Non-calorie Pairogenwere added to the culture media and their effects onthe proliferation of cultured human keratinocytes,melanocytes, and fibroblasts were tested. More-over, the author investigated the synergistical effectsof FFC and natural factors included in Non-caloriePairogen on the proliferation of human fibroblastsin culture.

MATERIALS AND METHODS

Cell Culture —— Normal human epidermal ker-atinocytes (NHEK) derived from primary cul-tures of neonatal foreskin were purchased fromKurabo (Osaka, Japan). They were maintainedin serum-free HuMedia-KG2 (Kurabo) containing10 µg/ml insulin, 0.1 ng/ml human recombinant epi-dermal growth factor (EGF), 0.5µg/ml hydrocor-tisone, 0.4% bovine pituitary extract, 50 µg/mlgentamycin sulfate, and 50 ng/ml amphotericinB. NHEK were seeded at a density of 3× 104

cells/35 mm polystyrene dishes (3× 103 cells/cm2;Becton Dickinson, Bedford, MA, U.S.A.) and cul-tured at 37◦C under a 5% CO2 atmosphere. Sub-confluent secondary NHEK cultured for 5 dayswere detached from the dishes by treatment with0.05% trypsin (Difco, Sparks, MD, U.S.A.) and0.02% ethylenediaminetetraacetate (Sigma Chemi-cal Co., St. Louis, MO, U.S.A.) in Ca2+-, Mg2+-freephosphate-buffered saline at 37◦C for 10 min. Thecells were collected by centrifugation and seeded atthe same density. This procedure was then repeated.

Normal human epidermal melanocytes(NHEM) derived from secondary cultures ofneonatal foreskin were purchased from Kurabo.They were maintained in serum-free melanoblast-proliferation medium-3 (MDMDF3)6) consistingof Ham’s F-10 medium (Gibco, Grand Island,NY, U.S.A.) plus 10 µg/ml insulin (bovine),0.5 mg/ml bovine serum albumin (fraction V), 1 µMethanolamine, 1 µM phosphoethanolamine, 10 nMsodium selenite, 100 µg/ml transferrin, 0.5 mMdibutyryl adenosine 3′ : 5′-cyclic monophosphate,2.5 ng/ml basic fibroblast growth factor (bFGF),100 U/ml penicillin G, 100 µg/ml streptomycin sul-fate, 50 µg/ml gentamycin sulfate, and 0.25 µg/mlamphotericin B. All these supplements werepurchased from Sigma except bFGF. Basic FGFwas purchased from Invitrogen (Carlsbad, CA,U.S.A.). NHEM were seeded at a density of 5× 104

cells/35 mm polystyrene dish (5.20× 103 cells/cm2)coated with type I collagen7) (Becton Dickinson),and maintained at 37◦C under a 5% CO2 atmo-sphere. Subconfluent tertiary melanocytes werecultured for 5 days, trypsinized, and seeded at thesame density. This procedure was then repeated.

Normal human dermal fibroblasts (NHDF) de-rived from primary cultures of neonatal foreskinwere also purchased from Kurabo. They weremaintained in 106S medium (Kurabo) supple-mented with 10 ng/ml human recombinant EGF,

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1µg/ml hydrocortisone, 3 ng/ml human recombi-nant bFGF, 10 µg/ml heparin, 2% fetal bovineserum, 100 U/ml penicillin G, 100 µg/ml strepto-mycin sulfate, 50 µg/ml gentamycin sulfate, and0.25 µg/ml amphotericin B (106S+medium). Apartfrom the antibiotics, the medium and supplementswere purchased from Kurabo. The added antibioticswere purchased from Sigma. NHDF were seededat a density of 2× 104 cells/35 mm polystyrenedish (2.08× 103 cells/cm2) and were cultured at37◦C under a 5% CO2 atmosphere. Subconflu-ent secondary fibroblasts were cultured for 5 days,trypsinized, and seeded at the same density. Thisprocedure was then repeated.

FFC can be prepared as follows.1) Ferric chlo-ride (FeCl3 · 6 H2O) is dissolved in a mixed solutionof 2 M ammonium formate, 1 M hydroxylamine,and 1 M formamide to a final concentration of 1 Mand then diluted with H2O to 10−8–10−14 mM. Anadditional 1 g of FeCl3 · 6 H2O per 10 ml solutionis dissolved in the solution, which is then grad-ually heated to 100◦C until FFC can be crystal-lized.1) The proportion of Fe(II) to Fe(III) was4 : 6 (10−8 mM solution), 6 : 4 (10−12 mM solution),and 7 : 3 (10−14 mM solution).1) Analysis using ion-exchange chromatography and X-ray diffractometrysuggests that Fe(II) and Fe(III) form dimers.1) Watercontaining FFC can be obtained by immersing FFCCeramic Beads R© (Akatsuka) in water. These beadsare prepared by heating hardened soil with FFC so-lution to approximately 800◦C. FFC Ceramic Beadsare made of porous ceramic treated with FFC so-lution. One day before the initiation of culture,200 g of FFC Ceramic Beads were immersed in 1 lof deionized and distilled water (DDW) for 24 hrin a glass bottle in a styrene foam box at roomtemperature. This DDW was referred to as FFC-DDW and had a concentration of 200 g/l. Vari-ous minerals in addition to FFC, such as Ca, Mg,K, Na, Fe, Cu, etc. were present in FFC-DDW.3) This FFC-DDW or control DDW was added tothe culture media (KG2, MDMDF3, and 106S+)at a dilution of 10−1. The final concentration ofFFC was 20 g/l, which was the most preferred con-centration.4, 5) Pairogen, Pairogen Gold, and Non-calorie Pairogen were added to the culture media(KG2, MDMDF3, and 106S+) at a dilution of 10−3.Low molecular weight collagen was prepared byenzymatic (collagenase) digestion of purified colla-gen from the scales of the Golden threadfin bream(Nemipterus virgatus). The concentration of colla-gen present in Non-calorie Pairogen was 0.019%.

Low molecular weight hyaluronic acid was pre-pared by enzymatic (hyaluronidase) digestion ofpurified hyaluronic acid from the fermented prod-ucts of lactic acid bacteria (Streptococcus zooepi-demicus). The concentration of hyaluronic acidpresent in Non-calorie Pairogen was 0.01%. Alow molecular weight collagen (0.019% solution)and hyaluronic acid (0.01% solution) were added to106S+ at 10−3 dilution. Alatsuka Co. generouslysupplied both the low molecular weight collagenand hyaluronic acid.Proliferation Assays —— The number of humankeratinocytes, melanocytes, and fibroblasts per 35mm dish was determined by phase-contrast mi-croscopy at 1, 3, and 5 days in culture. The cal-culation was based on the average number of cellsfrom 10 randomly chosen microscopic fields usingphase-contrast microscopy. The area covered was0.581 mm2.4, 5)

RESULTS

Effects of Pairogen, Pairogen Gold, Non-caloriePairogen, and FFC on the Proliferation of Cul-tured Human Keratinocytes

When human secondary keratinocytes were cul-tured in KG2, they attached well to the cul-ture dishes and many mitoses were observed from1 day. The cells proliferated well and reached sub-confluency (Fig. 1A) by 4 or 5 days. Pairogen(Fig. 1B), Pairogen Gold (Fig. 1C), Non-caloriePairogen (Fig. 1D), and FFC (Fig. 2) increased thenumber of keratinocytes and the number of their mi-toses from 1 day. These cultures reached near con-fluency by 4 or 5 days. Numerous keratinocytes andtheir mitoses were observed upon treatment with ei-ther these drinks or FFC (Fig. 1). The drinks andFFC almost doubled the number of keratinocytescompared with the control culture (Fig. 2A). Theproliferation-stimulating activity of FFC and thesedrinks was not affected after serially sub-culturingthe cells. No change in the stimulative effect wasobserved even in tertiary (Fig. 2B) or the fourth(Fig. 2C) cultures. All the drinks and FFC had asimilar effect on keratinocytes (Fig. 2).

Effects of Pairogen, Pairogen Gold, Non-caloriePairogen, and FFC on the Proliferation of Cul-tured Human Melanocytes

When lightly pigmented human tertiarymelanocytes were cultured in MDMDF3, they

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Fig. 1. Effects of Pairogen, Pairogen Gold, and Non-calorie Pairogen on the Proliferation of Human Epidermal Keratinocytes in KG2Human keratinocytes in tertiary culture were cultured in KG2 (A) or in KG2 with Pairogen (B), Pairogen Gold (C), and Non-calorie Pairogen (D)

at a dilution of 10−3. After 5 days, keratinocytes increased in number and large keratinocyte colonies are observed in control culture (A). By contrast, inthe cultures added with Pairogen (B), Pairogen Gold (C), or Non-calorie Pairogen (D), much more keratinocytes and their mitoses (arrows) are observedthan control (A). Phase-contrast microscopy. Bar, 100 µm.

Fig. 2. Kinetics of the Proliferation of Human Epidermal Keratinocytes in KG2 with or without Pairogen, Pairogen Gold, Non-caloriePairogen, and FFC

Human secondary (A), tertiary (B), and 4th keratinocytes were cultured in KG2 (Cont, ©) or in KG2 with Pairogen (Pair, •), Pairogen Gold (Gold,�), and Non-calorie Pairogen (Non, �) at a dilution of 10−3, or with FFC (�, 20 g/l). In the cultures added with Pairogen, Pairogen Gold, Non-caloriePairogen, and FFC, keratinocytes dramatically increased in number in the serial cultures. The number doubled in the cultures with the drinks and FFC.The data are typical examples of results of each passage. Experiments were repeated two times and similar results were obtained.

attached well to the culture dishes and few mitoseswere observed from 1 day. The cells proliferatedwell and reached sub-confluency (Fig. 3A) by 5 or6 days. Pairogen (Fig. 3B), Pairogen Gold (Fig. 3C),Non-calorie Pairogen (Fig. 3D), and FFC (Fig. 4)increased the number of melanocytes and theirmitoses from 1 day, and reached near confluencyby 5 or 6 days. Numerous melanocytes and their

mitoses were observed by treating the cultureswith these drinks or FFC (Fig. 3). The drinks andFFC almost doubled the number of melanocytescompared with the control culture (Fig. 4A). Theseproliferation-stimulating activity was not affectedafter serially sub-culturing the cells. No changein the stimulative effect was observed even in thefourth (Fig. 4B) and fifth (Fig. 4C) cultures. All

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Fig. 3. Effects of Pairogen, Pairogen Gold, and Non-calorie Pairogen on the Proliferation of Human Epidermal Melanocytes inMDMDF3

Human 4th melanocytes were cultured in MDMDF3 (A) or in MDMDF3 with Pairogen (B), Pairogen Gold (C), or Non-calorie Pairogen (D) ata dilution of 10−3. After 5 days, melanocytes increased in number and extended dendrites in control culture (A). By contrast, in the cultures addedwith Pairogen (B), Pairogen Gold (C), and Non-calorie Pairogen (D), much more melanocytes and their mitoses (arrows) are observed. Increaseddendritogenesis and cell expansion are also observed. Phase-contrast microscopy. Bar, 100 µm.

Fig. 4. Kinetics of the Proliferation of Human Epidermal Melanocytes in MDMDF3 with or without Pairogen, Pairogen Gold, Non-calorie Pairogen, and FFC

Human tertiary (A), 4th (B), and 5th (C) melanocytes were cultured in MDMDF3 (Cont, ©) or in MDMDF3 with Pairogen (Pair,•), Pairogen Gold(Gold, �), Non-calorie Pairogen (Non, �) at a dilution of 10−3, and FFC (�, 20 g/l). In the cultures added with Pairogen, Pairogen Gold, Non-caloriePairogen, and FFC, melanocytes dramatically increased in number in the serial cultures. The number doubled in the cultures with drinks and FFC. Thedata are typical examples of results of each passage. Experiments were repeated two times and similar results were obtained.

the drinks and FFC had a similar effect on themelanocytes.

Effects of Pairogen, Pairogen Gold, Non-caloriePairogen, and FFC on the Proliferation of Cul-tured Human Fibroblasts

When human secondary fibroblasts were cul-tured in 106S+, they attached well to the cul-

ture dishes and many mitoses were observed from1 day. The cells proliferated well and reachedsub-confluency by 4 or 5 days (Fig. 5A). Pairogen(Fig. 5B), Pairogen Gold (Fig. 5C), and Non-caloriePairogen (Fig. 5D) increased (Fig. 6) the numberof fibroblasts and their mitoses from 1 day, andthe cultures reached sub-confluency by 4 or 5 days.Numerous fibroblasts and their mitoses were ob-

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Fig. 5. Effects of Pairogen, Pairogen Gold, and Non-calorie Pairogen on the Proliferation of Human Dermal Fibroblasts in 106S+Human tertiary fibroblasts were cultured in 106S+ (A) or in 106S+ with Pairogen (B), Pairogen Gold (C), and Non-calorie Pairogen (D) at a

dilution of 10−3. After 4 days, fibroblasts increased in number in control culture (A). By contrast, in the cultures added with Pairogen (B), PairogenGold (C), and Non-calorie Pairogen (D), much more fibroblasts and their mitoses (arrows) are observed. Non-calorie Pairogen (D) was most effectivefor stimulating fibroblast proliferation. Phase-contrast microscopy. Bar, 100 µm.

Fig. 6. Kinetics of the Proliferation of Human Dermal Fibroblasts in 106S+ with or without Pairogen, Pairogen Gold, and Non-caloriePairogen

Human secondary (A), tertiary (B), 4th (C), and 5th (D) fibroblasts were cultured in 106S+ (Cont, ©) or in 106S+ with Pairogen (Pair, •), PairogenGold (Gold, �), and Non-calorie Pairogen (Non, �) at a dilution of 10−3. The increase in the number of fibroblasts in the cultures treated with Pairogen,Pairogen Gold, and Non-calorie Pairogen was greater than in control culture. Pairogen and Pairogen Gold doubled the number of human fibroblasts,though Non-calorie Pairogen tripled their number at all serial subcultures. Non-calorie Pairogen was most effective. The data are typical examples ofresults of each passage. Experiments were repeated two times and similar results were obtained.

served by treating the cultures with these drinksor FFC (Figs. 6 and 7). Pairogen, Pairogen Gold,and FFC almost doubled the number of fibroblastscompared with the control cultures (Figs. 6A and7A). By contrast, Non-calorie Pairogen (Figs. 6Aand 7A) almost tripled the number of fibrob-lasts compared with the control culture. Theproliferation-stimulating activity of these drinks and

FFC was not affected after serially sub-culturingthe cells. No change in the stimulative effect wasobserved even in tertiary (Figs. 6B and 7B), thefourth (Figs. 6C and 7C), and fifth (Fig. 6D) cul-tures. Pairogen/Pairogen Gold and FFC affected theproliferation rate to a similar extent, but Non-caloriePairogen was most effective at stimulating humanfibroblasts.

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Fig. 7. Kinetics of the Proliferation of Human Dermal Fibroblasts in 106S+ with or without FFC, Low Molecular Weight Collagen,and Hyaluronic Acid

Human secondary (A), tertiary (B), and 4th (C) fibroblasts were cultured in 106S+ (Cont, ©) or in 106S+ with Non-calorie Pairogen at a dilutionof 10−3 (Non, •), FFC (20 g/l, �), FFC + collagen (FFC + Coll, �), FFC + hyaluronic acid (FFC + Hya, �), FFC + collagen + hyaluronic acid (FFC+ Coll + Hya, �), collagen (Coll, �), hyaluronic acid (Hya, �), and collagen + hyaluronic acid (Coll + Hya, �). FFC and FFC + hyaluronic aciddoubled the number of human fibroblasts at all serial subcultures. Moreover, Non-calorie Pairogen, FFC + collagen, and FFC + collagen + hyaluronicacid tripled the number of fibroblasts compared with the control cultures at all serial subcultures. However, collagen, hyaluronic acid, or collagen +hyaluronic acid failed to increase the number of fibroblasts compared with control culture at all serial subcultures. The data are typical examples ofresults of each passage. Experiments were repeated two times and similar results were obtained.

Effects of FFC, Collagen, and Hyaluronic Acidon the Proliferation of Cultured Human Fibrob-lasts

The proliferation-stimulating effects of Non-calorie Pairogen on human fibroblasts exceededthose of Pairogen, Pairogen Gold, and FFC,suggesting that low molecular weight collagenor hyaluronic acid included only in Non-caloriePairogen is involved in stimulating fibroblast prolif-eration in cooperation with FFC. To clarify whichof these factors are responsible for the effects,low molecular weight collagen and hyaluronic acidwere supplemented to 106S+ with or without FFC.Although treatment with low molecular weighthyaluronic acid with or without FFC failed to ex-ceed the number of fibroblasts stimulated by FFC orcontrol DDW alone (Fig. 7), the combination of lowmolecular weight collagen with FFC dramaticallyincreased the number of fibroblasts to a level similarto that observed using Non-calorie Pairogen at sec-ondary (Fig. 7A), tertiary (Fig. 7B), and the fourth(Fig. 7C) cultures. Three combinations of FFC,low molecular weight collagen, and hyaluronic acidfailed to exceed the proliferation level elicited byFFC plus low molecular weight collagen. However,low molecular weight collagen alone, hyaluronicacid alone, or collagen plus hualuronic acid failedto exceed control proliferation level (Fig. 7). Theseresults suggest that FFC markedly stimulate the

proliferation of fibroblasts synergistically with lowmolecular weight collagen.

DISCUSSION

My previous study showed that FFC4) andFFC-containing Pairogen5) and Pairogen Gold5)

stimulated the proliferation of mouse epidermalkeratinocytes, melanoblasts, and melanocytes inserum-free primary cultures of neonatal skin. Thepresent study revealed that Pairogen, PairogenGold, and FFC equally stimulated the prolif-eration of human epidermal keratinocytes andmelanocytes derived from neonatal foreskin. Be-cause the proliferation-stimulating activity ofPairogen, Pairogen Gold, and FFC on human ker-atinocytes and melanocytes was similar, the vinegar,vitamin, or herbal components included in Pairogenor Pairogen Gold did not seem responsible for theadditional stimulative effects observed. These re-sults indicate that the proliferation of epidermalcells constituting the human skin is equally stimu-lated by the FFC in these drinks. This suggests thepossibility that the human epidermal function can beregulated by FFC. No difference was observed be-tween mouse and human cells. Therefore, it is pos-sible that the proliferation of human and mouse epi-dermal cells are stimulated by a similar mechanism.

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In the present study, FFC-containingPairogen/Pairogen Gold and FFC were shownto double the proliferation rate of cultured humanfibroblasts. By contrast, Non-calorie Pairogentripled the proliferation rate of cultured humanfibroblasts, while only doubling the proliferationrate of cultured keratinocytes and melanocytes. Thelow molecular weight collagen as included in Non-calorie Pairogen greatly stimulated the proliferationof fibroblasts in the presence of FFC. This levelof stimulation was comparable to that elicited byNon-calorie Pairogen. The low molecular weighthyaluronic acid, on the other hand, caused nosuch proliferative effect. Moreover, low molecularweight collagen alone failed to stimulate the prolif-eration of cultured human fibroblasts. Therefore, itis conceivable that FFC synergistically stimulatesthe proliferation of cultured human skin fibroblastswith low molecular weight collagen. Because theproliferation-stimulating activity of Non-caloriePairogen and FFC on human keratinocytes andmelanocytes was similar, low molecular weightcollagen did not cause a similar synergistic effecton the proliferation of human keratinocytes andmelanocytes. Therefore, low molecular weightcollagen may specifically stimulate the proliferationof human fibroblasts in cooperation with FFC.

It is known that fibroblasts produce and releasecollagen.8) Collagen levels gradually decrease in thedermis9) and the three dimensional structure of col-lagen fibers progressively disorganizes with age.10)

These decreases may be one of the causes of skinaging.11) Decrease in collagen content or increasein disorganized collagen fibers in the dermis mayresult in an increased appearance of wrinkles.10)

The decreased collagen content in the dermis mayresult from the reduced proliferative activity of der-mal fibroblasts12) or the reduced production of col-lagen by them.9) Because the reduced prolifera-tive activity of fibroblasts results in lowered col-lagen contents in the skin, the increased prolifera-tive activity (increased fibroblast number) by FFCas suggested in the present in vitro study mightcompensate the reduced collagen contents in agedskin with increased collagen synthesis. This hy-pothesis may be partly supported by the observationthat the increased fibroblast proliferation resultedin the increase in collagen content in the skin.8) Itis not known whether low molecular weight colla-gen stimulates the proliferation of dermal fibrob-lasts. In fact, this study showed that low molecularweight collagen alone failed to stimulate the pro-

liferation of cultured human dermal fibroblasts. Itshould be emphasized, however, that low molecularweight collagen can greatly stimulate the prolifer-ation of human dermal fibroblasts in the presenceFFC. It is possible that FFC changes the homeo-static conditions within fibroblasts to those prefer-able for the stimulation of low molecular weightcollagen. However, it remains to be investigated in afuture study which mechanisms are involved in thestimulation of fibroblast proliferation by FFC andlow molecular weight collagen.

In this study, Pairogen, Pairogen Gold, Non-calorie Pairogen, and FFC doubled or tripled theproliferation rate of keratinocytes, melanocytes, andfibroblasts, suggesting the possibility that the prolif-eration of all three cell types constituting the skinmay be coordinately regulated by FFC and thatskin homeostasis may be maintained by FFC. Itis possible that FFC may maintain this balance byregulating the proliferation of keratinocytes,7, 13, 14)

melanocytes,15–21) and fibroblasts.22–25)

Acknowledgements The author thanks Dr. or Mr.H. Kunoh, T. Nishimura, H. Tagata, and M. Iinomiof the Institute for Biological Process Researchof Akatsuka Co., Mie, Japan for valuable discus-sions and for generous supply of low molecularweight collagen and hyaluronic acid included inNon-calorie Pairogen.

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