Original Contribution

Topical antioxidant application augments the effects of intense pulsed light therapy
Bruce M Freedman, MD, FACS
Plastic Surgery Associates of Northern Virginia, McLean, VA

Summary

Background There has been great interest in improving the efcacy of nonablative technologies by combining them during facial skin rejuvenation. The purpose of this study was to determine whether the addition of topical polyphenolic antioxidants to an intense pulsed light (IPL) treatment regimen augmented the effects of facial IPL treatments. Methods Thirty female volunteers, ages 3452, with skin phototypes 13 were randomly assigned into three groups: group A (n = 10) received three full-face IPL treatments spaced 3 weeks apart; group B (n = 10) underwent 6-weekly full-face treatments of a pneumatically applied topical polyphenolic antioxidant solution; group C (n = 10) received the combination of the three full-face IPL treatments and the six full-face topical antioxidant applications. Skin biopsies, skin polyphenolic antioxidant levels, and skin moisture content levels were obtained and clinical efcacy variables were noted prior to and following the treatment period. Results Compared to group A, group C demonstrated signicantly greater epidermal and papillary dermal thickness, decreased lipid peroxide concentration, increased skin moisture content, and increased polyphenolic antioxidants levels (P < 0.05). There was qualitative improvement in hydration, texture, and pore size. Compared to group B, group C demonstrated signicantly greater papillary dermal thickness (P < 0.05), and qualitative improvement in reduction of ne lines, reduction of hyperpigmentation, and skin dullness. group B and group C had equivalent polyphenolic antioxidant levels, lipid peroxide concentration, and epidermal thickness. Conclusion The addition of polyphenolic antioxidants to an IPL regimen improved the clinical, biochemical, and histological changes seen following IPL treatment alone. These data support the use of multimodal therapy to create synergy and to optimize clinical outcomes in nonablative facial skin rejuvenation. Keywords: intense pulsed light, topical antioxidants, facial rejuvenation

Introduction
In 1994, intense pulsed light (IPL) technology became commercially available for use in treatment of vascular
Correspondence: Bruce M Freedman, MD, 8180 Greensboro Drive #1015, McLean, VA 22102, USA. E-mail: bfreedman58@aol.com Presented at the ASLMS Annual Meeting, Washington, DC, April 15, 2009. Accepted for publication July 26, 2009

anomalies of the skin. Over the following decade, IPL technology has undergone a series of modications and innovations that have broadened its clinical use. IPL has since become a widely accepted and reliable technique for the treatment of photodamaged skin. IPL systems utilize high intensity ashlamps that emit pulses of polychromatic light in the 5151200 nm wavelength spectrum, utilizing the mechanism of selective photothermolysis to target dermal chromophores. The

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resulting thermal injury affects the dermal vasculature and initiates a cascade of inammatory events that includes broblastic proliferation and an increase in collagen production.1 This type of dermal remodeling is well suited for nonablative facial rejuvenation. Numerous studies have conrmed that a series of IPL treatments can decrease facial rhytides, improve skin texture, correct irregular pigmentation, and reduce telangiectasis.2,3 The employment of different nonablative technologies has recently been promoted to improve results and reduce recovery time. Typically, topical compounds such as photosensitizing agents or exfoliating acids have been used in conjunction with microdermabrasion or IPL to provide multimodal therapy.4,5 Recently, there has been great interest in exploring the effects of topical antioxidants on the skin. Botanically derived extracts from green tea, rosemary, and horse chestnut seeds contain polyphenolic compounds that posses signicant antioxidant properties. These polyphenols (i.e., the epicatechins) have demonstrated an ability to prevent ultraviolet radiationinduced lipid peroxidation, DNA damage, and carcinogenesis.69 Clinically, topically applied polyphenolic antioxidants have been shown to decrease inammation, prevent erythema, and reverse photoaging.1012 The purpose of this study was to determine whether the addition of topically applied polyphenolic antioxidants could augment the effects of IPL therapy in facial rejuvenation.

epigallocatechin, ursolic acid) was pneumatically applied to the face at 180 mm Hg. The solution contained no humectants or hydrating agents. Group C (n = 10) received the combination of the three full-face IPL treatments at 3-week intervals and the six full-face topical antioxidant applications at 7- to 10-day intervals. During those weeks when both treatments were administered, the antioxidant application was performed immediately prior to the IPL treatment. Skin care products such as antioxidants, triretinoin, and glycolic acid agents were avoided 6 weeks prior to and during the treatment period. In all groups, digital photographs, 2 mm full-thickness preauricular skin biopsies, skin moisture content, and skin polyphenolic antioxidant levels were obtained prior to treatment (control) and 1 week following the last treatment session. Side effects such as persistent erythema and blistering following treatment were also recorded.
Skin antioxidant levels

Materials and methods

Thirty female volunteers, with Fitzpatrick skin types I-III, aged 3852, were randomized into three groups. All participants consented to partake in a prospective study to evaluate the effects of IPL and antioxidant application on facial skin in accordance to the guidelines of the 1975 Declaration of Helsinki. Group A (n = 10) received three full-face IPL treatments spaced 3 weeks apart delivered by a Sciton Prole System using the Broad Based Light module (Sciton Corporation, Palo Alto, CA, USA) set at the following parameters: 16 J cm2, 10 ms, 560 nm lter. This corresponded to a calculated uence rate of 2500 W cm2. Group B (n = 10) underwent six full-face treatments at 7- to 10-day intervals with the HydraFacial Wave System (Edge Systems Corporation, Signal Hill, CA, USA). Each treatment consisted of one pass over the entire face with the spiral tip hand piece of the crystal free microdermabrasion unit set at 180 mm Hg. A polyphenolic based antioxidant serum containing polyphenolic avonoids and polyphenolic diterpenes (e.g.,

Skin polyphenolic antioxidant levels were obtained from the left cheek using a noninvasive optical device (Biophotonic Scanner, Pharmanex, Provo, UT, USA). This technology employed laser energy at 473 nm and 10 mW power to stimulate molecules containing carbon-carbon double bonds generating an optical ngerprint. The emitted backscattered light was captured by a sensitive light detector, which was then processed and calculated using a Raman scattering spectroscopic technique that has been validated in humans in vivo.13 Using a spectral Raman peak at 1520 cm)1, a linear relationship has been established between antioxidant concentration and Raman intensity, indicating that absolute Raman intensity counts are a biomarker for skin antioxidant levels.14,15
Lipid peroxidation

The skin biopsies for lipid peroxide analysis were frozen until assayed. Lipid peroxidation was assessed by determining the concentration of cutaneous lipid peroxides as described by Sorg et al.16 Briey, skin samples were homogenized in methanol containing butylated hydroxytoluene. The homogenates were sonicated and centrifuged and the supernatant separated into two 500 mL aliquots. One aliquot was incubated with 50 mL of 10 mM triphenylphosphine in methanol while 50 mL methanol was added to the other aliquot. A 500-mL mixture containing 25 mM sulfuric acid, 200 mM ammonium ferrous sulfate, 100 mM xylenol orange,

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and 4 mM butylated hydroxytoluene in 90% methanol was then added to both aliquots; optical density was read at 585 nm 1 h later. The specic lipid peroxide signal was obtained by subtracting the nonspecic signal (triphenylphosphine) from the global signal. Lipid peroxide concentration was determined using cumene peroxide as a standard.
Histological assessment

inuence outcome. Therefore a two-sided paired t-test was used to identify statistical differences in epidermal and papillary dermal thickness, skin moisture content, lipid peroxide concentration, skin polyphenolic antioxidant levels, and side effects between groups. A P-value of less than or equal to 0.05 was used to declare statistical signicance. The Wilcoxon signed ranks test was used to evaluate the change from baseline (control) in the clinical efcacy variables. Statistical signicance was set a priori at P < 0.05.

The skin biopsies for histology were xed in a 10% buffered formaldehyde solution, embedded in parafn, and cut in 4-lm sections. Sections were stained with standard hematoxylin and eosin for light microscopy. The slides were reviewed in a blinded fashion to evaluate epidermal and papillary dermal thickness as well as epidermal and dermal cellular and extracellular elements. The slides were examined with an Olympus Microscope and precision measurements were performed using an Olympus (Olympus Corporation, Tokyo, Japan) Micrometer at 40 magnication.
Skin moisture content

Results
After the treatment protocol, the epidermal thickness in group B increased to 75 9 lm, and in group C increased to 80 8 lm. These values were statistically greater than the control epidermal thickness of 50 5 lm (P < 0.05). However, the epidermal thickness in group A did not increase statistically after treatment. In addition, the epidermal thickness in group C was equivalent to that found in group B. After the treatment protocol, the papillary dermal thickness in group A increased to 370 20 lm, in group B increased to 360 30 lm, and in group C increased to 475 40 lm. These values were statistically greater than the control papillary dermal thickness of 285 20 lm (P < 0.05). Also, the papillary dermal thickness in group C was statistically greater than that in both groups A and B (P < 0.05). The Raman intensity count, a reection of skin polyphenolic antioxidant levels, averaged 16 000 2000 in the control tissue. In group A (IPL alone), the Raman intensity count decreased signicantly to 12 000 2000, while in group B (antioxidant alone) it increased signicantly to 24 000 3000 (P < 0.05). In group C (IPL plus antioxidant), the Raman intensity count averaged 20 000 2000, a value statistically greater than in both the control and group A (P < 0.05). However, this value was statistically less than that in group C. Lipid peroxide concentration in group A increased signicantly to 260 30 nmol g from a control value of 60 10 nmol g (P < 0.05). Lipid peroxide concentration in groups B and C did not increase relative to control and was signicantly less than that found in group A. In group A skin moisture content was signicantly reduced to 35 6 IU from the control level of 45 5, whereas in group B skin moisture increased signicantly 71 5 (P < 0.05). However, in group C the skin moisture content was 59 9, a level greater than in control and group A, but less than in group B. These data are depicted in Table 1.

Water content detection was obtained utilizing interdigital capacitance polyimide lm sensor technology.17 Skin moisture content was measured by calculating skin capacitivity using a noninvasive skin probe (MoistSense Skin Sensor, Moritex Corporation, Tokyo, Japan). This methodology was based on determining the dielectric constant or relative permittivity of the skin; all measurements were taken under xed environmental conditions of 23 C and 50% relative humidity.
Clinical assessment

A panel of three independent medical clinicians examined each of the patients before and after the study period. Efcacy variables were scored on a 09 scale (0 = none, 9 = severe) for the following skin attributes: (1) ne lines; (2) pore size; (3) hyperpigmentation; (4) dullness; and (5) skin texture.18 Differences in clinical outcome for each skin attribute were tabulated as changes from mean score baseline (control) in each patient.
Statistical analysis

The Pearsons chi-square test was used to compare treatment groups A, B, and C with respect to age, gender, and skin types. These parameters were found to be similar indicating that the patients had been effectively randomized such that the subject variables did not

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Table 1 Results comparing data between groups before treatment (control); after IPL (group A); after antioxidant application (group B); and following IPL plus topical application of polyphenolic antioxidant solution (group C)
Control Epidermal thickness (microns) Papillary dermal thickness (microns) Skin polyphenolic antioxidant level (Raman intensity units) Lipid peroxide concentration (nmol g) Skin moisture content (International units) 50 285 16 000 60 45 5 20 2000 5 5 Group A 55 370 12 000 260 35 7 20* 2000* 30* 6* Group B 75 360 24 000 65 71 9*, 30* 3000*, 10 5*, Group C 80 475 20 000 80 59 8*, 40*,, 2000*, 20 9*,,

*P < 0.05 vs. Control; P < 0.05 vs. Group A; P < 0.05 vs. Group B.

Figure 1 Mean decreases from baseline score in the efcacy variables. , Group A; , Group B; , Group C.

Changes in the clinical efcacy variables are depicted in Figure 1. At the end of the study periods groups A, B, and C showed a signicant improvement from baseline in ne lines (P < 05). However, group C demonstrated statistically greater improvement than groups A and B (P < 0.05). Pore size in all groups was reduced relative to control, but equivalent between groups. Following treatment, groups A, B and C showed a signicant improvement from baseline in hyperpigmentation (P < 0.05). The improvement in groups A and C was statistically greater when compared to the improvement observed in group B (P < 0.05). Clinically, at the end of the study period, all groups showed a signicant improvement from baseline in skin texture (P < 0.05). However, group C demonstrated statistically greater improvement than groups A and B (P < 0.05). Side effects, particularly persistent erythema and blistering, differed between the groups. The side effects in group A were statistically greater than those noted in groups B and C (Table 2). The minor blistering noted in group A resolved without scarring.

Discussion
Minimally invasive technologies, such as IPL, have become increasingly popular in the treatment of

photoaged skin. This trend has resulted from the overall safety, reduced recovery time, and versatility of these modalities. These factors have a broad appeal to clinicians and patients, indicating that the number of nonablative procedures will continue to grow. In spite of the utility of IPL in facial rejuvenation, there are some questions concerning the predictability of results. Published reports indicate a varied response to IPL treatment. Bitter1 observed a 1090% improvement in rhytides, while Goldberg and Samady19 reported significant clinical improvement in only 9 out of 30 patients after a series of IPL treatments. It has been suggested that in some patients the repetitive photo-insult may induce microscopic changes analogous to the dermal scarring observed with ultraviolet associated photodamage.3 In these patients an exuberant inammatory response to high intensity visible light exposure could lessen the overall benets seen with IPL therapy. In an effort to deliver optimal clinical improvement with a reliable safety prole, recent research has focused on combining multiple modalities to accomplish these goals. This study sought to determine whether the addition of topical polyphenolic antioxidants could enhance the clinical efcacy of IPL therapy. Recently, there has been an increased focus on antioxidants and their role in skin health. Several antioxidants, including the botanically extracted polyphenolic compounds, have demonstrated anti-aging properties when applied topically. These compounds have been shown to scavenge superoxide anions and other radicals, prevent lipid oxidation, reduce antigenotoxicity, and elevate the antioxidative capacity in the skin.20,21 Collagen synthesis in the papillary dermis and decreased elastosis have also been attributed to topical antioxidant application.22 Fujimura et al. demonstrated that the polyphenols in horse chestnut seed extract induced contraction forces in broblasts resulting in decreased periorbital rhytides.23 Other studies have suggested that polyphenolic antioxidants can increase broblast density and collagen deposition during the wound healing process.911,24 The effects have been purported to translate clinically into increased

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Table 2 Side effect prole

Group A Persistent erythema Blistering Total side effects 3 2 16% (5 30)* Group B 0 0 0% Group C 1 0 3% (1 30)

Each subject in groups A and C received three IPL treatments for a total of 30 IPL exposures. *P < 0.05.

collagen production, ultraviolet radiation protection, and reduction of ne lines. The mechanism and timing of antioxidant delivery was designed to achieve optimal results. Lee et al. demonstrated that the ux and skin deposition of topical antioxidants increased by approximately 20 times in microdermabrasion-treated skin.25 Therefore, the antioxidants in this study were delivered to the skin via a crystal-free microdermabrasion technique. Other research has shown that applying polyphenolic antioxidants to microdermabraded skin resulted in measurably increased levels of polyphenolic antioxidants in the dermis.24 The polyphenolic antioxidants in this study were applied immediately prior to each IPL treatment and also between IPL treatments to preserve and maintain the antioxidative capacity of the skin. A pretreatment approach was employed based on ndings that polyphenolic antioxidants applied prior to ultraviolet and visible light exposure decreased measurable DNA damage in human broblasts and keratinocytes.7 In addition, it has been show that antioxidants applied topically after irradiation may not decrease oxidative stress.22 This study demonstrated that the topical application of polyphenolic antioxidants during IPL therapy enhanced the known effects of IPL treatment. The benets conveyed by the polyphenolic antioxidants in this study appear to be both reparative and preventative. Polyphenolic antioxidants application enhanced the cellular mechanisms activated in the skin during IPL therapy. Papillary dermal thickness and skin moisture content, ostensibly an indicator of skin barrier function, were measurably increased when polyphenolic antioxidants were applied to the skin during the course of IPL therapy. Clinically, ne lines, texture, pore size, and overall skin appearance were improved to a greater extent in skin treated with the polyphenolic antioxidants. These ndings most likely accounted for the histological improvement in the skin architecture as well as the improvement in skin efcacy variables seen following polyphenolic antioxidant addition to an IPL regimen.

Figure 2 Lipid peroxide concentration before treatment (control), after IPL (group A); after antioxidant application (group B); and following IPL plus topical application of polyphenolic antioxidant solution (group C).

Other mechanisms by which the polyphenolic antioxidants appeared to exert their impact was through their anti-inammatory and anti-oxidative properties. This was manifest in this study through a reduction in the adverse effects associated with IPL therapy. Mahmoud et al.20 reported that visible light could induce DNA damage through the generation of reactive oxygen species. Likewise, Sorg et al.16 demonstrated that the moderate dose of visible light delivered during IPL therapy increased lipid peroxide concentration in vivo.16 In this study, the pneumatic topical application of polyphenolic antioxidants during IPL therapy mitigated the lipid peroxide concentrations observed during a course of IPL therapy (Figure 2). Furthermore, the addition of polyphenolic antioxidants to an IPL regimen decreased the incidence of erythema and blistering (Table 2). These ndings corroborate current views that topically applied antioxidants exert anti-inammatory effects. In conclusion, the addition of polyphenolic antioxidants to an IPL regimen improved the clinical, biochemical, and histological changes seen following IPL treatment alone. As such, polyphenolic antioxidants may confer a protective effect on facial skin exposed to high-intensity visible light radiation and reduce deleterious effects of IPL therapy. These data support the use of multimodal therapy to create synergy and to optimize clinical outcomes in nonablative facial skin rejuvenation.

Acknowledgments
The author would like to acknowledge Dr. James Henry, Department of Pathology, Reston Hospital Center for his assistance with the histological analysis and Jacqueline D Higgins for her assistance with manuscript preparation and technical support.

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