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A randomized, controlled comparative study of the wrinkle reduction benefits of a cosmetic niacinamide/peptide/retinyl propionate product regimen vs. a prescription 0·02% tretinoin product regimen
Tretinoin is considered the benchmark prescription topical therapy for improving fine facial wrinkles, but skin tolerance issues can affect patient compliance. In contrast, cosmetic antiwrinkle products are well tolerated but are generally presumed to be less efficacious than tretinoin.
To compare the efficacy of a cosmetic moisturizer regimen vs. a prescription regimen with 0·02% tretinoin for improving the appearance of facial wrinkles.
An 8-week, randomized, parallel-group study was conducted in 196 women with moderate to moderately severe periorbital wrinkles. Following 2 weeks washout, subjects on the cosmetic regimen (n=99) used a sun protection factor (SPF) 30 moisturizing lotion containing 5% niacinamide, peptides and antioxidants, a moisturizing cream containing niacinamide and peptides, and a targeted wrinkle product containing niacinamide, peptides and 0·3% retinyl propionate. Subjects on the prescription regimen (n=97) used 0·02% tretinoin plus moisturizing SPF 30 sunscreen. Subject cohorts (n=25) continued treatment for an additional 16 weeks. Changes in facial wrinkling were assessed by both expert grading and image analysis of digital images of subjects’ faces and by self-assessment questionnaire. Product tolerance was assessed via clinical erythema and dryness grading, subject self-assessment, and determinations of skin barrier integrity (transepidermal water loss) and stratum corneum protein changes.
The cosmetic regimen significantly improved wrinkle appearance after 8 weeks relative to tretinoin, with comparable benefits after 24 weeks. The cosmetic regimen was significantly better tolerated than tretinoin through 8 weeks by all measures.
An appropriately designed cosmetic regimen can improve facial wrinkle appearance comparably with the benchmark prescription treatment, with improved tolerability.
Topical tretinoin is considered a benchmark treatment for the mitigation of fine facial wrinkles.1–3 Use of tretinoin cream (0·02%) has demonstrated a benefit after 24 weeks of treatment.1 However, there are several side-effects (e.g. erythema and desquamation) associated with tretinoin use, especially during the first few weeks, which may cause patients to discontinue use.4 One study showed that concomitant use of a niacinamide-containing moisturizer with tretinoin therapy augmented the response to tretinoin and improved the stratum corneum, decreasing tretinoin-associated side-effects.5
In contrast to the irritancy potential of retinoid therapies, cosmetic and cosmeceutical antiwrinkle products are generally well tolerated by the skin and are pleasant for patients to use. While there are few published reports of direct comparative studies,6 it is generally presumed that such products do not have clinical efficacy comparable with that of prescription topical therapies such as tretinoin.
To determine whether significant wrinkle reduction efficacy comparable with that of a prescription could be achieved using cosmetic products, a treatment regimen was developed containing niacinamide, the palmitoyl peptides palmitoyl-lysine-threonine and palmitoyl-lysine-threonine-threonine-lysine-serine (Pal-KT and Pal-KTTKS, respectively),7–12 and a less irritating retinyl ester commonly used in over-the-counter and cosmetic products (retinyl propionate).13,14 A study was conducted to compare the efficacy of this cosmetic niacinamide/peptide/retinyl propionate (NPP) product regimen vs. a prescription 0·02% tretinoin product regimen for improving the appearance of facial fine lines and wrinkles. Product regimen tolerability was also assessed.
Materials and methods
Study design and subjects
An 8-week, randomized, parallel-group facial appearance study was performed in Cincinnati, OH, U.S.A. from February to August 2008 in 196 women aged 40–65 years who were neither pregnant nor lactating. Eligible subjects had Fitzpatrick skin types I–III and moderate to moderately severe periorbital wrinkles on both sides of their face. Wrinkle severity was assessed using a six-point ordinal photonumeric scale (Fig.1). Subjects preconditioned their face for 2 weeks using a mild facial cleanser (Olay® Foaming Face Wash; Procter & Gamble Company, Cincinnati, OH, U.S.A.) ad libidum and a facial moisturizer (Olay® Complete All Day Moisture Lotion Sensitive Skin SPF 15; Procter & Gamble Company) twice daily in place of their regular cleansing and moisturizing products. After the preconditioning period, subjects were randomly assigned to either the NPP regimen or the tretinoin regimen for 8 weeks of treatment. A computer-generated randomization list was drawn up by the study statistician and was given to the study site for treatment allocation. Prior to study start, 25 self-selected subjects (cohort) on each product regimen agreed to continue treatment for an additional 16 weeks. Written informed consent for participation in this study was obtained from all subjects in accordance with the Helsinki II declaration, and the protocol was approved by an Institutional Review Board (Schulman Associates IRB, Inc., Cincinnati, OH, U.S.A.).
Subjects were instructed to dose both product regimens as per the manufacturer’s package instructions or, when appropriate, investigator guidance. Subjects assigned to the NPP regimen used a daytime sun protection factor (SPF) 30 lotion (Olay® Professional Pro-X Age Repair Lotion SPF 30; Procter & Gamble Company) in the morning, and a night cream (Olay® Professional Pro-X Wrinkle Smoothing Cream; Procter & Gamble Company) in the evening, both over the entire face. In addition, these subjects applied a wrinkle treatment (Olay® Professional Pro-X Deep Wrinkle Treatment; Procter & Gamble Company) twice daily, that is, prior to the daytime SPF 30 lotion and the night cream, on areas of concern, as determined by the subject. Table 1 shows the ingredient list for the test products. All the test products in the NPP regimen contained niacinamide, the peptides Pal-KT and Pal-KTTKS, and carnosine in a moisturizing base. The daytime SPF 30 lotion also contained a broad-spectrum sunscreen (homosalate, avobenzone, ensulizole, octocrylene) and vitamins C and E (sodium ascorbyl phosphate and tocopheryl acetate). The wrinkle treatment contained 0·3% retinyl propionate. The subjects randomized to the tretinoin regimen applied 0·02% tretinoin in an emollient base (Renova®; Neutrogena Corporation, Los Angeles, CA, U.S.A.)15 to the entire face every other evening and a manufacturer-recommended SPF 30 moisturizing sunscreen (Neutrogena Healthy Defense® Daily Moisturizer SPF 30; Johnson and Johnson) every morning for the first 2 weeks; after the initial 2-week period, subjects used the 0·02% tretinoin every evening. Subjects of childbearing age in the tretinoin regimen group had monthly urine pregnancy tests during the study. To assess compliance, the products were weighed at baseline and at each visit. Subjects and study personnel dispensing product were not blinded to treatment.
Improvement in the appearance of fine lines and wrinkles was measured by expert visual grading of high-resolution digital images using the Rapid Evaluation of Anti-aging Leads (REAL 3.0) system16 taken at baseline and at 8 and (in the cohort) 24 weeks. Three trained expert graders independently assessed changes in the appearance of fine lines and wrinkles around the eyes by comparing identified baseline and post-treatment images side-by-side using a ± eight-point ordinal scale. The expert graders and other assessors were blinded to the treatments. Dermatologists on the study advisory panel evaluated a set of representative study images for concurrence with the outcomes observed in expert grading. Images of the infraorbital/crow’s feet areas were also taken at baseline and at 8 and 24 weeks with the commercially available VISIA CR imaging system (Canfield Scientific, Fairfield, NJ, U.S.A.) and analysed for changes in periorbital wrinkle area fraction (wrinkle area divided by assessment area).
In addition, each subject completed a self-assessment questionnaire containing 14 questions evaluating nine efficacy attributes (fine lines and wrinkles around the eyes, evenness of skin tone, red blotchiness, age spots, evenness of skin texture, skin firmness, skin radiance, skin hydration, and overall appearance) using a 10-point continuous scale at baseline and at 2, 4, 6 and 8 weeks in the entire study population and additionally at 16 and 24 weeks in the cohort.
Subject tolerability to each regimen was measured by expert clinical grading of erythema and dryness using a six-point scale and by a subject self-assessment questionnaire using a four-point scale at baseline and at 2, 4, 6 and 8 weeks in the entire population and additionally at 16 and 24 weeks in the cohort. Skin barrier integrity was measured by transepidermal water loss (TEWL) at two sites on the face using a Vapometer SWL-2 (Delfin Technologies, Ltd, Kuopio, Finland).
D-Squame® tape strips (CuDerm Corporation, Dallas, TX, U.S.A.) from the right cheek were collected at baseline and at 8 and 24 weeks, and analysed for the amount of soluble protein, human serum albumin (HSA), involucrin, keratin 6, keratins 1, 10 and 11, fibronectin and cortisol via immunoassay (SkinMAP; Millipore, St Charles, MO, U.S.A.).17 Levels of keratin 6, fibronectin and cortisol from over 90% of the subjects were below detection limits; therefore, reliable comparisons could not be made for these three analytes.
The primary analysis variable was expert visual grading for the change in appearance of fine lines and wrinkles at 8 weeks. For post-treatment analysis, expert visual grades for the change in appearance of fine lines and wrinkles, change from baseline for wrinkle area fraction, erythema, dryness, self-assessment efficacy questions, and protein analytes from D-Squame® tape strips (normalized to total protein and log transformed) were all analysed, separately, via an analysis of covariance model with age and baseline wrinkle grade as covariates using SAS version 9.1 software (SAS Institute, Cary, NC, U.S.A.). Self-assessment tolerability questions were analysed using χ2 tests. Results were considered significant if P<0·05 (two-sided). Ninety-three subjects would permit detection of a mean wrinkle grade difference of 0·28 at 95% significance (two-sided) with an assumed SD of 0·95 and 80% power. Assuming a 5% drop-out rate, 98 subjects for each group (196 in total) were required.
Subject accountability and baseline characteristics
Of the 97 tretinoin regimen subjects and 99 NPP regimen subjects who started the study, 93 and 97 subjects, respectively, completed the study. One subject in the tretinoin regimen group withdrew due to redness and dryness; three subjects in the tretinoin regimen group and two in the NPP regimen group were withdrawn due to noncompliance. Five subjects (three in the tretinoin regimen group; two in the NPP regimen group) were excluded from the REAL image analysis due to image quality issues (facial expressions, etc.). Table 2shows the demographics and baseline values of the two balanced groups.
After 8 weeks of treatment, the appearance of facial fine lines and wrinkles improved in both groups as measured by expert visual grading of the REAL images (P=0·05, tretinoin regimen; P<0·01, NPP regimen). The NPP regimen gave significantly greater improvement than the tretinoin regimen (P<0·01; Fig. 2). A significantly higher percentage of subjects on the NPP regimen (58%) was judged to look better after 8 weeks of treatment, defined as being given the minimum positive grade of +1 on each side of their face by at least two of the three graders, compared with the tretinoin regimen (41%, P=0·03). Also, a significantly higher percentage of subjects on the NPP regimen responded substantially to treatment (mean grade ≥ 2) as compared with the tretinoin regimen (28% vs. 13%, P=0·02). For perspective, a +2 grade would indicate visible improvement in at least one to three small lines and/or 15–25% of areas of crepiness or cross-hatching. Examples of subjects showing substantial responses to each treatment regimen are shown in Figure 3.
As determined by analysis of the VISIA CR images, both product regimens also significantly reduced periorbital wrinkle area fraction at week 8 relative to baseline (P<0·01 for both). The mean percentage reduction from baseline in wrinkle area fraction was greater for the NPP regimen group vs. the tretinoin regimen group (17% vs. 11%, P=0·06, Fig. 4). The change from baseline of relevant efficacy self-assessment questions at 4 and 8 weeks is shown in Table 3. Both groups noticed significant improvement from baseline in overall skin appearance, fine lines and wrinkles, and eye lines and wrinkles at both time points. After 4 weeks, subjects in the NPP regimen group noticed a significant improvement from baseline in overall skin feel and uneven skin texture, and both groups noticed a significant improvement in these assessments at 8 weeks. By 8 weeks, both groups noticed improvement from baseline in deep wrinkles. The self-assessed improvements in overall skin appearance, overall skin feel, and eye lines and wrinkles were significantly greater for the NPP regimen group than for the tretinoin group after both 4 and 8 weeks, and for uneven skin texture after 4 weeks.
After 8 weeks of treatment, the stratum corneum barrier in the tretinoin regimen group was significantly compromised, with TEWL increasing by 5·9 g m−2 h−1 from baseline (Fig. 5). TEWL values in the NPP regimen group remained essentially unchanged from baseline. There was a significant difference in change in TEWL between the two groups at 8 weeks (P<0·01).
After 2 weeks of treatment, both product regimen groups exhibited a significant increase in erythema from baseline (Fig. 6). After 4 weeks of treatment, erythema continued to be significantly increased in the tretinoin regimen group and was significantly higher compared with the NPP regimen group (P=0·01). Erythema in the NPP regimen group was lower at 4, 6 and 8 weeks compared with 2 weeks and was no longer statistically significantly different from baseline. At 6 and 8 weeks, erythema did decrease in the tretinoin regimen but remained significantly higher than at baseline.
At 2, 4, 6 and 8 weeks, the tretinoin regimen group showed significantly more skin dryness from baseline (Fig. 6). There was no significant change from baseline in skin dryness in the NPP regimen group at 2, 4, 6 and 8 weeks. In addition, the skin of subjects in the tretinoin regimen group was significantly drier than the skin of subjects in the NPP regimen group at 2, 4, 6 and 8 weeks (P<0·01 for all).
The results of the subjects’ self-assessments of product tolerability were consistent with the expert erythema and dryness results (data not shown). Compared with the NPP regimen group, after 2 weeks of treatment significantly more subjects in the tretinoin regimen group experienced itchiness, redness/rash and peeling/flaking, which is consistent with known side-effects of tretinoin.4 Symptoms continued in the tretinoin regimen group for the entire 8-week study with a peak between 4 and 6 weeks. Subjects in the NPP regimen group who reported any irritation at any time point generally reported experiencing it only a ‘slight amount’.
There were significant differences between the product regimens for each of the four stratum corneum protein analytes after 8 weeks of treatment (Table 4). Soluble protein, HSA and involucrin levels were significantly increased from baseline in the tretinoin regimen group. In contrast, in the NPP regimen group, soluble protein and HSA decreased from baseline, and involucrin was essentially unchanged from baseline. Keratins 1, 10 and 11 significantly decreased in the tretinoin regimen group but were significantly increased in the NPP regimen group.
Cohort 24-week results
Of the 50 subjects (n=25 in each group) who continued on treatment for an additional 16 weeks, 48 (25, tretinoin regimen; 23, NPP regimen) completed the entire 24 weeks. As seen in Figures 2 and 4, after 8 weeks treatment the mean improvements in the two cohorts were not significantly different by either expert visual grading of the REAL images or by image analysis of the VISIA CR images. The mean cohort responses were not consistent with those determined in the full study population, as the mean tretinoin response was higher in the cohort than in the full population, while for the NPP cohort the mean response was lower. This is likely to be attributable to the self-selection of the cohort subjects, which was made prior to the start of treatment. Wrinkle severity at enrolment, however, was not significantly different between the cohorts, nor was it significantly different between the cohorts and the full populations. For both regimens, the appearance of facial fine lines and wrinkles continued to improve from 8 to 24 weeks as measured by both visual grading and image analysis (P<0·01 for both). Consistent with the 8-week results for the cohort, after 24 weeks of treatment there were no significant differences between the NPP regimen and the tretinoin regimen by either measure (P=0·74, P=0·77, respectively). There was a statistically significant difference in change in TEWL between the two groups at 8 and 24 weeks (P<0·01, Fig. 5). After 24 weeks of treatment, the stratum corneum barrier was not different from baseline in the tretinoin regimen group (P=0·47) but was significantly improved in the NPP regimen group (P<0·01).
After 16 and 24 weeks of treatment, the subset of the NPP regimen group exhibited a nonsignificant decrease from baseline in erythema and dryness (P>0·21, data not shown). After 16 weeks of treatment, erythema in the subset of the tretinoin regimen group was not different from baseline, but at 24 weeks of treatment there was a significant decrease in erythema. Skin dryness in the tretinoin regimen cohort was not significantly different from baseline after 16 and 24 weeks of treatment.
In general, subjects in both groups continued to notice an improvement in their skin from baseline based on the self-assessment questionnaire (data not shown). In addition, the NPP regimen subjects did not report any significant irritation after 16 and 24 weeks of treatment; the tretinoin regimen subjects did report significant peeling and flaking at 16 weeks but no significant irritation after 24 weeks (data not shown).
After 8 weeks, protein analyte changes in the cohort were consistent with those in the full population (Table 4). After 24 weeks, protein changes in both cohorts generally improved relative to 8 weeks. None the less, soluble protein levels remained significantly higher than baseline in the tretinoin regimen group and significantly lower than baseline in the NPP regimen group. Levels of HSA were similar to baseline in the tretinoin regimen group (P=0·59) but significantly reduced relative to baseline in the NPP regimen group. Involucrin levels remained not significantly different from baseline following 24 weeks use of the NPP regimen; however, levels of involucrin in the tretinoin regimen group decreased between 8 and 24 weeks, which is consistent with the TEWL and irritation data showing accommodation to tretinoin treatment with longer term use. Overall, the NPP regimen group had significantly lower levels of soluble protein, HSA and involucrin and significantly higher levels of keratins 1, 10 and 11 than the tretinoin regimen group at 24 weeks.
To our knowledge, this is the first of its kind long-term clinical study comparing a cosmetic anti-ageing regimen against a recognized prescription topical treatment for improving the appearance of facial wrinkling. The NPP regimen, which contained cosmetic ingredients known to affect the appearance of wrinkles, along with moisturizing and skin barrier building components, comparably improved the appearance of fine lines and wrinkles relative to the prescription tretinoin regimen as measured by both expert grading and image analysis in a long-term study, with better tolerability. Improvements in skin texture with topical niacinamide treatment have been reported and linked to enhanced barrier function,7 and a peptide used in the NPP regimen has been shown to reduce the appearance of fine lines and wrinkles without adverse effects on skin barrier.10 The beneficial impact of the NPP regimen ingredients is consistent with the results seen for TEWL, protein analyte changes, and the clinical data on erythema and dryness. Multiple studies have established the beneficial effects of niacinamide for improving barrier function of the stratum corneum as measured by TEWL.18–20 In the present investigation the NPP regimen had less of a positive effect on TEWL at 8 weeks vs. baseline than one would expect from the literature given that all three products contained niacinamide. The muted benefit on TEWL was probably a consequence of the wrinkle treatment product also containing retinyl propionate.
Another observation is that barrier function restoration and stabilization of stratum corneum repair mechanisms may occur at different rates, as TEWL values in the tretinoin cohort had returned to baseline by week 24 but some marker protein levels had not.
We recognize that there are limitations in this study. Tretinoin is more irritating to the skin and the products tested in the NPP regimen are more moisturizing. Additionally, while the evaluators were blinded to treatment, the subjects were not which could have affected the subjects' self-assessments. Finally, a larger 24-week cohort group size would have provided more statistical power for more robust conclusions. These limitations notwithstanding, the study data, from both objective and subjective assessments, provided consistent results and showed that the new NPP regimen improved the appearance of fine lines and wrinkles similarly to the tretinoin regimen, with fewer side-effects.
The results of this study show that the efficacy of a prescription product for improving the appearance of facial fine lines and wrinkles can be achieved with an appropriately designed cosmetic regimen, while providing additional benefits in aesthetics, skin tolerance and potential patient compliance.
The authors acknowledge the assistance of Karen Driver in the writing and preparation of the manuscript. The Dermatologist members of the Olay Professional Alliance for Skin Care Innovation (Drs Vince Bertucci, Doris Day, Zoe D. Draelos, Pearl E. Grimes, Stephen H. Mandy, Maritza I. Perez and Susan H. Weinkle) participated in the research design and contributed to the critical evaluation of the intellectual content of this manuscript.
Human Growth Factor Cream and Hyaluronic Acid Serum in Conjunction with Micro Laser Peel
The present study investigated the use of a novel hyaluronic acid serum in combination with a cream comprising a mixture of human growth factors in conjunction with the micro laser peel procedure for skin rejuvenation. After preconditioning the face with the hyaluronic acid serum followed by the cream twice daily for one month, 15 female volunteers between 35 to 65 years of age with demonstrable facial wrinkling received a micro laser peel on the entire face using an erbium-doped yttrium aluminium garnet laser. Immediately following the laser procedure, the subjects applied the test products twice daily until the second laser peel one month later. Immediately following the second procedure, the subjects reapplied the test products for another month. In the large majority of subjects, erythema or edema, crusts or erosions, and transitory stinging or burning sensations after the micro laser peel were minimal or mild when the skin was treated with the serum followed by the cream. The micro laser peel in conjunction with the test products helped to significantly improve hyperpigmentation, wrinkles, and texture as compared to before treatment. This study with the micro laser peel device demonstrated that a novel hyaluronic acid serum combined with the human growth factor cream can be successfully used for skin rejuvenation in conjunction with light-to-medium invasive laser skin treatments.
Today, a large variety of cosmetic procedures including light and laser therapies are used for nonsurgical skin rejuvenation.1–3 The field of nonsurgical skin rejuvenation has been inspired by patient expectations for minimal risk and rapid recovery.1 The recovery time for nonsurgical skin regeneration depends on the procedure; generally there is minimal downtime after nonablative treatments and significant downtime after ablative procedures, such as CO2 laser resurfacing. Earlier studies indicate that topical skincare regimens can be beneficial in reducing postprocedure symptoms.4 With the goal to assess a topical skin care regimen that would reduce procedure-related signs and possibly enhance outcome of the procedure, the present study reports on a skin care regimen consisting of the combined application of a high content hyaluronic acid serum with a cream containing a mixture of human growth factors and cytokines. A slightly invasive laser procedure for skin rejuvenation, known as a micro laser peel, was performed; the device was adjusted to remove up to 20µm of skin. This erbium-doped yttrium aluminium garnet (Er:YAG) laser is an adjustable, minimal-to-full, epidermal, laser peel that removes the outermost layers of the skin as a function of the laser settings. Depending on skin removal and the patient, adverse events are commonly limited to transitory erythema, edema, crusts and erosions lasting for several days after the laser procedure.5
The serum was formulated with 1% high molecular weight (about 1000kDa), pharmaceutical grade, bioengineered hyaluronic acid, while the human growth factor and cytokine mixture was obtained through a biotechnology process using cultured human fetal fibroblasts. The fibroblasts originated from a dedicated cell bank, which was established for the development of wound healing products. The test cream with the growth factor and cytokine mixture has previously been studied for the treatment of adverse events associated with photodynamic therapy.6 Furthermore, the cream was intensively studied as effective skin care regimen for aged facial skin when used alone7–9 and was reported to be beneficial in scar management after surgery.10
Hyaluronic acid (HA; also known as hyaluronate or hyaluronan) is a high molecular weight, negatively charged (polyanionic) polysaccharide and is naturally present in virtually all vertebrate tissues and fluids including the skin's extracellular matrix. Through its complex interactions with matrix components and cells, HA has versatile roles in skin related to both its physicochemical and biological properties. HA is well known for its excellent water-holding capacity.11 Further, HA supports tissue architecture of extracellular matrix, governs skin elasticity, is involved in cell migration and differentiation processes during wound healing and inflammation, and might act as an antioxidant due to restriction of movement of reactive oxygen species.12 There is also some evidence that HA and growth factors (i.e., fibroblast growth factor) act synergistically to accelerate healing.13 HA was shown to interact with growth factors, thereby protecting them from degradation by proteases.14 Finally, HA is used as a topical drug delivery system for localized delivery of certain drugs to the skin.15 Therefore, due to HA's specific properties in relation to growth factors, topical products with concentrated HA in combination with human growth factors might be particularly well suited to help improve recovery and outcome after cosmetic light and laser therapies.
Study design. The three-center study was conducted according to the Declaration of Helsinki as approved by the Independent Institutional Review Board (Plantation, Florida). After obtaining informed consent, 15 Caucasian, female volunteers between 35 and 65 years of age, in good general health, not nursing or pregnant, of Fitzpatrick skin type 1 to 4, and with moderate-to-severe facial photodamage underwent the treatment regimen. Excluded were subjects with any active or history of skin disease affecting the face; subjects who received microdermabrasion, glycolic or less than 20% trichloro acetic acid (TCA) peel, or hydroquinone-containing products on the face within one month prior to the beginning of the study; subjects who received nonablative laser, light, or radio-frequency treatments within three months prior to the beginning of the study; and subjects who received retinoids, dermabrasion, or greater than 20% TCA peel, Botox®, or filler injection, or ablative laser or cosmetic surgery within six months prior to the beginning of the study.
Micro laser peel. The subject received two micro laser peels (MicroLaserPeel™ Profile Contour 2940nm Er:YAG laser system; Sciton, Palo Alto, California) separated by one month on entire face adjusted to up to 15µm during the first treatment and up to 20µm during the second treatment. Topical 5% lidocaine (LMX 5 Cream; Ferndale Laboratories, Ferndale, Michigan) was provided 30 minutes prior to treatment.
Micro laser peel skin care regimen. The subjects applied a 1% sodium hyaluronate serum (Hyalis; Neocutis, San Francisco, California) followed by an oil-in-water-based skin cream containing a proprietary mixture of human growth factors and cytokines called processed skin cell proteins (Bio-Cream—Bio-restorative Skin Cream with PSP®, Neocutis) one month prior to the laser peel in order to condition the skin before the procedure and after each laser peel for a total of two months twice daily (morning and evening). Before the application of the test products, the subjects were asked to cleanse the skin using a gentle cleanser (Cetaphil® Gentle Skin Cleanser, Galderma Laboratories, Fort Worth, Texas). During the daytime, when sun protection was required, the subjects were additionally asked to use a sun protection factor (SPF) 45 sunscreen (Coppertone® Oil Free Sunsceen Lotion SPF 45; Schering-Plough HealthCare Products, Memphis, Tennessee). Gentle cleanser and sunscreen were also provided to the subjects at the beginning of the study.
Evaluations. Clinical assessment of recovery (erythema, edema, crusts, and erosions) and improve-ment of signs of skin aging (hyperpigmentation, wrinkle, texture, pore) were assessed using 0 to 3 or 0 to 4-point visual scoring systems (Tables 1 and 2). In addition, clinical photography was taken under standardized conditions before, three days after the micro laser peels, and at the end of the study period using the OMNIA imaging system (Canfield Scientific; Fairfield, New Jersey). Subjects were further asked whether they tolerated the test products well, whether the products helped to keep skin moist, soothe or calm skin, or reduce general discomfort (pain, burning, stinging, etc.) after the micro laser peel. Possible answers were “Yes,” “Not sure, but seems likely,” or, “No.”
All 15 subjects averaged 51±9 years of age (between 36 to 64 years) completed the study. One subject, however, refused to undergo the second micro laser peel since she felt that downtime after the procedure was significant and not acceptable. This subject remained in the study and continued using the test products, but did not receive a second micro laser peel. One subject missed the evaluation three days after the second laser peel (Day 33).
Adverse events and tolerability. The subjects did not experience any adverse events in relation to the test products. One subject had severe periorbital edema occurring one day after the first laser procedure (15 joules). This adverse event was related to the micro laser peel procedure and not to the study products. While the use of the test products was continued, the edema reaction resolved after about five days using ice cooling without any medicinal intervention. This subject tolerated the second laser peel, which was set at a lower laser intensity (10 joules), without the occurrence of any adverse events. One subject developed asthma and one subject a tooth and root canal abscess during the study period; both adverse events were not related to the study products or the laser procedure. Eighty-seven percent of the subjects after the first peel, and 91 percent after the second peel reported to tolerate the test products well when used starting immediately after the laser procedure for three days. The few subjects who did not report tolerating the test products well mentioned slight stinging or burning sensations after application of the test products. The questionnaire did not allow distinguishing whether the HA serum, the growth factor and cytokine skin cream, the cleanser, or the sunscreen were the cause of these transitory sensory sensations. All subjects (100%) reported to tolerate the test products well one month after each procedure.
Clinical assessment of recovery. On average, erythema was mild involving up to one-third of the face (corresponding to score 2), whereas edema was minimal, scant rare (score 1) three days after the micro laser peel. After the peel, mild crusts and erosions were readily seen (3–5 lesions of 3mm or less; score 2). With the exception of some minimal, scant rare erythema (score 1) seen in 27 percent of subjects after the first peel and 40 percent after the second peel, edema and crusts and erosions completely disappeared within one month after the procedure in all subjects. As judged from the slightly decreased intensity of erythema, edema, and crusts and erosions, the second peel appeared to be better tolerated despite the fact that the subject generally received a more intense peel removing up to 20µm. The detailed results are shown in Figure 1.
Clinical assessment of signs of skin aging. Wrinkles, hyperpigmentation, and pores were reduced, and skin texture improved after the micro laser peels (Figure 2). The improvements in the signs associated with skin aging were particularly pronounced one month after the second peel (Day 60). In average, signs of wrinkles were reduced by 16 percent, pores by 30 percent, and hyperpigmentation by 36 percent, whereas texture improved by 45 percent as compared to baseline after the two-month study period. These improvements are illustrated in the photographs of one subject taken before and at the end of the study period (Figure 3).
Subject questionnaires. Eight or more out of 10 subjects (≥80%) reported that test products helped or seemed likely to help keep skin moist, sooth or calm skin, and reduce general discomfort after the first and second laser peel. Furthermore, when asked at the end of the study, 90 percent of subjects responded favorable or very favorable toward using the test products again when undergoing another micro laser peel in the future.
Appropriate skin care in conjunction with cosmetic procedures, such as light and laser therapies, represents an important aspect in nonsurgical skin rejuvenation by helping to minimize downtime and enhance outcome of the procedures. This study with the micro laser peel device suggests that the use of a combination of a high content (1%), high molecular weight and pharmaceutical grade hyaluronic acid skin serum with an oil-in-water cream containing a proprietary mixture of human growth factors and cytokines is generally well tolerated and can be successfully used for postprocedural skin care after cosmetic and dermatological procedures, such as light-to-medium invasive laser skin treatments. The properties of hyaluronic acid that protect growth factors from degradation by proteases,14 may make the use of hyaluronic acid and growth factor topical products an especially promising combination for treating open skin wounds where protection of growth factors from protease activity can be beneficial.16 This hypothesis remains to be confirmed in additional, placebo-controlled studies since no active control group was used in this initial series of patients. This will further determine the potential benefits of the treatment regimen versus other treatment modalities and skin care routines.
Glycolic acid peel therapy – a current review
Chemical peels have been time-tested and are here to stay. Alpha-hydroxy peels are highly popular in the dermatologist’s arsenal of procedures. Glycolic acid peel is the most common alpha-hydroxy acid peel, also known as fruit peel. It is simple, inexpensive, and has no downtime. This review talks about various studies of glycolic acid peels for various indications, such as acne, acne scars, melasma, postinflammatory hyperpigmentation, photoaging, and seborrhea. Combination therapies and treatment procedure are also discussed. Careful review of medical history, examination of the skin, and pre-peel priming of skin are important before every peel. Proper patient selection, peel timing, and neutralization on-time will ensure good results, with no side effects. Depth of the glycolic acid peel depends on the concentration of the acid used, the number of coats applied, and the time for which it is applied. Hence, it can be used as a very superficial peel, or even a medium depth peel. It has been found to be very safe with Fitzpatrick skin types I–IV. All in all, it is a peel that is here to stay.
Alpha-hydroxy peels have been popular in dermatological practice for years and are well-established. They have practically no downtime, and are usually superficial or medium depth peels. They have therapeutic as well as cosmetic benefits when used on skin.1 Glycolic acid (GA) obtained from sugarcane is used in the most common alpha-hydroxy acid peel.2
GA has the smallest molecular weight amongst all the alpha-hydroxy acids. It penetrates skin easily, making it a popular peel agent.3 GA has two carbon atoms: one carbon atom is with a carboxyl group and the other carbon atom is with a hydroxyl group. GA is extremely hydrophilic. The pH of a non-buffered solution ranges from 0.08–2.75.4 Previous authors have recommended the use of a buffered or partially neutralized GA, which is safer than free GA.5 Glycolic acid peels are commercially available as free acids, partially neutralized (higher pH), buffered, or esterified solutions.6 They are available in various concentrations ranging from 20%–70%. The higher the concentration and lower the pH, the more intense the peeling will be.7 In general, gel formulations have a slower penetration time and are easier to control.8
Fabbrocini, in 2009, classified glycolic peels as: very superficial (30%–50% GA, applied for 1–2 minutes); superficial (50%–70% GA, applied for 2–5 minutes); and medium depth (70% GA, applied for 3–15 minutes).8 GA peels have antiinflammatory, keratolytic, and antioxidant effects. GA targets the corneosome by enhancing breakdown and decreasing cohesiveness, causing desquamation.9 The intensity of GA peel is determined by the concentration of the acid.10 GA peels need to be properly neutralized in order to stop acidification of the skin.11
Material and methods
Before starting a series of GA peels, the status of the skin should be assessed for the occurrence of any dry or scaly patches on skin, open sores that may have become acidified through the use of GA/tretinoin creams.12Priming the skin with hydroquinone, or topical retinoids, before performing a peel has been found to increase peel efficacy and reduce the risk of postinflammatory hyperpigmentation.13 After the skin has been cleansed and degreased, GA solution is applied using cotton buds or a brush in a sequential manner starting from the forehead to the left cheek, chin, right cheek to cover the entire face. Care is taken to protect the eyes and the corners of the nose and lips. The peel is neutralized within 3–5 minutes, or when uniform erythema is seen. If frosting is observed in any particular area before the set time or end-point, it is important to neutralize the peel immediately. It is always better to start with a low concentration (20% GA) and increase the concentration and application time during subsequent sessions.3 Peeling is repeated once every 15 days for 4–6 months until the desired result is achieved.14
Mechanism of action
Glycolic acid peels have antiinflammatory, keratolytic, and antioxidant effects. GA targets the corneosome by enhancing breakdown and decreasing cohesiveness, causing desquamation.9 The intensity of peel is determined by the concentration of the acid, the vehicle used to carry it, the amount of acid applied, and the technique used.10
GA peels need to be properly neutralized to stop acidification of the skin. Applying acid to the skin saturates the ability of cells to resist acidification; excess acid must be neutralized to avoid burning. Alpha-hydroxy acid peels can be neutralized with water or with basic solutions, such as ammonium salts, sodium bicarbonate, or sodium hydroxide.11
Acne, acne scars, melasma, postinflammatory hyperpigmentation, photoaging, and seborrhea are indications for chemical peeling.15–19 GA may be used in acne also to normalize keratinization and increase epidermal and dermal hyaluronic acid and collagen gene expression.20
Acne and acne scars
In a study by Wang et al, Asian patients with skin type IV with acne were treated with 35% GA and 50% GA peels, once in 3 weeks for 10 weeks. There was significant resolution of comedones, papules, and pustules. The skin texture improved and follicular pore size reduced. Most of the patients were found to have brighter and lighter looking skin. Consistent and repetitive treatment with GA was needed for the apparent improvement of acne scars and cystic lesions. Only a small percentage of patients (5.6%) developed side effects, in the form of postinflammatory hyperpigmentation, mild skin irritation, and exacerbation of herpes simplex infection. Hence, GA peels were found to be ideal as an adjunctive treatment for acne.21
In another study by Atzori et al, 70% GA was used in comedonic acne, papulopustular, and nodular/cystic acne. While comedones improved rapidly, papulopustular acne improved after six peel sessions, and nodulocystic lesions required ten sessions at three-week intervals. A significant improvement of coexisting post-acne superficial scarring was noted. Patient tolerance and compliance were both found to be excellent.22
Kim et al did a comparative study using 70% GA and Jessner’s Solution (resorcinol 14%, lactic acid 14%, and salicylic acid 14%, in an alcohol base), respectively, in two groups of patients with acne. Three peel sessions were done for each group. Though acne improved in both to the same extent, there was more exfoliation seen in the Jessner’s Solution group. Hence, GA was found to be the better tolerated of the two peels for the treatment of acne in Asian patients.23
Grover and Reddu conducted a study of 41 patients with Fitzpatrick Skin Type III–V, of whom 16 patients had acne. Initially, they used 10% GA for a period of 1–2 minutes, then gradually increased the duration to 5 minutes and concentration to 30% GA. All patients had greasy skin with high sebaceous activity. The severity of acne was graded on a four-point scale, which revealed that mild to moderate acne was seen in eight patients, severe acne in seven patients and one patient had nodulocystic acne. Apart from comedones, papules, and pustules, a significant number of patients had scarring and pigmentation. After undergoing peels with GA, the therapeutic response was good in 75% of patients, on the basis of both patient and observer assessments. Patients with postinflammatory hyperpigmentation and scarring showed excellent improvement. Significant decrease in the number of comedones and papulopustules was observed in patients with mild to moderate acne. However, the patient with nodulocystic acne lesions did not respond well to therapy. Compared to the 90% response seen by Wang et al,21 75%–78% of patients with acne, excepting the patient with nodulocystic acne, showed a good-to-fair response with GA peels.24
Kessler et al compared 30% GA versus 30% salicylic acid (SA) peels in 20 patients with mild to moderate acne, using a split-face design. Peels were performed every 2 weeks for a total of six treatments. Both peels improved acne. However, the authors found that the SA peel had better sustained efficacy (ie, number of acne lesions, and improvement rating by blinded evaluator) and fewer side effects than GA, presumably due to the greater lipophilicity of SA. The authors agreed with the impression that SA peels are better tolerated than GA peels in acne patients.25
A study by Erbağci and Akçali concluded that a 70% GA peel performed every 2 weeks resulted in significant improvement in atrophic acne scarring, as compared to 15% GA cream used daily. In one group of 23 patients, GA peels were performed bi-weekly with increasing application time and acid concentration from 20%–70%. A second group of 20 patients was instructed to use 15% GA cream twice daily for a period of 24 weeks. It was seen that six patients, using 70% GA, showed significantly better results than daily use of 15% GA for 24 weeks (P < 0.05). Furthermore, apparently good responses were observed in the peel group only (P < 0.01).26
In the author’s experience, GA peels are excellent for use on Indian skin. Patients with Fitzpatrick’s skin types III–IV with post acne pigmentation are treated with 20% GA peel for the first three sessions. The concentration of GA is then increased to 35% in the subsequent five to six sessions. There is an interval of 15 days between sessions. Patients respond well, with clearance of lesions, in around eight to ten sessions, without any adverse effects (Figures 1 and and22).
Lin and Tham studied the use of GA peels in ten Asian women. Concentrations of 20%–70% GA were administered every 3 weeks, either alone or in combination with a topical regimen of 2% hydroquinone plus 10% GA. There was significant improvement (P < 0.06) in melasma and fine facial wrinkling in patients who received the combination of creams and peeling.27
Kalla et al did a comparative study of 55%–75% GA versus 10%–15% trichloroacetic acid (TCA) peels in 100 patients with recalcitrant melasma. The peels were conducted at 15-day intervals in both groups. It was seen that the response to TCA was rapid, and produced better results than GA. However, relapse was more common in the TCA group (25%) than in the GA group (5.9%).28
In a study by Javaheri et al, peeling was performed upon 15 Indian females with melasma, using 50% GA, once-monthly for 3 months. An improvement in Melasma Area Severity Index (MASI) score was observed in 91% of patients (P < 0.01). A better response was seen in patients with epidermal melasma, compared to those with mixed melasma (P < 0.05).29
In a study by Sarkar et al, 20 Indian patients received serial GA peels (30% GA for the first three sittings; 40% GA for the next three sittings), combined with the modified Kligman’s formula (2% hydroquinone, 0.025% tretinoin, and 1% mometasone). A further 20 Indian patients received only the modified Kligman’s formula, with no peeling. In both groups, a significant decrease in the MASI score was observed from baseline to 21 weeks (P < 0.001). However, the GA peel group showed more rapid and greater improvement (P < 0.001).30
A comparative study by Hurley et al was done on 21 Hispanic women with bilateral, epidermal, and mixed melasma to assess the efficacy of 4% hydroquinone cream versus 4% hydroquinone cream combined with GA peels. Patients received GA peels (20%–30% GA) every 2 weeks to one side of the face only, in addition to twice-daily application of 4% hydroquinone cream to the other side of the face. Pigmentation was measured objectively using a Mexameter® (Courage + Khazaka electronic GmbH, Cologne, Germany) and the MASI, and measured subjectively using a linear analog scale and physician and patient global evaluation. Both sides of the face showed a reduction of pigmentation, and there was no significant difference.31
The concentration of GA used by Hurley et al was low (20%–30%), compared to the 30%–40% GA used by Sarkar et al.32 This could be a reason for the difference in the results they observed.
In a study by Khunger et al, patients with melasma were treated with a 70% GA peel on one half of the face, while the other half was treated with a 1% tretinoin peel. A significant decrease in the modified MASI score was observed on both facial sides from baseline to 6 weeks, and then from 6 to 12 weeks (P < 0.001).33 A study by Kligman found the two peels to be equally effective and well tolerated.34
In 15 cases of melasma (epidermal: 80%; dermal: 13.3%; and mixed: 6.6%), 52.5% GA concentration was applied for 3 minutes. There was good to fair response in patients with epidermal and mixed melasma, while no significant improvement was seen in dermal melasma.24
Serial GA peels (from 35%–50%, and 70% every second peel) plus combination topical therapy (azelaic acid and adapalene) in 28 women with melasma found better results in the group receiving chemical peel plus topical therapy (P = 0.048), but only when the GA concentration was 50% or higher.35
In another study, a triple combination cream consisting of fluocinolone acetonide 0.01%, hydroquinone 4%, and tretinoin 0.05% was used in an alternating sequential treatment pattern, cycling with a series of GA peels, for the treatment of moderate to severe melasma. Spectrometry measurement of the difference in melanin between involved and uninvolved skin confirmed that hyperpigmentation was significantly reduced at 6 and 12 weeks, compared with baseline (P < 0.001), with evaluations showing improvement of 90% or more by week 12.36
In another study, 10 patients with melasma were treated with a triple combination of tretinoin 0.05%, hydroquinone 4%, and mometasone furoate 0.1%. Serial GA peels were performed at three-weekly intervals, with application times from 2–6 minutes, depending on tolerance and erythema. Glycolic acid of 57% concentration, with 55% free acid, and pH 2.3 was used on the face in gradually increasing durations of application. Significant reduction in melasma was noted after four peels. One patient developed irritation and hyperpigmentation, while one developed persistent erythema. No other side effects were reported.37,38
In a comparative study of 10%–20% TCA versus 20%–35% GA peels for the treatment of melasma, similar improvement was seen with both peels. However, the GA peel was seen to be associated with fewer side effects than the TCA peel, and gave the added benefit of facial rejuvenation.39
In another similar study of 15% TCA peel versus 35% GA peel for the treatment of melasma, there was no statistically significant difference in efficacy. Both peels significantly reduced MASI scores, and both were found to be equally effective in the treatment of melasma. It was also seen that adverse effects were more common with TCA than with GA peels.40
In a pilot study by Burns et al, postinflammatory hyperpigmentation was treated with a series of GA peels in skin types IV–VI. No adverse effects were reported in dark skin, and the GA peel proved to be efficacious.41 This echoed the study by Grover and Reddu, in which skin types III–V showed overall improvement of skin texture in almost all patients.24
In the author’s experience with cases of postinflammatory hyperpigmentation in skin types III and IV, a series of 35% GA peels has produced good results. All patients are instructed to use sunscreen before and after the peel. Priming is done with a combination of mometasone and tretinoin 0.025% creams for 3 weeks before the peel. The patient is asked to stop the creams 1 week before the peel. Post-peel care, in the form of sun protection, and avoidance of facial scrubs, steam, and sauna is advised. Complete resolution of postinflammatory hyperpigmentation is commonly seen after six to eight peel treatments (Figures 3 and and44).
Photodamage, in the form of dyschromias, actinic keratoses, solar lentigines, and fine wrinkling has also shown improvement with a combination of GA and TCA peels.42,43 In a study of 50% GA peels by Newman et al, there was improvement in mild photoaging of skin. Other significant improvements were noted, including decreases in rough texture and fine wrinkling, fewer solar keratoses, and slight lightening of solar lentigines. Histologic analysis showed thinning of the stratum corneum, granular layer enhancement, and epidermal thickening. Some specimens showed an increase in collagen thickness in the dermis.44,45 Glycolic acid peels do not affect deep wrinkles or deep pigmentations.3
GA peels have frequently been combined with other peels and treatments, to give better results. In a study by the author, microneedling was combined with 35% GA peel to treat acne scars in patients with skin type III–IV. Microneedling was performed six-weekly, and 35% GA peel was performed 3 weeks after each microneedling session. There was significant improvement in superficial and moderately deep atrophic box scars and rolling scars. In addition, there was improvement in skin texture and reduction in post-acne pigmentation.46
GA and TCA peels are performed sequentially in cases of postinflammatory hyperpigmentation, post-acne pigmentation, and melasma. This combination has been found to produce a deeper and more uniform peel than TCA used alone.47
Combining Jessner’s Solution and GA for the treatment of photoaged skin, actinic keratoses, and rhytides resulted in a uniform GA peel, but the risk of overpeel and scarring are high, especially in dark-skinned individuals.48–50 GA has been combined with 5-fluorouracil to treat actinic keratosis. Pretreatment of the skin with 5-fluorouracil 5% increases the efficacy of the treatment and shortens the healing time.51
GA peeling has also been used in combination with microdermabrasion, for the treatment of acne vulgaris and superficial acne scars, in order to increase treatment efficacy and achieve treatment goals within a shorter time. Alpha-hydroxy acid peels decrease corneocyte cohesion, making the abrasion more efficient.3However, combining GA peels with microdermabrasion at the same session could lead to postinflammatory hyperpigmentation in skin types III–VI. Therefore, care should be taken with darker skin types.52 Briden et al reported good patient satisfaction when using superficial GA peels with microdermabrasion for photoaging.53
In a study on photodamaged skin, an intense light that combined a narrow-band (405–420 nm) blue light (antiinflammatory) emission with a near-infrared (850–890 nm) emission was applied to the skin. Concomitant glycolic peels were performed, and daily Vitamin C cream was given. Results showed significant improvements in pore size, rhytids, and radiance.54 GA peeling is also combined with Vitamin C in cases of melasma and postinflammatory hyperpigmentation. In a patient with melasma, 70% GA peeling was performed on one side of the face, along with iontophoresis with nanosome vitamin C; 70% GA peeling alone was performed on the other side of the face. It was seen that both sides improved, but that the side treated with iontophoresis and nanosome Vitamin C showed better results.55 Superficial GA peels can also be used with botulinum toxin and fillers in order to obtain overall improvement in wrinkles, skin tone, texture, radiance, and clarity. In one study, the interval between peels and fillers was 1 week. The peel was administered after injecting botulinum toxin during the same visit, or the procedures were separated by one or more days to minimize the potential for side effects.8,56,57
The GA peel is time-tested. Proper selection of patients, timing of peel, and neutralization on-time should ensure good results and no side effects. Superficial peels using alpha-hydroxy acids increase the epidermal activity of enzymes, leading to epidermolysis and exfoliation.10
The minor side effects reported are: erythema, stinging sensation, sensation of pulling of facial skin, mild burning, and transient postinflammatory hyperpigmentation. Unbuffered GA can cause erosive blisters and scarring.40,58 In rare cases, hypopigmentation, persistent erythema, and flare-up of pimples have been reported.24,36,59
Epidermolysis may occur if the patient has used topical retinoids, anti-acne creams, or skin lighteners in days prior to the peel. Other causes are excessive facial scrubbing before the peel, and pre-existing dry skin. It is important to stop priming agents around 1 week before the peel. Sun exposure after the peel may cause postinflammatory hyperpigmentation.60 There has been one report of contact urticaria with a GA peel.61
Glycolic acid is the alpha-hydroxy acid used most commonly by dermatologists for chemical peeling. It is used in concentrations from 20%–70% in all skin types. It should be used in lower concentrations initially, and then the strength of the peel should be gradually increased in subsequent sessions. There should be a minimum interval of 2 weeks between two treatment sessions. Peel neutralization is extremely important and it depends on erythema seen. However, in dark skin, it may be difficult to appreciate erythema. In such cases, it is better to time the peel between 3–5 minutes and judge the desired end-point depending on the time.2 The longer the duration, the deeper the depth of the peel. Pre-peel priming and post-peel care are equally important. Depending on the condition, tretinoin, kojic acid, and hydroquinone can be used for pre-peel priming. In order to avoid postinflammatory hyperpigmentation and scarring in dark-skinned patients, it is important to avoid facial scrubs, depilatory creams, waxing, bleaching, microdermabrasion, and laser hair removal for at least 1 week before the procedure. After the peel, the patient should be advised to avoid sun exposure, facial scrubs, loofahs, picking at the peeling skin, taking steam inhalation, and using creams that cause exfoliation. The patient must be instructed to use a broad spectrum sunscreen during the day, and a moisturizer at night. The GA peel is safe for a correctly selected patient when the correct technique is used. It does not produce any systemic toxicity. It can be combined with other treatment modalities for better cosmetic outcomes.
How Much Do We Really Know About Our Favorite Cosmeceutical Ingredients?
To date, we are unaware of a review that has investigated common cosmeceutical ingredients in order to answer the three specific questions proposed by the father of cosmeceuticals, Dr. Albert Kligman. It is the goal of this review to gather all the published scientific data on five common cosmeceutical ingredients, answer the three major questions about the scientific rationale for their use, and ascertain how much we really know about consumers' favorite cosmeceutical ingredients.
Most of the research concerning cosmeceutical retinoid ingredients is based upon the effects of retinoic acid on the skin. Clinical trials concerning retinol and retinaldehyde are scant and lacking in statistical evaluation for significance. There is research substantiating the effects of kinetin in plants and also in-vitro antioxidant effects. However, proof of anti-aging activity remains elusive, and the clinical efficacy of kinetin is based on limited data. Niacinamide is the ingredient investigated that most closely upholds the “Kligman standards” of cosmeceutical-ingredient analysis. With the available scientific evidence on topical niacinamide, clinicians are able to adequately answer questions about permeability, mechanism, and clinical effect. Both green tea and soy have been popularized commercially based on their antioxidant effects, yet there is a paucity of clinical studies concerning their efficacy as topical anti-aging agents. It may be that soy and green tea are better at preventing the signs and symptoms of skin aging than actually reversing them. Since cosmeceutical products are claiming to therapeutically affect the structure and function of the skin, it is rational and necessary to hold them to specified scientific standards that substantiate efficacy claims.
Increasingly, patients are looking for cost-effective noninvasive methods to improve the appearance of their skin. Many patients are turning away from prescription medications and medical procedures and are instead considering over-the-counter (OTC) treatments, such as cosmeceuticals. Factors that cause patients to switch to OTC products include lack of adequate insurance coverage for prescription products used to treat photoaging, the need to pay for physician visits to obtain prescription products, and failure to discern “fiction from fact” related to unsubstantiated claims made by some OTC anti-aging skin care products. A cosmeceutical product by definition is a cosmetic product in which the active ingredient is meant to have a beneficial physiological effect due to an enhanced pharmacological action when compared with an inert cosmetic.1
Every day in clinical practice, dermatologists see patients who ask them if cosmeceutical products are beneficial. Accurate advice is a challenge that requires knowledge of the structure and function of human skin and the available scientific data, which may or may not support the efficacy of a given cosmeceutical product. As dermatologists, it is very hard for us to keep up with the latest evidence on the popular cosmeceutical ingredients due to the vastness of ingredients, the multitude of commercially available products and their efficacy claims, and the validity or accuracy of data gleaned from in-vitro and clinical studies, if available.
What questions need to be asked when scientifically evaluating the potential therapeutic merits of a cosmeceutical
According to Dr. Albert Kligman, when asked to evaluate a new cosmeceutical product that claims a beneficial physiological effect, it is important to ask three questions:
- Can the active ingredient penetrate the stratum corneum (SC) and be delivered in sufficient concentrations to its intended target in the skin over a time course consistent with its mechanism of action?
- Does the active ingredient have a known specific biochemical mechanism of action in the target cell or tissue in human skin?
Are there published, peer-reviewed, double-blind, placebo-controlled, statistically significant, clinical trials to substantiate the efficacy claims?1 (Table 1.)
To date, we are unaware of a review that has investigated common cosmeceutical ingredients in order to answer these three specific questions. It is the goal of this review to gather the available published scientific data on five common cosmeceutical ingredients, to answer the three major questions described above, and to ascertain how much we really know about our consumers' favorite cosmeceutical ingredients.
The first question addresses whether the active ingredient penetrates the SC. The SC is an effective barrier to transepidermal water loss (TEWL) and to the penetration of exogenous substances. Typically, the substances that cannot penetrate the SC easily include: proteins, sugars, peptides and nucleic acids with molecular weights greater than approximately 1000kDa, and highly charged molecules.1
In addition, it is important to determine if the ingredient is delivered in sufficient concentrations to its intended target in the skin to produce the desired effect. The minimum concentration of ingredient required to produce the desired effect is termed the threshold concentration and is important for evaluating cosmetic formulas.
If the agent meets the first criterion, that is penetration of the SC in an amount exceeding the threshold concentration, the second question follows intuitively. The second question asks if there is a known biochemical or pharmacological mechanism of action for this active ingredient to substantiate the marketing claim. Most pharmacologically active ingredients have mechanisms of action on cellular components that regulate physiological functions (i.e., inhibition or activation of enzymes involved in signal transduction or gene expression).2 Surprisingly, some popular cosmeceutical ingredients have mechanisms that are currently unknown and require further research. If a viable biochemical or pharmacological mechanism of action exists, and the product can reach its target at sufficient concentrations for a long enough duration, the product deserves further clinical experimentation, preferably a randomized, double-blind, vehicle-controlled clinical trial.
Clinical trials should include the use of noninvasive instrumentation, which measures characteristics such as TEWL, corneometry, skin elasticity, colorimetry, surface replica analysis, and other techniques that can be used to assess efficacy claims. Pretreatment and post-treatment photography alone are not adequate because there are major pitfalls in standardized clinical photography. In addition, it is difficult to measure appearance clinically because it is highly subjective and endpoints are difficult to establish. The pitfalls of published clinical trials to date are that many of the reports are anecdotal, have been performed on small groups of patients, and statistical significance has not been well established.
Another way to demonstrate the effects of a product is histology. The histological changes of photoaged skin are well documented. Demonstration of reversal of histological features of photoaging is another approach to substantiate efficacy claims for the reversal of photoaging.3
This next section presents a summary of the published scientific data of five popular cosmeceutical ingredients. Specifically, this article reviews the evidence on penetration of the SC, the mechanism of action, and the anti-aging effects on skin based on clinical studies.
What background information is available on cosmeceutical retinoids
Retinoids consist of natural and synthetic derivatives of vitamin A that can be found in both prescription medications and OTC cosmeceuticals. Retinoic acid (RA) as well as the synthetic napthalene derivatives adapalene, tazarotene, and bexarotene are registered prescription drugs. The topical cosmeceutical retinoids include retinyl esters, retinol, retinaldehyde, and the group of oxoretinoids.4
RA has been extensively studied and used as an effective topical treatment for photoaging, acne, and numerous other dermatological disorders. RA can, however, be irritating to the skin, limiting its use in some patients. Retinol (ROL) and retinaldehyde (RAL) are of interest to the scientific community and cosmetic industry as gentler yet still effective alternatives to RA. However, the other cosmeceutical retinoids, retinyl palmitate and retinyl-acetate, are retinoid esters and are not considered effective against photoaging and therefore are not discussed in this review.4,5
In the skin, ROL is oxidized into RAL, which in turn is oxidized into RA, the biologically active form of vitamin A. Topical cosmeceuticals containing ROL and RAL are theoreticized to work because once absorbed they are metabolized to RA, which induces pharmacological activity. Given this consideration, not only is the percutaneous absorption profile of ROL and RAL important to its effectiveness, but the metabolism of ROL and RAL to RA is just as important.
What data is available on the percutaneous absorption and metabolic activity of retinoids?
In general, retinoids are lipophilic molecules that can penetrate the epidermis.6 One study demonstrated the specific penetration characteristics of ROL and RAL in human skin in vivo by measuring levels of the skin enzyme cytochrome P-450-dependent RA 4-hydroxylase (CP450-RAH) (Table 2A).7 The enzyme CP450-RAH is induced by presence of RA in the skin and therefore its induction can be used as an indicator of ROL and RAL penetration and metabolism to RA. The study found a significant induction in this enzyme following topical application of ROL and RAL to human skin in vivo. After 48 hours of occlusion, both ROL and RAL (0.025% and greater) increased the enzyme activity significantly while lower concentrations did not cause significant induction.7 Interestingly, the increase in enzyme induction was not linear and higher doses of ROL and RAL only caused small increases in enzyme activity. However, it should be noted that at lower doses (0.01% and 0.025%), RAL was a greater inducer of CD450-RAH than ROL.7 Given the results of this study, it seems the threshold concentration for adequate penetration and metabolism of RAL and ROL into RA is 0.025%. Hence, it is important that the concentration of RAL and ROL in cosmetic formularies be at least 0.025%. However, it must also be considered that concentrations above 0.025% may not induce greater penetration or metabolism to RA.Summary of penetration data
In another experiment, metabolism of ROL, RAL, and RA was studied utilizing in-vitro human skin and dermal fibroblasts.8 Radiolabeled ROL and RAL were applied either topically to the skin biopsies or to the culture media of the fibroblast suspension and the metabolites were identified by high-performance liquid chromatography (HPLC) after 24 hours of incubation. The skin cultures demonstrated a gradient distribution of the retinoids within the skin: 75 percent of absorbed activity was in the epidermis, 20 percent in the dermis, and 2 to 6 percent in the culture medium for the three retinoids tested. Of the epidermal extracts, 60 percent of applied ROL remained unmetabolized. The main ROL metabolites in the epidermis were retinyl esters (18.5%), RA (2%), RAL (1.6%), 13-cis-retinoic acid (1%), and polar compounds. The dermis yielded similar metabolites, but a higher proportion of polar compounds. RAL was also metabolized in the epidermis, with 43 percent of the absorbed radioactivity being RAL, 9 percent retinyl esters, 14 percent ROL, and 0.8 percent RA.8
Collectively, several studies have therefore demonstrated an absorption and metabolic capacity for topical ROL and RAL.7,8 Retinyl esters appear to be the major metabolite, while the formation of RA from these substances constitutes a small proportion of the metabolites formed. However, whether this conversion is sufficient for pharmacological activity is unknown. In-vivo studies may better quantify both metabolism and dose-response relationships.6
What are the potential mechanisms of action of retinoids?
ROL and RAL are metabolized to RA in the skin and therefore, are assumed to exhibit the same mechanism and clinical effects as RA (Table 2B). Of these effects, the surface roughness, mottled hyperpigmentation, and fine wrinkles demonstrate the most significant improvement with RA therapy.9Although ROL- and RAL-containing cosmeceuticals have not been shown to produce the magnitude of clinical results obtained with the prescription products that are approved and used to treat photoaging (tretinoin, tazarotene), many consumers appear to be pleased with their cosmetic results anecdotally.Summary of mechanisms of action and clinical significance
Hyperpigmentation. RA is thought to reduce mottled hyperpigmentation by enhancing epidermal cell turnover. Enhancing epidermal cell turnover decreases the contact time between keratinocytes and melanocytes and promotes a rapid loss of pigment through epidermopoiesis.10
Fine lines and wrinkles. RA therapy reduces fine lines and wrinkles by increasing the capacity of the epidermis to hold water through stimulation of glycosaminoglycan (GAG) synthesis and by stimulating collagen synthesis through increases in transforming growth factor (TGF-beta) and procollagen.9–12Furthermore, it is believed that RA may also retard or prevent further dermal matrix degradation by inhibiting the enzymes that break down collagen and preventing oxidative stress.11,13–17
Roughness. RA therapy reduces skin roughness by modulating the expression of genes involved in cellular differentiation and proliferation, hence promoting epidermal cell turnover.9,11 The effects are believed to be mediated through binding to retinoic acid receptors (RAR) and subsequent binding of these complexes to specific genes affecting gene transcription.18,19
What clinical studies are available on cosmeceutical retinoids?
There are extensive clinical trials investigating the anti-aging effects of topical RA (Table 2C). RA is considered to be one of most effective and well-substantiated compounds for treating the signs and symptoms of aging and/or photodamaged skin, including fine lines, hyperpigmented spots, and wrinkles.10,20,21 However, few studies have been reported for the OTC retinoids. Here, the authors present the results of studies investigating the anti-aging effects of RAL and ROL.Levels of evidence for substantiation of clinical data
Some studies have reported that RAL can produce significant clinical improvement in the appearance of fine and deep wrinkles.10,22 In one study, the efficacy of RAL 0.05% cream for the treatment of photoaging was compared to 0.05% RA and vehicle creams over an 18-week time period.22 A total of 125 patients (40 in the RA group, 40 in the RAL group, and 45 in the vehicle group) were studied. This study demonstrated that RAL can produce clinical improvement in fine and deep wrinkles.22 However, statistical significance was not reported and no intra-individual comparisons were made.
Topical ROL has been shown in vivo to have only a modest effect compared with topical RAL and RA while two randomized controlled trials showed a significant improvement in fine wrinkles after 12 and 24 weeks of treatment with the use of ROL versus placebo.10,23 One of the randomized, controlled trials was completed by Sorg et al10 who evaluated the effectiveness of topical ROL in improving the clinical signs of naturally aged skin in a randomized, double-blind, vehicle-controlled, left and right arm comparison study.10The study population comprised 36 elderly subjects (mean age 87 years) residing in two senior citizen facilities. Topical 0.4% ROL lotion or its vehicle was applied at each visit by study personnel to either the right or the left arm, up to three times a week for 24 weeks. Clinical assessment was made by using a semiquantitative scale and biochemical measurements from skin biopsy specimens. After 24 weeks, there were significant differences between ROL-treated and vehicle-treated sides for fine wrinkling scores (p<0.001). In addition, histology showed ROL treatment significantly increased GAG expression (p=.02 [n = 6]) and procollagen I (p=0.049 [n = 4]) compared with vehicle. This data provided support that topical ROL improves fine wrinkles via its metabolism to RA and subsequent increases in GAGs and collagen production.
What conclusions can be drawn from data on cosmeceutical retinoids?
After sunscreens, many believe topical retinoids are the most important drug class to combat and reverse the signs of aging. With more than two decades of experimentation, there is a vast amount of evidence that regular retinoid use over several months results in clinical improvement in skin texture, wrinkles, and pigmentation.24 However, most of this knowledge and experience is about prescription products containing RA and not about OTC retinoids, ROL and RAL. Of the limited data available concerning OTC retinoids, RAL seems to be the most efficacious.10,22 Therefore, those patients who are looking to decrease and or prevent the signs of aging without use of a prescription product should use RAL containing cosmeceuticals as they have the best scientific evidence supporting their efficacy in reducing the signs and symptoms of aging.
So what do we tell our patients concerning retinoid cosmeceuticals? The authors have presented sufficient evidence on cutaneous penetration and concerning mechanism of action and semi-sufficient evidence on the clinical anti-aging effects of ROL and RAL. They believe it is important to remind the patients that while the most effective anti-aging treatment is achieved with prescription retinoids, there is some evidence to support the use of RAL and ROL to decrease fine lines and wrinkles.
What is kinetin (N-furfuryladenine growth factor)?
What data is available on the percutaneous absorption of kinetin?
The authors were unable to find any studies investigating the percutaneous absorption of kinetin. However, kinetin has been shown to be nonirritating to the skin, easily formulated, chemically stable, and compatible with other formulation components.27
What are the potential mechanisms of action of kinetin?
Kinetin is shown to exert its anti-aging effects in different systems including plants, fruit flies, and cultured human skin fibroblasts.28–31 Studies on human fibroblasts in vitro have demonstrated that kinetin may have the ability to delay the onset of age-related changes as well as decrease the severity of these changes.23 These age-related changes include the alteration in cell size and shape, growth rates, cytoskeletal structure, macromolecular synthesis, and quantity of lipofuscin. The delay of age-related cellular characteristics were most pronounced in cultures where kinetin was continuously present. It was noted that some aging characteristics began to reappear upon removal of kinetin, and youthful characteristics in general were better maintained in younger cells compared to older cells. This suggests that continued use of kinetin is necessary to maintain results and that there may be additional benefit to starting kinetin at a younger age.32 However, the mechanism by which kinetin exerts its effects on human fibroblasts remains elusive.
Antioxidant effects. Investigators have surmised that the mechanism of action that results in age retardation may involve the genes that influence aging and may involve kinetin acting as both an inhibitor of reactive oxygen species (ROS) formation and a scavenger of ROS.28,33 Many studies have shown kinetin to be a powerful antioxidant. Kinetin has the ability to mimic superoxide dismutase (SOD) activity, activate both SOD and catalase expression, and quench ROS.28,29,35,36 Also, kinetin has been shown to prevent the oxidation of unsaturated fatty acids and inhibit the in-vitro oxidation of DNA.36,37 Also, Verbeke et al38 demonstrated that kinetin can inhibit the oxidation and glycation/glycoxidation of proteins. By inhibiting the oxidation and glycation/glycoxidation of proteins, kinetin inhibits the formation of advanced glycation/glycoxidation end products (AGE). These results strengthen the view that kinetin is a powerful antioxidant with significant biological properties and useful potential for the prevention of oxidative damage.38
Other effects. Although clinical studies of topical kinetin have suggested improvement in skin texture, a decrease in hyperpigmentation, and a decrease in TEWL, there appears to be no reported mechanisms for how kinetin improves skin texture, fine wrinkles, hyperpigmentation, and/or the SC permeability barrier.
What clinical studies are available on kinetin?
One open-label study about the clinical safety and efficacy of kinetin 0.1% lotion on human skin was published by McCullough et al.39 Ninety-six subjects with photodamaged skin showed improvement in the categories of skin texture, color, blotchiness, and fine wrinkles after 24 weeks of twice-daily application of 0.01%, 0.05%, or 0.10% kinetin. Average improvements ranged from 17 to 63 percent over baseline. In addition, the results of this study suggest that kinetin improves barrier function of the SC, as evidenced by a mean decrease in TEWL after 14 weeks of use. Hence, this demonstrated that topical kinetin (0.01%–0.1%) can partially improve some of the clinical signs of mild-to-moderately photodamaged skin, such as skin texture, fine wrinkles, skin color, and blotchiness, and can help restore normal skin barrier function with 12 to 24 weeks of topical application.39
Another study investigated the anti-aging effects of topical kinetin 0.03% in combination with niacinamide 4% versus niacinamide 4% alone in Asians. Amasino et al26 found that the combination of kinetin and niacinamide and niacinamide alone effectively improved many of the facial aging signs in Asians. This combination of ingredients reduced the number of hyperpigmented spots and red blotchiness as well as increased SC hydration status with more persistence than the formula containing niacinamide alone. The authors believe that this indicates a decisive role for kinetin in the formulation and that kinetin plus niacinamide may be used as an adjunctive therapy for anti-aging purposes of the skin.27
What conclusions can be drawn from data on kinetin?
In summary, kinetin is a relatively new ingredient in the cosmeceutical world with anti-aging potential given its anti-aging role in plants and its in-vitro antioxidant effects. In order to recommend kinetin, the authors feel that the scientific evidence supporting kinetin is still lacking in permeation studies and mechanistic discoveries as to how this growth factor reduces hyperpigmentation, improves skin barrier function, and improves skin texture. There is modest support of its efficacy based on clinical studies.
What background information is available on niacinamide and nicotinic acid?
While the nutritional value of niacin (vitamin B3) may be well recognized, the skin care benefit of topically applied niacin is a recent discovery based on recently published studies. Niacin (vitamin B3) has two potential forms that can be used in cosmeceuticals: niacinamide (nicotinamide) and nicotinic acid. It is debatable as to whether these two forms of niacin are interchangeable as topical cosmeceuticals. Some studies claim that niacinamide and nicotinic acid are readily converted into each other in vivo40 while other studies speculate that niacinamide and nicotinic acid may have very different pharmaceutical activities despite having identical vitamin activities.41 In other words, nicotinic acid may have more benefits than topical niacinamide on the skin due to the fact that in addition to having the vitamin effects on skin (increasing levels of niacinamide adenosine dinucleotide [NAD]), it may also have drug-mediated effects on skin via interacting with nicotinic acid receptors present in the skin.41–44 Yet, the disadvantage of using nicotinic acid as a topical cosmeceutical is its unpleasant side effect of vasodilation that results in skin flushing. This is an effect that is not harmful but intensely disliked by most patients.45,46 In contrast to nicotinic acid, niacinamide does not cause skin flushing nor does it cause changes in blood pressure, pulse, or body temperature.47 Due to the decreased number of side effects of topical niacinamide compared to nicotinic acid, the effects of niacinamide as a topical cosmeceutical agent have been studied more to date.
Niacinamide, also known as nicotinamide, is the precursor of important cofactors niacinamide adenosine dinucleotide (NAD) and its phosphate derivative, niacinamide adenosine dinucleotide phosphate (NADP). These cofactors and their reduced forms (NADH and NADPH) serve as reduction-oxidation (redox) coenzymes in more than 40 cellular biochemical reactions. Thus, niacinamide has the potential to exert multiple effects on skin and is a promising anti-aging cosmeceutical ingredient.
What data is available on the percutaneous absorption of niacinamide?
Feldmann et al48 highlighted the possibilities for the topical application of niacinamide because they were able to prove sufficient percutaneous penetration into human skin.48,49 In addition, several other studies have used increased levels of NAD in skin cells after the topical application of niacinamide as evidence of percutaneous penetration.50
What are the potential mechanisms of action of niacinamide?
Studies have shown that niacinamide has the potential to act as an antioxidant, can improve epidermal barrier function, decrease skin hyperpigmentation, reduce fine lines and wrinkles, decrease redness/blotchiness, decrease skin yellowness (sallowness), and improve skin elasticity.51,52 The mechanisms by which niacinamide provides this array of skin benefits is not completely understood, but the role of niacinamide as a precursor to the NADP family of coenzymes may play a significant role in all of these improvements.50
Antioxidant capacity. Niacinamide increases the antioxidant capacity of skin after topical application by increasing the reduced forms (NADPH), which have potent antioxidant properties.53–55 This is probably the most well-studied anti-aging effect of niacinamide.
Epidermal barrier function. Niacinamide may improve the skin barrier function in two ways: first, by its ability to upregulate the synthesis of ceramides as well as other SC intercellular lipids, and second, by stimulating keratinocyte differentiation.56,57 Ceramides and other intercellular SC lipids are known to play a central role in the structural and functional integrity of the epidermal barrier function. The responsible mechanism for the increase of ceramide synthesis in niacinamide-treated, cultured keratinocytes was found to be based on the upregulation of serine palmitoyltransferase, the rate-limiting enzyme in sphingolipid synthesis. The increase in ceramide synthesis has been confirmed in an in-vivo trial after topical application of 2% niacinamide emulsion for four weeks applied twice daily.56 The elevation of ceramides after treatment with niacinamide is associated with an improved barrier function as evidenced by a reduced TEWL and an increase in the cutaneous resistance to potential harmful topical agents.56 The second mechanism likely responsible for improved barrier function is a stimulation of keratinocyte differentiation seen both in cell cultures in vitro and in-vivo studies conducted by Tanno et al.56,57 In cell cultures, more rapid keratinocyte differentiation following treatment with niacinamide was established.56 In particular, it was possible to determine an influence on keratin, K1. K1 is a basic keratin synthesized mainly in the lowest layers of the stratum spinosum. The functional limitations of aging skin include reduced “turnover of the epidermis” (i.e., a slower epidermal cell cycle) due to a deficiency of NADP in aging cells.58,59Tanno et al also demonstrated an in-vivo improvement in epidermal barrier function with improved keratinocyte differentiation after the application of topical niacinamide. Once again, the improved barrier function was evident by a decrease in TEWL and increase in SC moisture content. Similar results were also obtained by Ertel et al.57 In conclusion, it seems that topical application of niacinamide increases NADP levels, which in turn stimulates keratinocyte differentiation. This results in a thicker SC, which is not only associated with an improved barrier, but is also associated with greater hydration retention capacity in the SC.59
Erythema and blotchiness. The mechanism by which redness/blotchiness is improved may be related to the improved skin barrier function for reasons discussed above.50,60,61 Increased barrier function may mean less irritation and redness when the skin encounters environmental insults, such as detergents and soaps, and hence less reddening of the skin. However, this theory has not been substantiated.
Yellowing of skin. The yellowing of skin that comes with aging may be a result of glycation of proteins in the skin called the Maillard reaction. The Maillard reaction is a spontaneous oxidative reaction between protein and sugar that results in cross-linked proteins (Amadori products) that are yellowish-brown in color and are fluorescent.62–64 These proteins can accumulate in the skin matrix components, similar to collagen, in response to oxidative stress as we age. Published data show a fivefold increase in collagen oxidation products in human skin from age 20 to 80.65 Since NADH and NADPH are antioxidants and their levels can be increased with niacinamide, a possible effect of topical niacinamide is inhibition of oxidative processes, such as protein oxidation, glycation, and the Maillard reaction, and hence the inhibition of skin yellowing.66–68
Fine lines and wrinkles. Multiple mechanisms may be involved in the ability of niacinamide to reduce the appearance of fine lines and wrinkles. The first to consider is that niacinamide may have the ability to increase dermal collagen and protein production. The development of wrinkles is a result of the decrease in epidermal cell layers and dermal components from a reduction in protein and collagen synthesis. Reduced protein synthesis is reflected in the levels of keratin, fillagrin, and involucrin in the skin. Keratin deficiency has an effect on the epidermal cell structure and its water-binding capacity. Fillagrin is an antecedent of natural moisturizing factor (NMF) and hence affects skin hydration. Involucrin is seen as significant for the cell envelope and structure of the SC. In summary, the effects of reduced collagen and protein synthesis are poor skin structure and reduced skin elasticity as well as a decrease in epidermal barrier function with a reduction in SC hydration. In studies on cell cultures, Oblong et al58 found that in aging cells it was possible to prove that niacinamide, as a precursor of NAD/NADP, stimulated collagen synthesis and the epidermal proteins keratin, fillagrin, and involucrin.51,58 In addition, another study was able to show niacinamide's ability to increase dermal matrix collagen production.66
The second mechanism that may be relevant to decreasing the appearance of wrinkles is the ability of niacinamide to reduce excess dermal glycosaminoglycans (GAGs). This is a controversial theory because both the elevation and depletion of dermal GAGs are associated with photodamaged or wrinkled skin.52,69 What is known is that the presence of GAGs is required for normal structure and function of the dermal matrix and increasing the levels of GAGs can increase the moisture content of skin. Testing has indicated that niacinamide reduces excess production of GAGs in old human dermal fibroblasts, thus supporting the potential involvement of this mechanism in reducing the appearance of fine lines and wrinkles.51,70 Given the above analysis and scientific data, it seems more likely that an increase in dermal proteins (including collagen) may play a bigger role in reducing fine lines and wrinkles than decreasing the level of GAGs.
Hyperpigmentation. Topical niacinamide may be effective in decreasing epidermal hyperpigmentation and reducing pigmented spots as we age.71 Hakozaki et al71 showed that the reduction of cutaneous pigmentation, surprisingly, was not due to the direct influence of niacinamide on melanin synthesis by melanocytes. Instead, they showed that niacinamide reduced melanosome transfer from melanocytes to surrounding keratinocytes in a co-culture system, although the specific mechanism remains unknown.71This was supported by a study using 5% niacinamide moisturizer, which provided 35 to 68 percent inhibition of melanosome transfer from melanocytes to keratinocytes.71
What clinical studies are available on niacinamide?
Tanno et al56 showed a reduction in pigmentation as a result of niacinamide. Eighteen Japanese women with hyperpigmentation were treated on one side of the face with 5% niacinamide and on the other side with vehicle only. The pigmentation change was evaluated qualitatively and quantitatively using high-resolution digital images and subjective judgments. In both forms of evaluation, it was found that after eight weeks of treatment there was significant lightening of hyperpigmentation on the side treated with niacinamide when compared with the effect of the vehicle (p<0.05).56
In a separate study also reported by Tanno et al performed with 120 Japanese women, comparisons were made among a sun protection factor (SPF) 15 cream with and without 2% niacinamide and the relevant vehicle. As a result of niacinamide treatment, there was a lightening of the skin after four and six weeks, which was noted to be markedly better than the formula without niacinamide.56
It is theoreticized that niacinamide may improve the texture of skin by speeding up epidermal turnover hence functioning as a mild exfoliant.72 Using a multiple angle reflectance spectrophotometer in an in-vivotest of the back of the hand, Matts and Solenick73 established a beneficial effect for the topical application of niacinamide in smoothing the skin surface structure. This study demonstrates that the long-term application of 2.5% niacinamide can correct the skin surface damage that results from aging. These results were statistically significant compared with the influence of the vehicle alone (p<0.05).59 In agreement with the above, another clinical trial using 3.5% niacinamide cream was compared with placebo for four weeks and demonstrated a 14.8-percent reduction in skin roughness (p=0.05).56,74,75
One of the best randomized, double-blind, split-face, placebo-controlled, clinical trials published on the anti-aging effects of topical niacinamide was done by Bissett et al.51 In this study, 50 white females with clinical signs of photodamage applied 5% niacinamide to half of the face and its vehicle control to the other half twice daily for 12 weeks. Analyses of the data revealed a variety of effects related to improvements in skin appearance for topical niacinamide including reductions in fine lines and wrinkles, hyperpigmented spots, red blotchiness, skin sallowness, and improvement in skin elasticity.51 Matts and Solenick later confirmed the results of Bissett et al with 5% and 2% niacinamide.59 The results also demonstrated that the anti-aging effects of niacinamide were dose dependent.
What conclusions can be drawn from data on niacinamide?
The topical use of niacinamide for anti-aging has proven to be effective not only when there are signs of a niacin deficiency. Despite the recent discovery of the cosmetic benefit of niacin for the skin, there have been sufficient studies completed to answer all three “Kligman questions.” It is the opinion of the authors that niacinamide is one of the best studied cosmeceutical ingredients for anti-aging. However, further research is required to uncover the specific mechanisms of niacinamide in the skin and to optimize the concentration of niacinamide in cosmeceutical formulations.
Is there additional information on nicotinic acid?
As mentioned earlier, a major obstacle in the topical delivery of therapeutic amounts of nicotinic acid to any tissue is its ability to cause a peripheral vasodilation that leads to a skin flushing response. While this effect is not harmful, it is intensely disliked by most patients.45,46 This issue may be avoided by using the long chain ester derivative of nicotinic acid, myristyl nicotinate (MN), which is able to deliver large amounts (in concentrations near 5%) of nicotinic acid without flushing.76,77
The advantage of using nicotinic acid over niacinamide is its drug-mediated effect on skin as a result of its ability to interact with nicotinic acid receptors present in the skin.42–44 Nicotinic acid receptors are G protein coupled receptors that when stimulated lead to an increase in skin leptin, which in turn activates several signaling pathways to enhance epidermal differentiation and stimulate wound healing.78,82
In a clinical study by Jacobson et al,76 MN increased skin cell NAD by 25 percent (p=0.0001) demonstrating effective delivery. Relative to placebo, MN treatment of photodamaged facial skin increased SC thickness by approximately 70 percent (p=0.0001) and increased epidermal thickness by approximately 20 percent (p=0.001). In two separate studies, MN treatment increased rates of epidermal renewal by six (p=0.003) to 11 percent (p=0.001) and increased the minimal erythemal dose by 8.9 (p=0.07) and 10 percent (p=0.05) relative to placebo. MN treatment also resulted in reductions in the rates of TEWL of approximately 20 percent relative to placebo on cheeks and arms of study subjects.
The above data demonstrates that topical nicotinic acid preparations can enhance epidermal differentiation and barrier function, suggesting that it may be effective in the treatment of photodamaged skin and other conditions (such as atopic dermatitis) with skin barrier impairments. However, it is hard to compare these results with the results presented above on niacinamide since not all of the same attributes were monitored (such as redness, yellowing, wrinkling, etc). Further investigation is needed to compare the efficacy of MN to niacinamide. Both niacinamide and nicotinic acid have significant numbers of published studies to answer all three major questions to date. However, there is more data available on the anti-aging effects and mechanisms of topical niacinamide.
What background information is available on soy?
Soybeans are known to contain many components with biological activity in the skin.83 The major components of soy are phospholipids (45–60%) and essential fatty oils (30–35%) while the minor components include the most active compounds, such as isoflavones and the proteases soybean trypsin inhibitor (STI) and Bowman-Birk inhibitor (BBI).84–86 In this review, the authors will focus on the effects of topical isoflavones and their ability to reduce ROS, stimulate collagen synthesis, increase moisture in the skin and also the proteases STI and BBI,83,87–94 and reduce skin hyperpigmentation.
What data is available on the percutaneous absorption of soy?
The most plentiful isoflavones in soy are genistein and daidzein. The permeation of isoflavonoids through the skin barrier is poorly investigated. However, the data that is available indicates that the compounds of this group can permeate through the SC and can reach viable layers of the epidermis and dermis.95,96 The permeation rate of soy through the SC is dependent upon the isoflavonoid's structure and vehicle composition.97 In general, the ionized form of a compound has lower percutaneous absorption compared to non-ionized form. This is due to the lipophilic nature of the SC.98,99 This theory explains why genistein in a completely neutral condition (pH 6) shows higher skin accumulation compared to the ionized form (pH 10.8). The same result was observed for permeation profiles of daidzein.
What are the potential mechanisms of action of soy?
Soy has been purported to exhibit a few potential modes of action, which may correlate with topical treatment of photoaging.
Antioxidant effects. Free radical formation plays a crucial role in aging skin.100 A clinical study by Sharma et al101 found that soy isoflavones (genistein and daidzein) have a fourfold mechanism of action to fight oxidative process in the skin.101 In this study, soy isoflavones were shown to raise cellular glutathione (GSH) content and glutathione S-transferase (GST) activity (p<0.05), prevent antioxidant enzyme depletion, decrease H2O2 formation (p<0.05), and prevent ornithine decarboxylase (ODC) induction and DNA degradation (p<0.01). Given the wide array of antioxidant effects, the authors speculate that soy isoflavones may not act as antioxidants themselves but instead affect cell signaling processes that increase the skin's own antioxidant capabilities. However, there is a lack of evidence supporting soy isoflavones as a cell-signaling molecule, and the antioxidant nature of soy isoflavones cannot be sidelined completely because of the polyphenolic structure. Given its structure, genistein can donate hydrogen atoms to deleterious oxygen free radicals and form less reactive phenoxy radicals in the process. Further research is required to determine soy's true antioxidant mechanism in skin. However, the topical application of soy isoflavones has been shown to increase the antioxidant capability of skin compared to control via increases in the four mechanisms mentioned above. In one study, genistein was able to decrease the H2O2 increment in human keratinocytes caused by ultraviolet B rays by 71 percent and daidzein almost completely inhibited H2O2 production by UVB. Daidzein was expected to be a less effective antioxidant due to its chemical structure; however, this was not the case in this present study.102Thus, the authors conclude that soy isoflavones may have inhibitory activity against oxidative damage and may be capable of preventing the biochemical alteration associated with aging.101
Phytoestrogen effects. The primary metabolites of soy isoflavones, genistein and daidzein, have been identified in various studies in animal and human cell cultures as phytoestrogens. Phytoestrogens are plant compounds with a weak estrogenic effect. In soy, genistein and daidzein are present as glycosides, which do not have estrogenic activity, and only upon conversion to their free isoflavone form do they demonstrate phytoestrogen activity.103 Phytoestrogens, just like estrogen, work by coupling with estrogen receptors (ERs) in the cell's nucleus. Two types of receptors, alpha and beta, have been identified and both are present in the skin.104 One study demonstrated a higher affinity of genistein for ER-beta;105 however, another study reported a higher affinity of genistein for ER-alpha agonist activity.106 In comparison with genistein, estradiol has 700-fold more ER-alpha and 45-fold more ER-beta activity.107 Even though phytoestrogens are weak estrogens, soy may contain as much as 1/1000 of its content as phytoestrogens. Therefore, circulating levels of phytoestrogens may be high and the subsequent biological effect may be great.
Several studies have shown that postmenopausal women have a measurably thinner dermis and less collagen compared to premenopausal women.108,109 Topical estrogen is able to retard skin thinning and collagen loss in postmenopausal women because estrogen receptor levels are highest in the granular layer of skin. Therefore, the phytoestrogens, genistein and daidzein, have a similar potential to retard skin thinning and collagen loss due to estrogenic stimulation.116 Further research is needed to compare the estrogenic activity of whole soy versus genistein and daidzein in their free isoflavone form.
Collagen synthesis effects. In other studies, soy isoflavones were investigated for their potential to stimulate collagen synthesis.111,112 One study demonstrated in vitro that genistein was able to increase collagen (COL1A2) gene expression in in-vitro human fibroblasts.112 Another study tested four different formulas in terms of their capacity to stimulate de novo collagen synthesis.111 Depending on the respective method of extraction, the four formulas contained different amounts of isoflavones. The isoflavone daidzein did not elicit any effect, but genistein was able to stimulate collagen levels in human dermal fibroblasts (HDF). Interestingly, the effect on collagen status did not correlate with the isoflavone content of the respective formulas. In other words, the formula with the highest amount of isoflavones did not display the highest efficacy in terms of collagen stimulation. In this study, the specific soy extract that was the most effective contained 10 to 12 percent genistein. The authors speculate that compounds other than isoflavones may play an important role for the collagen stimulatory effect. This fits well with their observation that purified isoflavones stimulated collagen synthesis to a lesser extent than whole topical soy. Therefore, it appears that the pronounced collagen stimulation of soy is only in part because of the isoflavones genistein and daidzein and other ingredients in soy, such as saponins, may play a larger role.111
Potential effects on breast cancer. Due to soy's potential as a phytoestrogen, there is some concern regarding an increased risk of breast cancer and uterine cancer with the use of soy products. However, data concerning this risk is contradictory. On one hand, the majority of breast cancers are estrogen dependent and the use of phytoestrogens could potentiate cancer cell growth, while on the other hand, epidemiological data suggest a protective effect of soy against the development of cancer (including breast cancer).113 The authors of this review speculate that soy's protective effect may be due to its antioxidant abilities while potential carcinogenic ability may be related to binding to estrogen receptors. Although the amount of phytoestrogens that are systemically absorbed from a topical preparation is likely very low, it would be prudent to avoid these products in a person with active breast cancer.116
Glucosaminoglycan effects. Finally, it has been shown that soy isoflavones can increase levels of GAG and specifically hyaluronic acid (HA) in aging skin although the exact mechanism of action, as far as we are aware, has not been discovered. HA is an anionic, nonsulfated GAG distributed widely throughout the skin. Hyaluronic acid is important for tissue repair and maintaining skin hydration.111 It is widely accepted that the HA content in skin, as well as all the GAG content, declines with age.114,115 Given the importance of HA for connective tissue overall, and the proven decline of HA in aged skin, there appears to be a necessity for compounds that stimulate HA production in aging skin. More research needs to be completed to demonstrate the efficacy of soy in stimulating HA synthesis and how clinically this increase in HA affects the appearance of skin.
Hyperpigmentation. Soybean trypsin inhibitor (STI) and Bowman-Birk protease inhibitors (BBI) in soy milk exhibit depigmenting activity and prevent UV-induced pigmentation in vitro and in vivo. It has been shown that STI and BBI inhibit the keratinocyte protease-activated receptor 2 (PAR-2), which is involved in the regulation of pigmentation.116–121 PAR-2 is expressed on keratinocytes and has been shown to increase keratinocyte phagocytosis.117,118 Several in-vitro and preclinical investigations have demonstrated that the modulation of PAR-2 activation facilitates keratinocyte-melanocyte contact and therefore enhances the transfer of melanosomes into the keratinocytes and produces reversible depigmentation.116,119
What clinical studies are available on soy?
Recently, a topical soy formulation was developed containing nondenatured STI and BBI.122 This is important because STI in particular is inactivated by heat and the processing of soybeans and soy milk can destroy their therapeutic effects, including depigmenting activity.123 Preliminary in-vivo human studies support the skin-lightening effect of nondenatured soy extracts.122,124–126 A study by Wallo et al83investigated the efficacy of a novel soy moisturizer containing nondenatured STI and BBI for the improvement of skin tone, pigmentation, and other photoaging attributes.83 Sixty-five women, with moderate facial photodamage, were enrolled in the 12-week, parallel, vehicle-controlled study. Efficacy was measured by clinical evaluation, colorimetry, digital photography, and self assessment. Improvement of skin tone was clinically defined as a reduction in mottled hyperpigmentation, lentigines, and blotchiness, with an increase in skin brightness (i.e., reflection of light from skin's surface). Improvement of skin texture was defined as a reduction in the surface roughness and/or an improvement in fine lines and wrinkling. The results showed that the novel soy moisturizer was significantly more efficacious than the vehicle in improving mottled pigmentation, blotchiness, dullness, fine lines, overall texture, overall skin tone, and overall appearance. Differences were marked from Week 2 to Week 12 for all above parameters (except dullness, which started at Week 4).83
Given STI and BBI's efficacy for treating hyperpigmentation, one might consider using whole soy for the treatment of melasma. Currently, there are conflicting views as to whether soy should be used to treat patients with melasma. On the one hand, one study found that treatment with topical soy twice daily for 12 weeks was shown to be effective for the treatment of melasma.127 On the other hand, high levels of estrogen have been shown to cause melasma and since soy has estrogen receptor bind capacity, it may be advisable to avoid soy in patients with melasma. Obviously further research is required; however, it should be noted that the isolated constituents of soy STI and BBI do not contain phytoestrogenic abilities and may be a good option for the treatment of melasma.
What conclusions can be drawn from data on soy?
All three of the “Kligman questions” are adequately answered for soy's protease inhibitor depigmentation effects. However, soy isoflavones, despite all the research into its antioxidant effects is lacking in-vivo, human, clinical trials with greater than 50 patients to prove the efficacy of soy isoflavones efficacy as an anti-aging topical cosmeceutical.
Given that there are two active ingredients in soy, isoflavones and protease inhibitors, there appears to be a need for specific labeling of topical soy products. As a physician evaluating a product, it is important to know whether the product contains whole soy, the isoflavones genistein and daidzein, or soy protease inhibitors STI and BBI in the nondenatured form.
What background information is available on green tea?
White, green, and black teas are derived from the leaves and buds of the tea plant (camellia senensis), with different varieties dependent on the type of processing and antioxidation or fermentation.128,129 Black tea is the most processed (fermented), while white tea recently replaced green tea as the least processed. The main active ingredients in green tea are polyphenols, also known as catechins, which include epicatechin, epicatechin-3-gallate (ECG), and epigallocatechin-3-gallate (EGCG).128,129 The largest catechin and most active antioxidant in any tea is EGCG. Green tea has the highest concentration of EGCG.130
Polyphenols comprise 30 to 35 percent of the dry weight of the green tea leaf.107 These polyphenols are believed to have anti-aging effects through decreasing inflammation and acting as a scavenger of free radicals. In addition, compounds found in green tea have been shown to influence biochemical pathways important in cell synthesis and responses of tumor promoters.131 In this review, the authors focus on the antioxidant and anti-inflammatory attributes of green tea, as these are the factors that most contribute to green tea's potential as an anti-aging cosmeceutical.
What data is available on the percutaneous absorption of green tea?
The formulary of green tea polyphenols as active ingredients in topical products remains a challenge in the cosmeceutical industry. Green tea polyphenols, as with most antioxidants, are highly unstable and easily oxidized in an ambient environment. Equally difficult is ensuring that the active ingredient penetrates the epidermis and that it stays in the skin long enough to exert its desired effect. EGCG is inherently hydrophilic, limiting its penetration in human skin. Thus, green tea extract is among the more difficult botanicals to formulate.132 Only when these formulation challenges are met can this topical antioxidant be effective.
After reviewing available product information, it appears that there is little standardization regarding the minimal concentration of green teas in cosmeceuticals, and many products lack active ingredient characterization. Some authors feel that 5% green tea extract is an effective concentration.132 However, the flaw in this logic is that it is the amount of green tea polyphenols and not the amount of green tea in a product that should be considered when evaluating a product. Many authors recommend products that contain polyphenols in the 90-percent range, which turns the product brown. This brown color does not indicate that the product has oxidized, as is the case when vitamin C products darken.53,116
What are the potential mechanisms of action of green tea?
Tea polyphenols have been shown to exhibit antioxidant and anti-inflammatory activities, which may potentially exert clinical benefits.
Antioxidant activity. Tea polyphenols are strong antioxidants.133 The polyphenols in tea have demonstrated the ability to quench the following ROS: singlet oxygen, superoxide radical, hydroxyl radical, hydrogen peroxide, and peroxyl radical.133–138 In addition, tea polyphenols have been shown to limit UV-induced lipid peroxidation in skin and reduce the oxidation of proteins in a free radical-generating system in vitro.139,140
Anti-inflammatory and collagen synthesis effects. Free radicals are known to promote oxidation of nucleic acids, proteins, and lipids and can damage intracellular structures including DNA.141,142 Free radicals also up-regulate transcription factors, such as activator protein 1 (AP-1) and nuclear transcription factor-kappa B (NF-kB).132 AP-1 is responsible for production of metalloproteinases that breakdown existing collagen, contributing to skin wrinkling.132 NF-kB up-regulates transcription of pro-inflammatory mediators, such as interleukin (IL)-1, IL-6, IL-8, and tumor necrosis factor-alpha.143 Acting through the cell surface, these pro-inflammatory mediators further activate AP-1 and NF-kB, resulting in more damage. It is the sum of these events that are responsible for skin aging.144
We now know that green tea and EGCG in addition to being effective free-radical scavengers, down-regulate UV-induced expression of AP-1 and NF-kB and suppress metalloproteinase and age-related collagen cross-linking in mice.139,145–149 In addition, it has been shown in vitro that green tea polyphenols inhibit the activity of collagenase and increases collagen biosynthesis rate of human fibroblasts.91
Photoprotectant effects. Thus taken together, a multitude of scientific evidence exists supporting the notion that green tea extract may improve aging skin through its antioxidant and anti-inflammatory capabilities. These scientific theories were put to the test in studies published by Elmets et al.150 In their study, human skin was pretreated with either green tea extract or one of its constituents and then exposed to two minimal erythema doses of solar stimulated light. Application of green tea extract and/or one of its constituents resulted in dose-dependent inhibition of UV-induced erythema. EGCG and ECG were the most efficient components in suppressing UV-induced erythema when tested individually. It was also shown that green tea extract can reduce the DNA damage that occurs after UV radiation through mechanisms discussed above.107,132 Thus, it appears that topical application of green tea extract and some of its components may be useful for mitigating the adverse effects of sunlight on human skin, such as photoaging.
In another study, topical green tea was shown to provide photoprotection anywhere from 24 hours up to 72 hours. It reduced the number of sunburned cells by 66 percent when applied 30 minutes prior to UVB exposure and when applied at 1- to 10-percent concentrations. A dose-dependent inhibition of ultra-violet-induced erythema was evident.150 Green tea extract also prevented psoralen-UVA photodamage with pre-and post-treatment by reducing erythema, hyperplasia, and hyperkeratosis.151
What clinical studies are available on green tea?
To date, we are only aware of one randomized, double-blind, controlled, clinical trial involving topical green tea extract. This study was completed by Chiu et al152 and consisted of 40 women with moderate photoaging. Eighteen subjects took 300mg green tea supplements twice a day and applied green tea cream twice daily; whereas, the other 18 subjects used a placebo cream and a placebo supplement twice daily. All subjects used the same sunscreen and cleanser. Experts were blinded and subjects were graded based on wrinkling, roughness, course rhytids, skin laxity, pigmentation, and other stigmata associated with photoaging at baseline and at eight weeks. At the end of eight weeks, investigators found no statistically significant clinical improvements in women using the green tea products. Because trends toward improvement were seen in the green tea group, investigators postulate that a longer study might be necessary to demonstrate efficacy. However, an improvement in the elastic tissue content of treated specimens (p<0.5) was observed upon histological examination of skin biopsies.152
What conclusions can be drawn from data on green tea?
Although it has been shown that green tea is able to influence mechanisms on skin that are beneficial for anti-aging, via its antioxidant and anti-inflammatory properties, there has yet to be a clinical trial to show a significant clinical improvement in the signs of aging with the topical application of green tea. Even so, green tea products are widely used by consumers despite the lack of evidence. For this reason, a healthy dose of skepticism is appropriate for dermatologists regarding the usefulness of many cosmeceuticals touting green tea until a split-face, double blind, randomized trial involving several concentrations of green tea extract or EGCG is conducted.
The term cosmeceutical was created to define a subgroup of topical products with “drug-like” effects on skin and to differentiate this subgroup from purely cosmetic products and topical prescription drugs. Since cosmeceutical products are claiming to affect the structure and function of skin it makes sense then to hold cosmeceutical products to higher standards of scientific substantiation than cosmetic products. In our opinion, these higher standards should include at minimum being able to substantiate the three major questions proposed by Dr. Albert Kligman, as discussed earlier. Thus, there needs to be a clear understanding that the ingredient penetrates into skin, that it has a defined mechanism of action, and that it has specific clinical effects with continued topical use.
As discussed here in this review, even these five very popular cosmeceutical ingredients fall short of the “Kligman standards” for cosmeceutical ingredients. Most of the research concerning cosmeceutical retinoid ingredients is based upon data related to RA's effect on the skin, and clinical trials concerning ROL and RAL are scant and lacking in statistical confirmation. Kinetin is supported by research substantiating its effects in plants and antioxidant effects in vitro, yet its anti-aging mechanism of action remains elusive. In addition, the clinical efficacy of kinetin has only been demonstrated with one study. Niacinamide is probably the closest ingredient investigated in this review to satisfy the three major questions in cosmeceutical ingredient analysis. With the available scientific evidence concerning the topical application of niacinamide on skin, we are able to adequately answer questions about permeability, mechanism, and clinical effect. Both green tea and soy are well known for their antioxidant effects, yet there is a conspicuous absence of clinical studies concerning the efficacy of soy and green tea as topical anti-aging cosmeceutical ingredients. It may be that antioxidants, such as soy and green tea, are better at preventing the signs and symptoms of aging than actually reversing the signs of aging. Perhaps clinical trials investigating the prevention of aging would show better efficacy for these two ingredients.
When evaluating a cosmeceutical ingredient, it is important for physicians to access the scientific literature, not only in medicine, but also in basic pharmacology and biochemistry, to verify that the claims made for active cosmeceutical ingredients are backed by a well-defined body of evidence. It was the goal of this review to present and analyze the research available on five popular cosmeceutical ingredients as to their ability to permeate the skin, their mechanisms, and their clinical effects. It is the authors' hope that physicians will be able to use the information to answer patients' questions concerning cosmeceutical products with a clearer understanding of what we really know about these ingredients.