Adilson Costa1, Elisangela Samartin Pegas Pereira1, Raquel Fávaro1, Margareth de Oliveira Pereira1, Paula Luz Stocco1, Elvira Cancio Assumpção1, Fernanda Sayuri Ota1, Stephanie Selma Barros Langen1
Keywords: SKIN AGING, DIETARY SUPPLEMENTS, VITAMIN C, GRAPE SEED EXTRACT
The skin aging process in humans is complex and driven by multiple causes including environmental and genetic fac- tors. 1-4 Exposure to UV radiation, which is the main environ- mental factor, results in morphological alterations, mainly in the dermis. In areas protected from the sun, the deepest morpholo- gic alterations occur in the epidermis.3 Photoprotected skin presents thin and delicate wrinkles (intrinsic aging). In contrast, skin that is frequently exposed to the sun is characterized by deep and well-marked wrinkles, a rough appearance and mot- tled pigmentation (photoaged skin). 1-4
Intrinsic skin aging is determined by genetic and hormo- nal factors. The physiological hormone decrease resulting from the aging process seems to be one of the most important factors for the aging of the skin and other organs. 4, 5
UV radiation damages human skin – affecting its color, tone and resistance – and causes premature aging. 3,6,7 Photoaged skin presents prominent alterations in the connective tissue''''''''s cellular component and extracellular matrix, with an accumula- tion of disorganized elastin in the deep dermis and a severe loss of collagen. 6 This process is a result of the activation of matrix metalloproteinases, which are responsible for the changes in the extracellular matrix of the connective tissue''''''''s collagen. 6 UV rays also attack keratinocytes and fibroblasts. 3 These alterations cause a number of molecular changes that lead to the destruction of extracellular collagen and halt collagen synthesis. 3
According to a theory developed by Denham Harman in 1956, 8,9 free radicals also have an important role in the aging process. Oxidative stress is caused by an imbalance between the formation of oxidants and the activity of antioxidant defense systems; free radicals are formed by the metabolism of oxygen. 9 Free radicals damage important skin structures such as cell membranes, DNA segments, collagen and elastic fibers, causing the clinically recognizable signs of skin aging. 8 The degradation of oxidized products is carried out by the proteasome, a multi- catalytic protease whose activity seems to decrease over an indi- vidual''''''''s lifetime, causing the incomplete degradation of oxidized proteins, an increase in protein aggregates and the acceleration of cellular dysfunction. 8-10
Oxidative reactions occur physiologically in the human body, but are nevertheless counterbalanced by the action of endogenous antioxidants in an individual''''''''s diet. When there is an imbalance in the oxidation-reduction state in favor of pro- oxidative reactions, cell damage takes place. This process is cal- led oxidative stress. 11
Antioxidant defense mechanisms prevent or limit the effects of oxidative stress, with the participation of endogenous enzymes such as the superoxide dismutase, catalase, glutathione peroxidase and other substances present in the diet, such as caro- tenoids, phenolic compounds, tocopherols and ascorbic acid. 11, 12
Carotenoids are naturally present in human skin; lycopene, a substance belonging into this group, is found in fruits and vegetables – especially in reddish ones such as tomatoes. 10, 12 When exposed to excessive UVB radiation, the skin protects itself forming erythema; nonetheless, this defense mechanism causes oxidative stress, interfering with the regulation of the genetic expression and damaging the DNA. 12 Oral supplemen- tation of lycopene – which has great antioxidant potential – seems able to provide protection against the erythema caused by UV radiation, with a subsequent reduction in oxidative stress. 12
Flavonoids, found in grapes, are polyphenolic com- pounds. 13, 14 In 1936, Rusznyák and Szent-György 13 showed that the flavonoids contained in citrus fruits decreased capillary per- meability and fragility in humans due to their antioxidant action. 13
Ascorbic acid (vitamin C) is essential for the synthesis of collagen and participates in the regeneration system of tocophe- rols (vitamin E), maintaining the plasmatic antioxidant poten- tial.11 Vitamin E is found in serum and in LDL particles, pro- tecting lipids from oxidation. Studies have shown its ability to reduce oxidative stress'''''''' biomarkers. 11, 14 An important source of ascorbic acid is acerola extract. The Biomarine Complex is rich in proteins and polysaccharides, and when present in oral sup- plements has been shown to improve the skin''''''''s structure. 15-17
In this manner, both clinical research on the use of oral supplements and the encouragement of their use are a growing practice in modern dermatology. This study evaluated, through subjective and objective assessments, the efficacy, safety and tole- rability of an oral supplement based on marine protein, concen- trated acerola and grape seed and tomato extracts, used for 360 days by 45 volunteers affected by photoaging.
A monocentric, phase IV, prospective, non-comparative, open clinical study was designed and approved by the Human Research Ethics Committee. It consisted of the use of Imedeen® Time Perfection (102.5 mg of Biomarine Complex® – compo- sed of marine proteins and polysaccharides –14.8 mg of Lycophence® GS – composed of lycopene and grape seed extract, and 30 mg of acerola extract (Ferrosan Laboratories S/A, Copenhagen, Denmark)), taken in the form of two daily tablets, ingested together, combined with the use of SPF 15 Episol ® sunscreen (Mantecorp Indústria Química e Farmacêutica Ltda., Rio de Janeiro, Brazil) on the face twice a day. The volunteers were instructed to use only the study suns- creen for 30 days prior to taking the oral supplement. The treat- ment lasted 360 days, and volunteers attended the research cen- ter monthly.
The clinical trial included 45 female volunteers, who sig- ned a term of free and informed consent. Study participants had a general dermatologic evaluation in order to verify that they met the inclusion criteria (aged 35-60; habitual users of SPF 15 facial sunscreen for at least 30 days prior to the beginning of treatment; Fitzpatrick phototype I to III; absence of known his- tory of allergic reaction to test products and seafood; absence of systemic and/or skin conditions that might interfere in the eva- luation of skin aging). The exclusion criteria included the pre- sence of other dermatoses, systemic conditions or use of medi- cations and/or products that interfered with the clinical evaluation of the study treatment; use of cosmetics in the area of the body being analyzed; use of oral supplement for photoaging; smoking; use of illicit drugs; intense sun exposure during the course of the study or in the 60 days prior to the study; preg- nancy or breastfeeding. Participants were excluded from the study if they failed to use any of the products in their full daily dose or less than 50% of the daily dose for more than seven con- secutive days or 15 non-consecutive days.
The volunteers underwent monthly skin ultrasound with 22 MHz probe (DUB®-USB, SkinScanner, Luneburg, Germany) on the face (left zygomatic region) and in the supe- romedial face of the left arm (4 cm below the lower limit of the axillary hair implantation line), and answered questionnaires regarding the evaluator physician''''''''s and volunteers'''''''' perceptions of the treatment''''''''s clinical efficacy and safety. The criteria evalua- ted by the subjective questionnaires were: wrinkles, fine lines, solar melanoses, other hyperchromias, erythema, hydration, radiance, sebum, smoothness, and overall appearance of the skin. Possible standardized answers were: total improvement, marked improvement, moderate improvement, slight improve- ment, unchanged, discreet worsening, moderate worsening, marked worsening and total worsening. The volunteers were also photographed (Canon® PowerShot G10, Oita, Japan) in their clinical evaluation.
Since the variables did not present a standard normal dis- tribution (Gauss curve) according to the Anderson-Darling test, non-parametric statistical tests were used. A significance level of p < 0.05, with 95% confidence intervals, was used. We used the test for equality of two proportions to analyze the questionnai- re responses, and Wilcoxon and Friedman tests to evaluate ultra- sound results.
Of the 45 volunteers, 33 (73.3%) completed the study. Seven dropped out for personal reasons, and five were removed due to the study''''''''s exclusion criteria (one pregnancy, one lumbar spine surgery, one dengue fever case and two cases of antibiotic use). There were no exclusions linked to the use of the study product.
Several changes considered statistically significant in all aspects (p < 0.05) were found in the efficacy questionnaire ans- wered by the evaluator physician (clinical assessment). According to the questionnaire''''''''s answers, in 360 days of use of the product, 6.1% of the volunteers showed improvement of wrinkles. Of these, 9.1% in fine lines, 12.1% in melanoses, 12.1% in other hyperchromias, 9.1% in erythema, 84.9% in hydration, 63.7% in radiance , 12.1% in sebum, 84.8% in smoothness , and 45.4% in the overall appearance of the skin. A significant improvement of the parameters was demonstrated after 30 days of product use, and the results were maintained after 330 days (Table 1, Graph 1 and Figure 1).
Patient reported results were similar to those of the clinical evaluation. The results after 330 days suggested improvement: 27.3% in wrinkles, 30.3% in fine lines, 21.2% in melanoses, 21.2% in other hyperchromias, 12.1% in erythema, 45.5 % in hydration, 42.4% in radiance; 18.2% in sebum , 42.5% in smoothness , and 48.6% in overall appearance (Table 2,Graph 2 and Figure 1).
In the ultrasound examination – the results of which were easily observed – it was concluded that there was a progressive increase in the values of dermal density in almost all visits, with improvements in the collagen and elastic fibers'''''''' pattern. Compared to the beginning of the study, at Day 360 was a sta- tistically significant increase in dermal density in both the face (132.3%, p < 0.001) and the left arm (51.9%, p < 0.001) – areas exposed to the sun and photoprotected (Tables 3 and 2), (raph 3 , and Figure 2).
Skin aging is caused by solar radiation and endogenous fac- tors.4 With the advancement of age, there is an increase in free radicals and a decrease in the skin''''''''s defense mechanisms, which accelerates skin aging. 3 Nevertheless, antioxidant products can attenuate that process.18 The availability of treatments that can stabilize or reverse the changes caused by aging is relevant to improving the population''''''''s quality of life and health. In this effort, nutraceuticals have arisen as a feasible option for systemi- cally treating photoaging.
Based on both the physician''''''''s and the volunteers'''''''' clinical assessments carried out in this study, oral supplementation con- taining marine protein and acerola, grape seed and tomato extracts was demonstrated to improve Fine lines, melanoses, other hyperchromias, erythema, radiance, sebum, smoothness, and the overall appearance of photoaged skin. A significant improvement in these characteristics was observed early in the treatment and was maintained throughout the study.
In 1998 Kieffer and colleagues 15 randomized two groups of volunteers to receive either placebo or the oral supplied used in this article for 12 months. Similarly to the findings of the pre- sent study, the authors observed improvement in a number of features of the skin (fine lines, global aging, hyperpigmentation and telangiectasia) through photographic assessment. Likewise, there was improvement in the self-assessment analysis and in skin density, which was measured by ultrasound (an increase in papillary and reticular dermis thickness was observed); there were no significant side effects. 15
The successful use of products enriched with Biomarine Complex in their formulation to treat the signs of skin aging was also described by Heule in 1992. He knew that improving photoaged skin required more than topical cosmetic action, so he conducted a pilot study with the Biomarine Complex. Objective and subjective improvements of the symptoms of aging skin were observed, including the attenuation of fine lines and skin pigmentation. The 90-day study included ultrasound examinations of the periocular region, which showed increased thickness of the epidermis and dermis (8.3% and 83.3%, respec- tively). 19
In 2011, Costa and colleagues 16 showed that the use of the product from the present study for 120 days was effective in improving aged skin features; statistically significant values were found in objective and subjective analyses. Through both the physician''''''''s and the volunteer''''''''s evaluations, the authors observed clinical improvement in wrinkles, fine lines, other hyperchro- mias, hydration, radiance, smoothness and overall appearance. In the corneometry examination, there were increases of 25.41% in the face and of 35.17% in the arm. In the pH test, there was a reduction of 10.37% and 10.10% in the face and arm, respec- tively. As a result, an improvement in the skin''''''''s hydration and a reduction (acidification) in the skin''''''''s pH (an ideal marker for hydrated skin) were observed. There was a significant reduction in seborrhoea, demonstrated by a 29.26% decrease in sebume- try measurements. According to the ultrasound examination, there was a gradual increase in measurements for the skin on the face and arm to 49.94% and 13.90%, respectively. All numerical parameters mentioned were statistically significant.
The skin''''''''s acidity is of crucial importance to its hydration; it controls the integrity and cohesion of the epidermis'''''''' stratum corneum. Acidity is of paramount importance for the epidermal antimicrobial barrier and in establishing the epidermic barrier''''''''s permeability. 17 The epidermal permeability function is explai- ned by the capacity of the billamelar lipid barrier''''''''s lipid secre- tor enzymes to be activated in acid pH, enhancing the integri- ty and cohesion of the stratum corneum and increasing the skin''''''''s hydration capacity. 17,20 In our clinical findings, we found improvement in the overall appearance of the volunteers'''''''' skin, a fact that was corroborated by the hydration capacity attributed to this nutraceutical combination, as described in pre- vious studies. 16
Vitamin C also has the ability to stabilize and reduce colla- gen''''''''s thermal sensitivity, stimulating collagen production in vitro and in vivo, and protect skin from photodamage. 21 These bene- fits can sustain a steady increase in dermal density obtained using the supplement, which is important for the repair of aged skin. There are reduced amounts of lycopene in dry skin, which is the most important sign of dehydration of the skin and of the early stages of wrinkle formation. 20, 22.23 Combined lycopene and vitamin C has the ability to sequester free radicals and defend the skin against damage caused by exposure to radiation. 24-26 The presence of lycopene in the nutraceutical is able to impro- ve hydration, which was verified in our results and in the above- mentioned findings of Costa and colleagues. 16
Oral ingestion of polyphenols prevents alterations in the epidermal barrier and improves the skin''''''''s protection against UVB; the grape seed extract contained in the study product hel- ped improve skin hydration, since it is rich in polyphenols. 22 High concentrations of flavonoids were found in these polyp- henols that, in in vitro studies, show higher antioxidant activity than that of vitamins E and C. 27 In the present study, the results demonstrated by the objective and subjective analyses reinforce our inference of how a product based on polyphenols has the ability to improve the appearance of aged skin, restoring hydra- tion and generating a greater tolerance to UVB rays. 22
The relevance and credibility of the present study are based not only on the long-term use of the compound, but also on the quality and reliability of the analysis of the results, which were substantiated in the volunteers'''''''' subjective analysis and the physician''''''''s clinical analysis, and were assisted by the high stan- dard of the tools used, such as digital photographic records and skin ultrasonography.
This study verified the high quality of an alternative treat- ment for photoaged skin, leading to the conclusion that the long-term use of the nutraceutical based on marine protein, acerola concentrate, and grape seed and tomato extracts is relia- bly effective and safe in improving aspects of cutaneous photoa- ging. The results were documented by photographic records, ultrasonography, and clinical and subjective evaluations.
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