Trending Anti-Aging Peptides

: The development of synthetic peptides for skin care dates to the 1980s. The cosmetic industry periodically launches new peptides, as they are promising and appealing active ingredients in the growing and innovative cosmetics market. In this study, trends in the use of peptides in anti-aging products were analyzed by comparing the composition of the products marketed in 2011 with products launched or reformulated in 2018. The scientiﬁc and marketing evidence for their application as active ingredients in anti-aging cosmetics was also compiled from products’ labels, suppliers’ technical data forms and online scientiﬁc databases. The use of peptides in anti-aging cosmetics increased by 7.2%, while the variety and the number of peptide combinations in products have increased by 88.5%. The most used peptides in antiaging cosmetic formulations are, in descending order, Palmitoyl Tetrapeptide-7, Palmitoyl Oligopeptide and Acetyl Hexapeptide-8. In 2011, the majority of peptides were obtained from synthesis, while in 2018, biotechnology processing was the dominant source. This study provides an overview of the market trends regarding the use of peptides in anti-aging products, providing meaningful data for scientists involved in the development of new peptides to identify opportunities for innovation in this area. existing scarcity


Introduction
The anti-aging market is expected to grow at an approximate 8% compound annual growth rate between 2018 and 2021, reaching a value of USD 271.0 billion by 2024 [1]. As the competition increases among cosmetic brands from the anti-aging market, new products claim to contain the ultimate innovations in order to stand out, often advertising new active ingredients.
Glutathione was the first biological peptide synthesized in the laboratory, and the development of new synthetic methods allowed for the synthesis of longer peptide chains, such as oxytocin and insulin [2]. Although peptides are distinguished from proteins by their shorter length, the cut-off number of amino acids to establish the classification as peptide is arbitrary [3]. According to the Food and Drug Administration (FDA), which is responsible for the regulation of health products, proteins are amino acid polymers with a specific defined sequence that is greater than 40 amino acids in size [4]. Peptides became popular in cosmetic products due to their bioactive properties, as they are able to interact with skin cells by multiple mechanisms, present high potency at low dosage and because their size is thought to achieve a moderate penetration into the upper skin layers. Due to their hydrophilic properties, chemical modifications such as esterification with alkyl chains, may be required to enhance penetration [5,6]. Furthermore, there are peptides whose structure is inspired by naturally occurring

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Signal peptides, which stimulate matrix protein production (such as collagen and elastin) and cell growth, amongst other cell metabolic functions (e.g., Palmitoyl Tetrapeptide-7, Palmitoyl Pentapeptide-4); • Carrier peptides, which may act as transportation facilitators for important substances or trace elements inside the cell, such as copper and magnesium (e.g., Tripeptide-1, GHK-Cu); • Neurotransmitter-inhibiting peptides, which may target expression wrinkles by inhibiting acetylcholine release at the neuromuscular junction by acting on distinct molecular targets (e.g., Acetyl Hexapeptide-8, Acetyl Octapeptide-3); • Enzyme-inhibiting peptides, which may reduce the activity of enzymes that participate in skin aging (e.g., soybean peptides which inhibit serine proteases, such as matrix metalloproteinases, (MMPs), and silk peptides, which inhibit tyrosinase).
Regarding safety, although some proteins are linked to skin allergy, including contact dermatitis, we were unable to find any evidence that peptides used in cosmetics may raise any concerns in this regard [12,13]. Moreover, protein hydrolysates, which contain bioactive peptides, are categorized as Generally Recognized as Safe (GRAS) by the FDA [7].
Nowadays, the "European glossary of common ingredient names for use in the labelling of cosmetic products" contains 848 entries with the word "peptide" [14]. Given the vast diversity of peptides available for cosmetic products, this study aimed to understand how the composition of anti-aging formulations has changed in recent years for these ingredients. For this purpose, an exploratory analysis of a pool of anti-aging products corresponding exclusively to multinational brands was performed. To our knowledge, this is the first study to describe the trends in the composition of cosmetic products on the market regarding anti-aging peptides.

Data Collection
Data were collected from anti-aging products from multinational manufacturers, marketed in parapharmacies and pharmacies from Portugal. Anti-aging cosmetics were included in the study if they exhibited on the label one of the following words: anti-wrinkle(s); anti-age/anti-aging; wrinkle repair; regenerator; aging; anti-slackening; firming. All the information available on the products' labels was collected from the manufacturers' websites. The data collection started in 2011 and was updated with products launched in 2018 or whose composition has been reformulated that year in order to avoid duplicate product analysis and to reflect the market trends. Cosmetics for application on the face, neck and eye contour were included, comprising more than 40 multinational brands. Following these criteria, 280 products were selected, 177 and 103 in 2011 and 2018, respectively.

Data Analysis
The peptides contained in the cosmetic products collected during the study were listed according to the International Nomenclature of Cosmetic Ingredients (INCI) and commercial names. Afterwards, the data were analyzed with respect to the following parameters.

Peptides' Prevalence and Combinations
The relative amount of cosmetic products containing peptides and the occurrence of combinations of these ingredients were assessed and expressed in percentage.

Top Peptides
The analysis focused on the twelve peptides with the highest usage frequency among the selected products in both years. The overall usage frequency for each peptide was determined by the sum of the number of products containing that specific ingredient in 2011 and 2018, and then they were ranked in descending order.

Peptides' Sources
Peptides were categorized according to their sources, based on the information provided by the manufacturer or mentioned in scientific literature.

Peptides' Mechanisms of Action and Scientific Evidence
All peptides found in the periods of analysis were categorized according to the mechanism of action [9,15]. The anti-aging benefits of each peptide were searched on the following online databases: PubMed, Google Scholar, Scopus, Cochrane and KOSMET, using the following keywords: "peptide INCI name" AND ("skin" OR "topical") AND aging. Given the innovative nature of these ingredients and the lack of peer-reviewed articles for some of the peptides found in marketed products, the manufacturers' technical data sheets and brochures were also analyzed.

Peptides' Prevalence and Combinations
Between 2011 and 2018, the percentage of anti-aging cosmetic products containing peptides was raised from 23.5% to 25.2% ( Figure 1). Furthermore, the number of products containing two or more peptides was also raised from 8.2% to 15.5%, showing an 88.5% increase.

Peptides' Prevalence and Combinations
The relative amount of cosmetic products containing peptides and the occurrence of combinations of these ingredients were assessed and expressed in percentage.

Top Peptides
The analysis focused on the twelve peptides with the highest usage frequency among the selected products in both years. The overall usage frequency for each peptide was determined by the sum of the number of products containing that specific ingredient in 2011 and 2018, and then they were ranked in descending order.

Peptides' Sources
Peptides were categorized according to their sources, based on the information provided by the manufacturer or mentioned in scientific literature.

Peptides' Mechanisms of Action and Scientific Evidence
All peptides found in the periods of analysis were categorized according to the mechanism of action [9,15]. The anti-aging benefits of each peptide were searched on the following online databases: PubMed, Google Scholar, Scopus, Cochrane and KOSMET, using the following keywords: "peptide INCI name" AND ("skin" OR "topical") AND aging. Given the innovative nature of these ingredients and the lack of peer-reviewed articles for some of the peptides found in marketed products, the manufacturers' technical data sheets and brochures were also analyzed.

Peptides' Prevalence and Combinations
Between 2011 and 2018, the percentage of anti-aging cosmetic products containing peptides was raised from 23.5% to 25.2% ( Figure 1). Furthermore, the number of products containing two or more peptides was also raised from 8.2% to 15.5%, showing an 88.5% increase.   (1) is represented in green, and the percentage of products containing two or more peptides (2+) is represented in orange. In 2018, there was also a greater diversity of peptides amongst anti-aging cosmetic products, as we were able to identify 29 different peptides versus the 14 peptides identified in 2011 (not illustrated), which represents more than a two-fold increase. Overall, 37 different peptides were used.
Cosmetics 2020, 7, x FOR PEER REVIEW 4 of 16 identify 29 different peptides versus the 14 peptides identified in 2011 (not illustrated), which represents more than a two-fold increase. Overall, 37 different peptides were used.
Tripeptide-1, Dipeptide-2 and Pimpinella anisium extract (apiacea peptides) were used in 2011 but did not appear in products launched in 2018.

Peptides Sources
The majority of peptides launched in 2011 are obtained through chemical synthesis ( Figure 3). Conversely, in 2018, there were more peptides that originated from biotechnology than synthetic ones. It is noteworthy that there is a significant amount of peptides found in both years whose source is unknown. In 2011, the most used peptides were Palmitoyl Oligopeptide, Palmitoyl Tetrapeptide-7 and Acetyl Hexapeptide-8. In 2018, the pole positions were occupied by Palmitoyl Tetrapeptide-7, Nicotiana benthamiana Hexapeptide-40 SH-Oligopeptide-1 and Palmitoyl Tripeptide-1.
Tripeptide-1, Dipeptide-2 and Pimpinella anisium extract (apiacea peptides) were used in 2011 but did not appear in products launched in 2018.

Peptides Sources
The majority of peptides launched in 2011 are obtained through chemical synthesis ( Figure 3). Conversely, in 2018, there were more peptides that originated from biotechnology than synthetic ones. It is noteworthy that there is a significant amount of peptides found in both years whose source is unknown.

Peptides' Mechanisms of Action and Scientific Evidence
Signal peptides were the most used in both years, followed by neurotransmitter-inhibiting and carrier peptides ( Figure 4). Contrary to the signal and carrier categories, the use of neurotransmitter-inhibiting peptides seems to be decreasing. We were unable to find enzyme-inhibiting peptides in this analysis.

Peptides' Mechanisms of Action and Scientific Evidence
Signal peptides were the most used in both years, followed by neurotransmitter-inhibiting and carrier peptides (Figure 4). Contrary to the signal and carrier categories, the use of neurotransmitterinhibiting peptides seems to be decreasing. We were unable to find enzyme-inhibiting peptides in this analysis. The information regarding all peptides present in the composition of the analyzed anti-aging products is summarized on Table 1.

Peptides' Mechanisms of Action and Scientific Evidence
Signal peptides were the most used in both years, followed by neurotransmitter-inhibiting and carrier peptides (Figure 4). Contrary to the signal and carrier categories, the use of neurotransmitterinhibiting peptides seems to be decreasing. We were unable to find enzyme-inhibiting peptides in this analysis. The information regarding all peptides present in the composition of the analyzed anti-aging products is summarized on Table 1. The information regarding all peptides present in the composition of the analyzed anti-aging products is summarized on Table 1. Anti-wrinkle and anti-sagging.
Randomized, controlled study ** [25] Cosmetics 2020, 7, 91 8 of 15 Increase in the levels of enzymes which are essential to break down and remove damaged cells.
Promotion of skin renewal and strengthening from connective tissue. Improvement microcirculation and skin density radiance.

Synthesis Unknown Aconitase
Increase in the enzymatic activity of aconitase and stimulates cell vitality Increase in cell energy production.

Discussion
This study explored the trending peptides used by the cosmetic industry in anti-aging formulations from the Portuguese market, which mainly encompasses multinational brands. From 2011 to 2018, the use of peptides in anti-aging products presented a small increase, reaching one fourth of the formulations on the market (Figure 1). However, the diversity of peptides almost doubled in these products, which reflects the growing importance of these ingredients in the period of analysis. The number of peptide combinations also followed this tendency. This observation may also be related with the use of peptide mixtures from ingredient suppliers, which often contain more than one peptide. There are 848 entries with the word "peptide" in the "European glossary of common ingredient names for use in the labelling of cosmetic products", but this study only identified 37 compounds, which corresponds to 4.4%. This may relate to the fact that this study is restricted to the anti-aging market and/or it may portray the lack of relevance of many peptides commercially available for cosmetic formulators.
In 2011, "Palmitoyl oligopeptide" was the most used peptide in cosmetic products, but this ingredient is hardly found in products released in 2018. This is possibly related to the fact that the term "Palmitoyl oligopeptide" was "removed" in 2013, as it has been used to designate two distinct molecules since its development in 1994. Those compounds were renamed as Palmitoyl Tripeptide-1 (Pal-GHK) and Palmitoyl hexapeptide-12 (Pal-KTTKS) in order to avoid misunderstandings. With this in mind, we found that the second most used peptide in 2018 was Palmitoyl Tripeptide-1, which demonstrates that this ingredient is still being used by formulators [40]. Curiously, the ingredient "Palmitoyl Oligopeptide" was still found in the composition of one product launched in 2018. Both matrikine-mimetic peptides act by stimulating fibroblast activity, thus increasing matrix protein and glycosaminoglycan synthesis in the dermis. Palmitoyl Tripeptide-1 has shown the ability to stimulate collagen synthesis in a human fibroblast culture and prevent its degradation after the exposure to UVA light [41]. Furthermore, Palmitoyl Tripeptide-1 has been tested in a clinical study involving 23 healthy female volunteers, at 4 ppm, promoting a small but statistically significant increase in skin thickness of about 4% [41]. Palmitoyl hexapeptide-12 is an elastin fragment used to stimulate collagen and elastin synthesis, as well as fibronectin and glycosaminoglycans. In a double-blind study, a group of 10 female volunteers performed two daily applications of an emulsion containing 4% Palmitoyl hexapeptide-12 or a placebo for one month. The application of the test product improved skin elasticity and firmness. However, the supplier did not provide the study methodology nor reported if these improvements had statistical significance [16].
The second most used peptide in 2011 was Palmitoyl Tetrapeptide-7, which in 2018 is ranking in first place. Palmitoyl Tetrapeptide-7 is a fragment of immunoglobulin G. Palmitoyl tetrapeptide-7 decreases IL-6 secretion and reduces inflammation after UVB exposure [42]. It is noteworthy that Palmitoyl Tetrapeptide-7 and Palmitoyl Tripeptide-1 are frequently used together in the ingredient Matrixyl™ 3000, which explains the usage percentages in 2018 [9].
Comparing the years 2011 and 2018, we found that the use of Acetyl Hexapeptide-8 (Argireline ® , also known as Acetyl Hexapeptide-3), is starting to decline, as it was not found in new products released in 2018. Notably, Acetyl Tetrapeptide-5 is being used more frequently in 2018 compared with 2011 [15]. Although both peptides act by inhibiting neurotransmission, it is unlikely that this finding reflects a direct replacement, as Acetyl Hexapeptide-8 is used to reduce wrinkles and Acetyl Tetrapeptide-5 is directed to minimize the appearance of dark circles and under-eye bags. There are several clinical trials involving Acetyl Hexapeptide-8. The anti-wrinkle efficacy of this peptide was accessed in a placebo-controlled clinical study, where an oil-in-water (O/W) emulsion containing 10% Acetyl Hexapeptide-8 was applied twice a day on the lateral preorbital region of 10 healthy women volunteers. Silicon imprints were obtained after 0, 15 and 30 days and analyzed by confocal laser scanning microscopy. The analysis showed that the test product decreased the depth of skin wrinkles by 30%, whereas the placebo provided a 10% reduction [25]. Later, a placebo-controlled clinical study investigated the possible synergism between Acetyl Hexapeptide-8 and tripeptide-10 citrulline in a study involving 24 women. The use of 10% Acetyl Hexapeptide-8 topically, twice daily for 60 days, promoted a significant decrease in transepidermal water loss (TEWL) [43]. Contrary to Acetyl Hexapeptide-8, we were unable to find clinical studies supporting the topical use of Acetyl Tetrapeptide-5.
Even though the molecules Cyclotetrapeptide-24 Aminocyclohexane Carboxylate, Acetyl Dipeptide-1 Cetyl Ester, Tripeptide-1 and Dipeptide-2 appear in the top 12, no clinical studies supporting their use were found in the literature.
As for Acetyl Dipeptide-1 Cetyl Ester, there is no evidence for its use as an anti-aging ingredient. However, it may be useful to reduce skin irritation, as it has shown a soothing effect on the stinging/burning induced by a 40 ppm capsaicin cream [28].
Notably, in 2018, we verified the emergence of SH-Polypeptides, which are single-chain synthetic peptides produced by biotechnology in living cells, such as those from tobacco plant (Nicotiana benthamiana) leaves [43]. These peptides aim to mimic human growth factors and cytokines, thus stimulating cellular growth, proliferation and differentiation after their recognition by keratinocytes' receptors. In this study, all SH-Polypeptides found on the market were derived from Nicotiana benthamiana, and there was only one cosmetic brand using this technology.
This study also provides evidence that from 2011 to 2018, biotechnology has gained increasing importance over chemical synthesis, becoming the dominant source for obtaining peptides for the cosmetic industry. However, it is not possible to draw a definitive conclusion, since there is a relevant amount of peptides whose source is unknown.
Regarding peptides' mechanisms of action, we found that signal peptides were the most prevalent in both years, followed by neurotransmitter-inhibiting and carrier peptides. The greater use of signal peptides may be related to the fact that these ingredients have more scientific evidence supporting their efficacy [8]. Contrary to other categories, the use of neurotransmitter-inhibiting peptides has decreased by 42.2%. Enzyme-inhibiting peptides seem to be of low relevance in this market. There are not many studies supporting the efficacy of these peptides, which may be the reason for this finding [9].
By analyzing Table 1, it is noticeable that most peptides have molecular targets located in the skin dermis. Among peptides' multiple biomolecular inspirations, growth factors and components from the dermis extracellular matrix stand out.
Aside from market trends, the choice of active ingredients for formulating cosmetic products should also rely on their efficacy. The majority of clinical studies available were performed by ingredient suppliers in order to generate ingredients' claims and are not usually published in peer-reviewed publications. Furthermore, the methodologies used to perform those studies are often undisclosed. This practice may be due to commercial and competitive reasons. Consequently, the scientific evidence supporting the efficacy of peptides is scarce in the literature and the technical documentation available to formulators. Additionally, clinical studies comparing the efficacy of peptides with similar mechanisms of action are missing both in commercial and scientific references. Moreover, for many ingredients, we were unable to find any studies which could support their efficacy.

Conclusions
Peptides are relatively new ingredients in the cosmetics field, whose chemical and biological properties encourage the industry to continually develop innovative compounds. The trends in the use of peptides in the cosmetic industry were not reported up to this date. In this study, peptides incorporated in anti-aging cosmetic products from 2011 to 2018 were disclosed and a trend towards increased use was noted, reaching 25% of the anti-aging products from the studied market. The variety of peptides used and the number of products containing peptide combinations have nearly doubled. Considering their mechanism of action, signal peptides are the most used ingredients, followed by neurotransmitter-inhibiting and carrier peptides. Furthermore, most peptides lack randomized clinical studies demonstrating their anti-aging efficacy in scientific literature, and there is an absence of systematic reviews summarizing the results from the existing studies. The scarcity of independent and