FRANCISCA RODRIGUES
Research Associate, REQUIMTE/LAQV, ISEP, Polytechnic of Porto, Portugal

Abstract

The demand for valorization of fruit by-products is stronger than ever due to environmental issues, being a challenge for economy and society. A panoply of studies has been carried out on fruit by-products to identify the phytochemicals present and evaluate its biological safety and efficacy for humans. The bioactive compounds present are mainly polyphenols, fatty acids, minerals, and vitamins, which provide skin beneficial effects, such as photoprotection, anti-aging and anti-inflammatory effects, among other activities. Therefore, the cosmetic field may take advantage of these high-added value ingredients. This paper aims to summarize the potential cosmetic applications of fruit by-products, with a case study of chestnut and kiwiberry by-products. A critical analysis of opportunities and challenges of these active cosmetic ingredients on the market is also provided.

On the last decades, the concerns related with food by-products, and their associated environmental problems, arise as a challenge not only for agro-food industries but also for consumers and governments (1). According to the most recent data, 88 million tonnes of food waste are annually generated in the European Union (EU) and the associated costs amount to 143 billion euros (2). Fruits and vegetables by-products and wastes represent 8% of the total amount, being the 5th highest contributors (3). Fruit losses, also known as fruit wastes, are mainly represented by fruits that are removed from the production line due to the absence of the caliber defined by authorities to be commercialized or due to the presence of superficial damages, while food by-products are composed by peels, pomace, flowers, stems, leaves, seeds, or pulps generated during the industrial production (4).
Most of these residues are not transformed due to technical limitations or difficulties into the market access, being discarded or redirected to non-food use (5). However, the Sustainable Development Goals defined by the EU aim to halve the per capita food waste at all levels by 2030, it is then urgent to find solutions to deal with this problem (6). These hot topics raise awareness of researchers for the bioactive composition of food wastes and by-products. Therefore, an implementation of circular economy aligned with the 2030 Agenda goals is expected by different industries, including the cosmetic one. On the other hand, consumers are more aware about the sustainability questions of the ingredients used in the cosmetic field. In parallel, the increase of the average life expectancy, particularly in developed countries, led to new paradigms related with beauty and, consequently, to the search of new natural compounds with anti-aging effects. Among the wide range of bioactive compounds, polyphenols are the most interesting due to health benefits, being found in fruit by-products (7-9). Various studies support the biological skin effects of polyphenols, such as antioxidant, anti-inflammatory, antimicrobial (10). Furthermore, natural compounds are normally associated by consumers to safety and efficacy, despite in some cases this idea is not completely true. Over the last years, research groups from various countries reported the rich content of fruit by-products that may provide skin beneficial effects, such as antiwrinkle, photoprotection, depigmentation, anti-cellulite, or anti-inflammatory activities, among others (9, 11).

EXAMPLES OF COSMETIC ACTIVE INGREDIENTS OBTAINED FRUIT BY-PRODUCTS
Many fruit by-products have been explored as source of active ingredients for cosmetic field. Among them, chestnut (Castanea sativa) (12-21), olive (Olea europaea) (22, 23), grapes (Vitis vinifera) (24), coffee (Coffea spp.) (25-27), apple (Malus domestica) (28, 29), citrus (Citrus spp.) (30) or kiwiberry (Actinidia arguta) (11, 31, 32) by-products arise as common examples thanks to their rich content in bioactive compounds. Our group has been particularly involved with chestnut and kiwiberry by-products projects (in deep cooperation with cosmetic industry) and, therefore, a focus on these matrixes will be provided.
Regarding chestnut (Castanea sativa) – Figure 1-, an enormous amount of by-products are generated during production, mainly shells, that are discarded as wastes (12). Shells are the principal industrial by-product, being composed by inner (6.3-10.1% of the chestnut weight) and outer (8.9-13.5% of the chestnut weight) shells (33). Their richness in polyphenols (mostly phenolic acids and tannins), vitamin E and amino acids have attracted the attention of the cosmetic field (18, 33), as well as other biological activities such as antibacterial, antioxidant and anti-inflammatory (18, 33). Shells are effective sources of natural antioxidants and deodorant compounds (16, 17), displaying a considerable skin hydration capacity and a protective effect from collagen degradation in keratinocytes (16, 17). Our team always work in a basis of sustainability, considering eco-friendly extraction techniques with low environmental impact, such as Subcritical Water Extraction (SWE), Ultrasound-Assisted Extraction (UAE) or Supercritical Fluids Extraction (SFE), among others. In one of our last studies, chestnut shells extracts prepared by these green techniques were explored with regards to antimicrobial activity, hyaluronidase and elastase inhibitory activities, in-vitro cytotoxicity on dermal cells (HaCaT and HFF-1) and skin permeation (using Franz cells coupled to human skin), imperative assays for the validation of new cosmetic ingredients (34). The results revealed good antimicrobial properties of the extracts as well as capacity to inhibit hyaluronidase (IC50 = 0.76–54.36 mg/mL) and elastase (33.56–82.70% at 0.4 mg/mL) activities, enzymes involved in the skin aging process. The ex-vivo assay in Franz cells has shown that the SWE extract to a higher polyphenols’ permeation (1061.6 μg/g dw), with ellagic acid being the major polyphenol permeating human skin (732.1–847.0 μg/g dw).
Considering safety aspects, 3D skin and ocular models assays were performed to ensure the absence of irritation of the most promising extract (SWE) and an in-vivo patch test confirmed the absence of irritation in humans based on the acute irritation index of 0.50 achieved 24 h after the patch removal. With these detailed assays our team validated, for the first time and in accordance with the European Regulation 1223/2009, a new cosmetic ingredient extracted from chestnut shells. We also developed and optimized a topical formulation with chestnut shells extract by a Quality by Design concept (Figure 1 (b)), focusing on a planned development that consider the vulnerabilities of the entire process through risk analysis tools and design of experiments (DoE) (35). Most recently (data not published) we performed clinical assays with a formulation containing chestnut shell extract in human volunteers that applied twice per day, for 56 days, the product. For that, biophysical techniques, including Corneometer®, Cutometer®, and PrimosPremium equipments were used, allowing to quantify the hydration, skin firmness, wrinkles depth, volume, and roughness. The results demonstrated that the active formulation led to a clear improvement of all parameters quantified, however without significant differences, which could be justified by the short period of use. A long-term use of the product is now being assessed.
Other chestnut by-products generated in lower amounts have also been studied by various groups. Among them, burrs, which represent 20% of the total chestnut weight (19), revealed to be a good source of polyphenols, vitamin E and amino acids with noteworthy antioxidant properties and protective effects on cellular membranes and DNA from oxidative damages, being able to protect living tissues against skin oxidative stress-mediated diseases as well as photoaging (19-21).

Leaves can also be considered as another by-product of the chestnut industry. According to different authors, leaves have antibacterial and antioxidant activities as well as protective effects against UV-mediated DNA damages and high effectiveness to scavenge ROS and Radical Nitrogen Species (RNS) (12-14). Excellent skin compatibility and safety has been reported for leaves extracts, with absence of genotoxic or phototoxic effects (14). On the other hand, in-vivo assays attested the absence of irritant effects in a patch test in human volunteers after 2 and 24 h of patch removal (12, 14).
Therefore, chestnut by-products are excellent sources of antioxidants that could be used as active ingredients in the skin healthcare field, particularly in the prevention or treatment of oxidative stress-related disorders.

Kiwiberry (Actinidia arguta) is a small grape-sized fruit, characterized by hairless skin and a pleasant aroma and flavor (Figure 2), that has a huge beneficial impact on human health, mainly due to the different biological effects associated with its consumption, such as antioxidant, anti-inflammatory, and antidiabetic activities (11). Worldwide, the kiwiberry production is exponentially increasing in different world regions, from Europe to Asia and North America, mainly due to the nutritional and healthy benefits associated with the fruit’s consumption (36). The fruit is rich in phenolic compounds (such as caffeoylquinic acid, chlorogenic, neochlorogenic and cryptochlorogenic acids, caffeic acid derivatives, catechin and derivatives), presenting different biological effects, such as antioxidant, antimicrobial, hemolytic or anti-diabetic activities (37-39). According to several authors, kiwiberry is a valuable source of vitamins (mainly vitamin C and A, but also E and B group (40)), as well as organic acids (particularly citric and succinic acids), minerals (41) and aminoacids (42). However, this fruit is extremely sensitive, leading to huge amounts of wastes. On the other hand, the leaves are removed to increase the solar exposure, being classified is some circumstances as a fruit by-product. Our team has been deeply involved in the valorization of these wastes and by-products, publishing different studies that attested its richness in bioactive compounds and their promising effects as cosmetic ingredients. In one of our last studies, we evaluated the potential of A. arguta leaves to inhibit elastase and hyaluronidase, achieving excellent results (65.62 ± 2.09% and 54.64 ± 4.17%, respectively). We also ensured the absence of cytotoxic effects in keratinocytes and fibroblasts of the MAE hydroalcoholic extract and proved the absence of irritation in 3D models, namely a skin (EpiSkin™) and an ocular (SkinEthic™ HCE, respectively) model. The ex-vivo skin permeation in Franz cells coupled to human skin showed a good permeability of 1-feruloylquinic acid, 5-caffeoylquinic acid and rutin. Lastly, in-vivo studies were performed through a patch test in human volunteers attesting the absence of allergic or irritative reactions. The M.I.I. determined 24 h after the patch removal was 0.10, allowing classifying the extract as non-irritating. A cosmetic formulation with the extract (Figure 2 (c)) was already developed by a mathematical model and tested in what concerns to stability, being now performed clinical studies to evaluate the anti-aging effects in human volunteers (data not published).

CHALLENGES AND FUTURE PERSPECTIVES
Figure 3 represents a SWOT diagram that aims to summarize the strengths, weaknesses, opportunities, and threats of employ fruit by-products as cosmetic ingredients.
The major strengths identified in the use of fruit by-products as cosmetic ingredients is their richness in bioactive compounds with skin biological effects (such as anti-aging, anti-inflammatory, anti-cellulitis, among others) as well as the possibility to convert them into high added value ingredients, generating profits for different industries in a circular economy approach. In contrast, the stability of the bioactive compounds and their safety assessment evaluation are the main weaknesses identified in this process, being a challenge to ensure them. On the other hand, the validation of fruit by-products extracts as new ingredients with proven safety and efficacy represent threats. A deep and impartial analysis of their composition, safety and efficacy by in-vitro and in-vivo assays should be performed for each new “candidate”, highlighting not only the benefits but, most importantly, the real potential effects considering the actual regulations and the in-vitro and in-vivo assays allowed by legislation (43). Particularly, the EU regulation is extremely restrictive and stablished a list of assays that must be performed to validate a new cosmetic ingredient.
Despite that, the animal assays are completely banned in Europe since 2004 for final products and 2009 for ingredients. Therefore, alternative in-vitro methodologies have been validated by the European Centre for the Validation of Alternative Methods (ECVAM) to ensure the assessment of new ingredients (43, 44). Despite the in-vitro assays already accepted by the EU regulation to validate new active ingredients, it is necessary to continue the search for more complex models that fully replace the skin metabolism and homeostasis. Nevertheless, these by-products represent an opportunity to achieve new ingredients with skin effectiveness due to the benefits described in the sections above. Therefore, the future perspective should be focused on scale-up processes that ensure the reproducibility of the bioactive compounds present in the extracts as well as the communication on the ecological impact of these new ingredients and their skin benefits to consumers.

FUNDING
This research was funded by project EXPL/BAA-GR/0663/2021—Kiwi4Health—Exploring the Eco-Innovative Re-Use of Kiwiberry, supported by national funds by FCT/MCTES and by the projects UIDB/50006/2020 and UIDP/50006/2020 through national funds. This work was also financially supported by projects PTDC/ASP-AGR/29277/2017 and 5537 DRI, Sérvia 2020/21 from Portuguese-Serbia Bilateral Cooperation, supported by national funds by FCT/MCTES and co-supported by FEDER throughout COMPETE 2020 - Programa Operacional Competitividade e Internacionalização (POCI-01-0145-FEDER-029277).

ACKNOWLEDGMENTS
Francisca Rodrigues (CEECIND/01886/2020) is thankful for her contract financed by FCT/MCTES—CEEC Individual Program Contract.

Figure 1. (a) Chestnut (Castanea sativa) and chestnut shells and burs; (b) cosmetic formulation with chestnut shell extract.

Figure 2. Kiwiberry (a), kiwiberry leaves (b) and cosmetic formulation with kiwiberry leaves extract (c).

Figure 3. SWOT analysis for possible cosmetic application of fruit by-products.

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ABOUT THE AUTHOR

Francisca Rodrigues is investigator at REQUIMTE/LAQV. She completed (2016) the PhD in Pharmaceutical Sciences in University of Porto and her research is focused on study new anti-aging cosmetic ingredients extracted from food by-products through in-vitro, ex-vivo and in-vivo assays. She works in collaboration with cosmetic industry, being the principal investigator of several projects and supervisor of PhD and MSc students.