J. Cosmet. Sci., 71, 11–21 (January/February 2020) 11 A New , Rapid Method for Examining Potential Skin-Brightening Ingredients Using Apple Slices JAMES V. GRUBER and JED RIEMER, Jeen International Corporation , Fairfi eld, NJ (J.V.G., J.R.) Accepted for publication October 12, 2019. Synopsis Darkening of fruits is the result of the oxidative activation of polyphenol oxidase converting low–molecular weight phenols present in the fruit body into quinone intermediates. Then, through polymerization, these reactive quinones convert to light yellow and red low–molecular weight melanin and, given enough time, to darker, higher molecular weight brown and black melanin. The process that occurs in the fl esh of cut fruit is very similar to the process that human skin cells use to make melanin: the oxidative activation of tyrosinase and conversion of tyrosine to dopaquinone and eventually to darker melanin. The conversion of the phenols by tyrosinase to quinones is the rate-limiting step in the biochemical manufacture of melanin. This article will discuss a new and cost effective way to screen skin-brightening ingredients by the use of apple slices as a model for skin using a chromameter to measure the change in color that occurs in apple slices over a short time course. Such measurements have been popularly used by food manufacturers to examine ingredients that inhibit fruit browning. Interestingly, as will be noted, many of the ingredients used commercially to inhibit food browning are also popular skin-brightening ingredients. We have found that a DermaLab (Cortex Technologies, Hadsund, Denmark) chromameter measuring the erythema index of apple slice darkening appears to be able to differentiate the benefi t of a formulation containing azelaic acid, a known skin-lightening ingredient, to minimize the darkening effects that occur in sliced apples. We will discuss how different apples behave differently when cut and how to best use the chromameter to analyze the changes that occur that can potentially help rapidly screen ingredients for their skin-brightening benefi ts. INTRODUCTION Development of new topical ingredients that can support skin-brightening and -calming claims is constantly expanding. In particular, because of the propensity of many Asian populations to seek ingredients that can help brighten complexion, ongoing research in this area is aggressive. The best way to confi rm whether an ingredient has potential skin- brightening benefi ts is to conduct a clinical screening on a population of individuals with darker skin. However, such robust clinical studies are typically quite expensive to run and can take time to note effective results. A more rapid method to investigate potential topical ingredient benefi ts involves irradiating volunteer’s skin with simulated solar radia- tion, UV energy, or possibly even high-energy visible light to cause a small spot that Address all correspondence to James V. Gruber at vincent@jeen.com .

JOURNAL OF COSMETIC SCIENCE 12 initially starts with an erythemal response (redness) and then over time moves to a tanned appearance, which can be used to investigate possible skin-brightening and -calming ingredients that might function to help reduce the Reactive Oxygen Species (ROS)-initiated skin effects of the energy exposure. Here, the problem is that this kind of study requires irradiating people to the point of redness, which some view as being problematic because it potentially exposes humans to harmful radiation effects. Again, this method can provide a more rapid way to investigate ingredients, but it is still somewhat limited because it requires humans and offers a limited number of testing sites (typically six). The use of skin explants and human skin tissue equivalents, such as MatTek’s Melanoderm (Ashland, MA), is a good way to test skin-brightening ingredients in vitro but requires a laboratory that can grow and handle human tissues or tissue equivalents, and the necessary spectropho- tometric analytical equipment to measure melanin expression. A much more rapid and convenient method has been developed that uses human- or mushroom-derived tyrosinase which is typically mixed with tyrosine and then analyzed using a UV-Visible spectropho- tometer to look for the development of melanin precursors (1). The method does allow for rapid screening, but it is a kinetic in situ assay that requires some skill in using the spec- trophotometer to gain meaningful data. Interestingly, the fact that mushrooms make a type of tyrosinase that can be purchased and used to analyze potential human skin-brightening ingredients raises an interesting point that the tyrosinase enzyme is highly conserved across humans, plants, fungi, and bacteria. In plants, tyrosinase is more often called poly- phenol oxidase (PPO) (2). The presence of PPO in fruits such as apples, pears, and bananas is the primary reason that, on opening the fruits, they begin to rapidly darken. Food browning is a very common problem for many commercial foods, which results in expensive disposal of vast amounts of food because the appearance of the food becomes undesirable for culinary consumption (3). Fruits are highly sensitive to the browning reaction, and popular fruits such as apples, pears, avocados, and bananas are especially sensitive to browning effects (4). A tremendous amount of food research has, therefore, been directed toward ways to minimize the browning of fruits and vegetables (5,6). Interestingly, from a chemical perspective, application of certain chemicals such as ascorbic acid, kojic acid, N-acetylcysteine, and 4-hexylresorcinol to sliced fruits to minimize the browning reaction is interesting because these popular food ingredients also turn out to be quite popular skin-brightening ingredients when used topically (7). Although the food science literature is replete with the use of such chemicals to minimize food browning, it has been noted that there is a very curious dearth in the literature on the possible use of fruit browning as a model examining melanin inhibition as it relates to skin. One article of interest by Zhao et al. examines the benefi ts of vanillyl cinnamate analogues on reducing the browning effects that occur in apples (8). Cinnamate derivatives are well known skin lightening ingredients. The fact that on slicing a fruit, such as apple, results in the immediate commencement of browning effects prompted us to examine more closely what, exactly, is happening inside the apple fruit that causes the browning to occur. In apples, an enzyme called PPO exists (9). This enzyme is called tyrosinase in fungi such as mushrooms and is functionally like human tyrosinase. In fact, PPO and tyrosinase are both categorized in the enzyme cataloguing system as EC 1.14.18.1. The enzyme is known as the rate-limiting catalyst in the conversion of phenols to quinones. In humans, the conversion of tyrosine to L-DOPA and then to dopaquinone are considered the rate-limiting steps in the skin-tanning response (10). Mushroom tyrosinase is commercially available