How to Predict AGEs Accumulation Slowdown Effect of a Cosmetic Ingredient? Two Steps In-Vitro System for Evaluating the Anti-AGE Impact of a New Blend

Advanced Glycation End-Products (AGEs), play a crucial part in advancing the process of cellular skin aging and its link to chronological age was re-assessed. AGEs accumulation alters cell structure and function of most types of skin cells, affecting skin’s mechanical and physiological properties, following the molecular transformations. Slowdown AGEs accumulation rate in skin, although a potent anti-aging strategy, is difficult and tricky. The lack of working methods for in-vivo and in-vitro measuring AGEs level compli-cates the evaluation and prediction of active ingredients’ ability to affect cellular AGEs accumulation. A two-step in-vitro systematic screening method is proposed and three different cosmetic active ingredients were selected for its demonstration, using BSA-Glucose and Collagen-Glucose predicting models. Candidates’ effects on AGEs accumulation were evaluated as standalone, and when formulated in a blend. Additionally, the potency of non-invasive au-to-fluorescence in-vivo measurement to detect AGEs levels among subjects of different ages was demonstrated. The results are presented in this work and the potential contribution of the proposed system to assist the desired inhibi-tion of AGEs accumulation in skin is discussed.


Introduction AGEs' Accumulation Phenomenon and the Methods for Its Measurement
The aging hypothesis of Advanced Glycation End-products (AGEs) was raised with the assertion that the accumulation of AGEs plays a crucial part in advancing the process of cellular aging [1] [2] [3]. AGEs are modified organic molecules, mainly proteins, lipids, and nucleic acids, that had passed non-enzymatic oxidizing glycation, with the active involvement of sugars [4]. Throughout life's passing years, AGEs gradually accumulate in cells, resulting in a chronic failure to preserve the balanced ratio of AGEs formation and their degradation [5]. Two main biochemistry pathways are involved in the cellular AGEs formation, one is based on the reaction of reducing sugars, especially aldoses and ketoses, and the other involves the impact of reactive carbonyl species with nucleophilic protein sites, catalyzed by metal ions [4].
Skin's chronic exposure to external oxidative stress, originated by UV radiation, cigarettes smoke, and urban pollution, was proven to dramatically boost the formation and accumulation of AGEs within skin cells. In addition, Intrinsic factors related to modern urban lifestyle, namely diets of highly processed and sugar-rich food, and little physical activity, are believed to increase AGEs accumulation [3] [6].
AGEs accumulation alters cell structure and function, affecting skin's mechanical and physiological properties, following the molecular transformations [2] [3]. Almost all skin cell types and components suffer from AGEs accumulation, keratinocytes, fibroblasts, immune cells, melanocytes, and extracellular matrix proteins (e.g. collagen and elastin) [7].
More than 20 different types of AGEs have been identified in the human body [8]. Due to their great heterogeneity, there is no one standard test for AGE measurement. Chromatographic, colorimetric, spectroscopic, mass spectroscopic and serological methods are used to determine AGEs content in biological samples [5] [9].
Simple in-vitro lab methods are needed, as a working tool, when searching ingredients, which affect AGEs accumulation in skin. The desired in-vitro method could predict the real glycation within human skin, on the one hand, and be easily performed, i.e., simple to apply, relatively short and not too expensive.
Our work investigates the prospect of achieving reduced AGEs accumulation within the skin, via active cosmetic ingredients, using two complementary in-vitro methods, BSA-Glucose and Collagen-Glucose for measuring AGEs accumulation, based on auto-fluorescence measurements [10] [11]. Additionally, we demonstrate the potency of non-invasive auto-fluorescence in-vivo measurement to detect AGEs levels among subjects of different ages. D. Cohen et al.

Study Population
All measurements were conducted on healthy human subjects from Hadassah Medical center, Israel (age ranged 26 -82) men and women, according to the Fitzpatrick scale with skin type II or III and with no visible skin abnormalities.
All studies were carried out following informed consent. The study was approved by the Institutional and Ministry of Health Ethics Committee.

Skin Auto-Fluorescence Measurements
In vivo Fluorescence spectroscopy was performed using a spectrofluorimeter (Fluoro-Max 4, JY Horiba, Edison, NJ, USA). The excitation source was a Xenon arc lamp. Measurements were performed by placing the fibreoptic probe in contact with the skin site of interest in human volunteers. Acquisition of emission spectra was the preferred method of measuring in vivo skin auto-fluorescence.
Serial fluorescence emission spectra were measured from healthy human volunteers (n = 11 -16). All measurements were performed on the abdomen.

Data Analysis
Correlations between age and fluorescent intensity values were calculated using Spearman's rho. A probability of p < 0.05 was considered statistically significant.

In-Vitro AGEs Accumulation Chemical Models
In-vitro AGEs accumulation was evaluated using two models: D. Cohen et al.

BSA-Glucose AGEs Accumulation Model
AGE-BSA was prepared by incubating a final concentration of 6 mg/ml BSA with 0.2 M D-glucose (Sigma Aldrich) in 50 mM PBS pH 7.4 in glass vials. Samples of tested ingredients were added to the glass vials, protected from light, and incubated at 37˚C for 14 days before auto-measurements.

Collagen-Glucose AGEs Accumulation Model
AGE-Collagen was prepared by incubating a final concentration of 1 mg/ml collagen (Sigma Aldrich) with 0.2 M D-glucose (Sigma Aldrich, U.S.A) in 50 mM PBS pH 7.4 in glass vials. Samples of tested ingredients were added to the glass vials, protected from light, and incubated at 37˚C for 14 days before auto-measurements.

Data Analysis
In-vivo measurements on human subjects: Correlations between age and fluorescent intensity values were calculated using Spearman's rho. A probability of p < 0.05 was considered statistically significant. In-vitro measurements: Four replicates experiments were analyzed unless otherwise noted. T-test assay was performed for statistical significance. A probability of p < 0.05 was considered statistically significant.

In-Vivo Evaluation on Human Subjects of AGEs Related Biomarkers Expression in Skin
Correlations between skin's content of AGE-affected vital molecules and chronological age were investigated on healthy volunteers aged 26 to 82 years. Special probes were attached to volunteers' abdomen skin. The contents of cross-linked collagen and elastin in the dermis were detected, using an auto-fluorescence intensity reader in specific emission spectra. As shown in Figure 1, a significant correlation between tested volunteers' age and the detected intensity of fluorescence was established in three tested AGE affected biomarkers, including pepsin and collagenase-digestible collagen cross-links (PDCCL, CDCCL), and cross-linked elastin (ECL) (p < 0.05 for PDCCL and CDCCL and p < 0.001 for ECL).

In-Vitro Evaluation of Protein-Glucose Models as Indicators for Slowing Down AGEs Accumulation, Following Treatments with Active Ingredients
Two in-vitro protein-glucose bio-reactivity models were used, one with BSA and the other with collagen as proteins. These BSA-Glucose and Collagen-Glucose models were served as acceleration models, enabling prediction of the possible slowdown effect of active ingredients in a few days, which generally takes years. As described in Methods, BSA, usually representing non-specific protein, was incubated with 0.2M glucose in pH-7.4 solution with Myrrh resin extract, Dead Sea Water (DSW), and Silybum extract or blended mixture.
In our work, three cosmetic active ingredients were collected and tested on BSA-Glucose and Collagen-Glucose accelerating models. Their AGEs accumulation was evaluated as standalone and when formulated in a blend, composed of three different ingredients.
Based on preliminary screening, Myrrh resin extract, Silybum extract, and Dead Sea water were selected and incubated with the BSA-Glucose model to measure AGEs accumulation. In addition, a blend comprised of three selected ingredients, was prepared and its effect on AGEs accumulation was tested using BSA-Glucose and Collagen-Glucose in-vitro models.
As shown in Figure 2  The blended mixture was tested with a collagen solution derived from bovine skin. The results showed that the blended mix reduced the AGEs accumulation by 81% compared to the untreated control, as illustrated in Figure 3.

Discussion
AGEs formation is a prolonged process, and therefore, long-lived proteins are its most vulnerable target molecules [3] [6]. In skin, a healthy and young appearance is highly dependent on the good functioning of these long-lived proteins, namely collagen, elastin, laminin, and fibronectin. The direct link between chronological age and cross-linked collagen and elastin, were re-assessed in this work. The accumulation of pepsin and collagenase-digestible collagen cross-links and cross-linked elastin were detected and found in a high correlation with the age of the volunteers, as presented in Figure 1. Reducing the formation rate of cross-linked collagen and elastin within the dermis is, therefore, one of the most valuable objectives, while aiming to affect age-related physiological skin changes.
Consequently, a proactive anti-aging strategy is proposed to slow down the unavoidable accumulation of AGEs within the skin. The AGEs accumulation slowdown strategy is associated with the protective and preventive approach, allocating most efforts to decelerate the process of biological skin aging, rather than fighting its undesired symptoms, when they appear.
Once synthesized within skin, AGE molecules tend to remain stable, and hence, the choice to enhance AGEs degradation ratio is not a real effective op- In order to develop skincare products, aiming to effectively treat AGEs accumulation, there is a need for a simple lab method, which could supply predicting data, enabling cosmetic researchers to screen various actives, and select the most promising ones. Furthermore, the desired method, is expected to help establish marketing anti AGEs claims, proving the AGEs attenuation effect of the tested D. Cohen et al.
active ingredient, as standalone, mixed in a blend with other actives, or when formulated in a final cosmetic product. Predicting AGEs' accumulation rate in the laboratory is a challenging task, since the process of their accumulation in skin is relatively slow, naturally involving chronic exposure, which takes months and years. Several accelerating lab methods have been proposed, using in-vitro protein-sugar biochemical models [5].
In our work, following early screening, three cosmetic active ingredients were selected and tested: Silybum marianum, Myrrh resin and Dead Sea water. Several scientific studies report that the plant Silybum marianum, which contains the active ingredient, silymarin, has a proven anti-AGEs capability [13]. Silymarin was found as an effective molecule to reduce the level of glycated albumin in plasma of diabetic rats and decrease the content of total level of AGEs Myrrh is a bush of desert flora, known to synthesize secondary metabolites, enabling its survival in the extreme conditions of the desert. Furthermore, Commiphora myrrha extract had presented anti-diabetic capabilities, when added as a food supplement to induced diabetic rats [16].
Dead Sea water, containing high levels of minerals, in addition to established therapeutic impacts to heal skin diseases, was proven for its contribution to improving skin smoothness, skin moisturizing level, and protecting skin against premature aging [17] [18].
To predict the capacity to slowdown the process of AGEs accumulation, the three selected cosmetic ingredients were tested, using two protein-glucose accelerating models, described in Methods, suggesting a distinct capability for each tested ingredient to contribute to the desired effect. A complex of the three selected ingredients had been blended, and its effect on AGEs accumulation rate was predicted, using both BSA-Glucose and Collagen-Glucose in-vitro models.
Based on the results, partially described in Figure 2 and Figure 3, an anti-AGEs patent was applied [19].
Due to its relative simplicity, performing BSA-Glucose tests is proposed as an initial filtering tool, enabling to perform a quick and inexpensive wide screening.
This is because Albumin is very abundand and represents general and water-soluble proteins. Collagen-Glucose is suggested as a complementary step, to be performed on a more precised list of candidates. We suppose that the Collagen-Glucose model could be considered as better representing the actual AGEs accumulation process that occurs in dermis, with the resulting crossed linked collagen.

Conclusions
The combination of both BSA-glucose and collagen-glucose methods is suggested as a two steps complementary system, starting with a wide screening step, on simple BSA-Glucose method and followed by more relevant to skin Collagen-Glucose model, allowing to predict anti-AGEs activity.

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Further work is needed to better understand the biological mode of activation of AGEs accumulation, and the involved steps, which may support slowing its rate within the skin.