Skin Care

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Introduction

Skin aging is a complex process involving both biological and molecular changes. Several phenotypes found in aged skin tissue are triggered by the presence of damaged molecules and organelles in the cells. Studies indicate that their proliferation is controlled by the autophagy process, which can result from three distinct mechanisms: microautophagy, chaperone-mediated autophagy (CMA), and macroautophagy. Of the three mechanisms mentioned, macroautophagy, through the lysosomes that inhabit human cells, is the most studied mechanism [8].

One of the responses to cellular aging is the accumulation of lipofuscin protein aggregates, lysosomes with excess cellular residues, due to reduced cellular recycling efficiency. Lipofuscin is a yellowish-brown pigment found in many cells of the body, such as keratinocytes, fibroblasts, neurons, myocytes, and hepatocytes. Such accumulation in the skin results in the appearance of age spots, different from each other in shape, size, color, and degree of projection. They are most frequently found on the body where the skin has greatest exposure to the sun, such as the face or back of the hands [9,10].

The homogeneity of skin tone, depending on the age of the individual, is also threatened by the accumulation of carbonyl proteins, which are generated by the attack of reactive oxygen species in the skin tissue [11]. 

Carbonylated proteins are products of the reaction between amino groups and reactive aldehyde compounds produced from lipid peroxidation, which is initiated by reactive oxygen species. Thus, in the skin, carbonyl proteins are more frequently detected in places exposed to the sun in the elderly, which contributes to the change in skin color to dark yellow due to extrinsic aging [12]. Carbonyl proteins also impact the proteasome system, as low-molecular-weight carbonyl proteins are degraded by proteasome activity. However, high-molecular-weight proteins are resistant to degradation and, therefore, inhibit the action of proteasomes [13]. The proteasome is a sophisticated and extremely efficient protein complex that acts through selective hydrolysis along with the ubiquitin molecule in signaling the degradation of numerous target proteins. Just like the autophagic process, proteasome activity catalyzes biological reactions for the digestion of damaged or malformed proteins [14].

The search for stimulators of recycling systems involves the study of numerous compounds, including plant extracts due to their rich chemical composition which, naturally, entails a wide possibility of biological functions. In this sense, our research group deepened its studies on the extract of Myrothamnus flabellifolia, the “resurrection plant” well known in traditional African medicine due to its ability to survive extreme dehydration for many years, as well as on the extract of coffee (Coffea arabica), an important source of antioxidant in the human diet, obtained from upcycling coffee seedcake. The phytochemical profile of the Myrothamnus flabellifolius 

leaf extract is composed of flavonoids, anthocyanins, alkaloids, steroids, terpenoids, triterpenes, cardiac glycosides, saponins, phlobatanins, tannins, polyphenols, and sugars, including trehalose, a compound that became widely known for its ability to induce cellular autophagy and mitigate diseases related to protein aggregation [15].Coffee extract, on the other hand, is rich in phenolic compounds and their derivatives (such as chlorogenic acids), alkaloids (especially caffeine), diterpenoid alcohols, carbohydrates, lipids, and volatile and heterocyclic compounds [16]. Lipofuscin is a peroxidized lipid-protein aggregate that accumulates during aging or under conditions of oxidative stress that can occur as a result of decreased antioxidant defenses or loss of repair functions. In other words, the presence of antioxidants is essential in protecting against oxidative stress [17].

Materials & Methods

Expression of mTOR and Parkin genes associated with Autophagy
Human, neonatal dermal fibroblast cultures (HDFn C-004- 5C, Cascade Biologics™, CA, USA) were incubated with 0.313 mg/mL (non-cytotoxic concentration) of MCE for 24 hours. Cell lysates were collected and submitted to RNA extraction using Purelink™ (Life Technologies) and quantified by spectrometry using NanoDrop Lite (Thermo Fisher Scientific), followed by RT-PCR analysis in StepOnePlus equipment (Applied Biosystems). Gene expression was performed using commercial kits (TaqManR Assays Gene Expression) for the following genes: mTOR (Mechanistic Target of Rapamycin Kinase) Hs00234508_m1; PARK2 (Parkinson protein 2) Hs01038322_m1 (Applied Biosystems). The test was performed using TaqManR RNA-to-CT™ 1-Step Kit (Applied Biosystems), under experimental conditions of 48°C for 30 minutes for reverse transcription, 95°C for 10 minutes for activation of AmpliTaq GoldR DNA Polymerase, followed by 50 cycles at 94°C for 15 seconds and at 60°C for 1 minute for denaturation and rewarming, respectively. The relative amount of mRNA was calculated by a mathematical model for relative quantification and real-time gene expression analysis and by the 2−ÄÄCT method.

Ubiquitin-Proteasome System Activity
Normal human dermal fibroblasts (NHDF) were cultured in NHDF culture medium (Sigma-Aldrich/Merck KGaA) overnight. Then, the culture medium was replaced by a fresh culture medium containing betulinic acid at 20 μg/ml (positive control) and MCE at 0.0375 mg/ml. An untreated cell group was cultured in a regular growth medium and incubated under the same experimental conditions (control group). Cell viability was previously verified by staining with Trypan Blue solution (Bio-Rad Laboratories Inc). After 24 h of incubation with each product, chymotrypsin-like proteasome activity was measured kinetically in all samples for 60 minutes. Measurements were taken every 2.5 min. The luminescence generated from proteasome cleavage of a peptide substrate added to the samples was quantified using Proteasome-Glo™ chymotrypsin- like cell-based assay kit (Promega Corp).

Lipofuscin Expression
Lipofuscin expression was performed by immunofluorescence in human, neonatal dermal fibroblast cultures (HDFn C-004- 5C, Cascade Biologics™, CA, USA) using antibody-enhanced detection of senescent cell reagent (SenTraGor™, Arriani Pharmaceuticals, Attica, Greece) and a Leica-DM 6000B fluorescence optical microscope coupled with a 2.8 MP Leica DFC7000T camera. Images were captured using LAS software (Leica Application Package v.4.12) and qualitatively evaluated by the intensity of fluorescence emitted by labeling with specific antibodies.

Clinical Trial
A randomized, single-blind, placebo-controlled clinical trial was performed with 34 subjects for 56 days. One group used a cream containing 2% w/w of MCE and the other group used a placebo, applying the solution to the face and hands twice a day. Age spots on the hand were evaluated through the analysis of standardized images captured with a Canon EOS 60D Digital Camera. Facial spots were evaluated using images captured with Visia CR equipment (Canfield Scientific, Inc.). The captured images were used to evaluate skin uniformity and luminosity, comparing the images taken before and after product use. A specific software program was used to analyze the images and the ‘total surface of spots’ and ‘Haralick Homogeneity Index’ parameters were used.

as a result, autophagy is induced. Jung et al. (2010) state that among the numerous proteins that regulate autophagy, mTOR (target of rapamycin) is the key component that maintains homeostasis between the autophagic process in response to the cell’s physiological conditions and environmental stress [20]. In other words, the absence of mTOR is key for the induction of cellular autophagy. As a result of autophagy induction, ATG13 and ULK1/2 proteins play a role in the biogenesis of phagophore and autophagosome [21], in addition to RB1CC1, a protein found in the initial and later events of autophagosome formation [22]. LC3 is a key protein in the regulation of autophagy and autophagosome biogenesis after becoming membrane-conjugated through interactions with ATG7 and other autophagic effectors [23]. At the same time, ATG9 plays a fundamental role in organizing the structure of the phagophore. In addition to the proteins involved in the formation of the autophagosome, it is also important to detect proteins that tag the compounds to be degraded. According to Narendra et al. (2008), PARK2, also known as the Parkin protein, is responsible for monitoring and selecting damaged mitochondria, in addition to mediating the process of sending them to autophagosomes and their subsequent degradation [24].

consequently there is less degradation of oxidized proteins and an increase in protein aggregation, which induces cellular degeneration and several diseases associated with aging [29]. That said, it is only possible to mitigate excessive “cellular waste” with high levels and effectiveness of these two systems: autophagy and proteasome. 

In addition to modulating the gene expression of autophagy-related markers, in vitro treatment of Normal Human Dermal Fibroblasts (NHDF) with 0.0375 mg/ml of MCE for 24 hours indicated that the ingredient was able to significantly stimulate proteasome activity (chymotrypsin-like proteasome activity) by 45% compared to the untreated control (Figure 3).

improvement (p < 0.001) in facial skin tone homogeneity after 28 days, when compared to baseline. Placebo showed an 11% improvement (p < 0.05). As for spot size, it was observed a 66% (p < 0.001) improvement with the use of the ingredient when compared to baseline. In the case of spots on the hands, the improvement is more significant after 56 days, being 63% (p < 0.05) for the ingredient compared to baseline. The placebo showed no significant improvement. As for spot size, there was an improvement of 26% (p < 0.05) compared to baseline. The placebo showed no significant improvement.