Evaluating the Revitalizing and Moisturizing Fruit-based active for skin vitality: scientific insights into the moisturizing power of apple juice

Despite the increasing utilization of Pyrus malus extracts in cosmetic formulations, comprehensive studies that correlate in vitro bioactivity with in vivo skin hydration effects remain limited. Therefore, this study aims to bridge this knowledge gap by integrating in vitro assays on human keratinocytes with in vivo clinical evaluations employing instrumental skin hydration measurements. The hypothesis is that the revitalizing cellular effects observed in vitro will correspond with enhanced skin hydration in vivo, thereby demonstrating a measurable and scientifically substantiated benefit for cosmetic application.

The study objectives were:

• To evaluate the metabolic and revitalising potential of PYRUS MALUS JUICE on human keratinocytes (HaCaT cells).
• To assess the short- and long-term moisturizing effects of a cosmetic formulation containing this extract, using validated biophysical tools in human volunteers.
• To establish a scientific bridge between in vitro mechanistic activity and in vivo cosmetic functionality.

Cell Line and Culture Conditions

Human immortalised keratinocytes (HaCaT) were selected as an established model for epidermal biology. Cells were cultured under standard conditions in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% foetal bovine serum (FBS), 1% penicillin-streptomycin, and 1% glutamine, maintained at 37°C in a 5% CO₂ humidified incubator.

For testing, cells were seeded into 96-well plates at a density of 1×10⁴ cells/well and allowed to adhere for 24 hours. Treatment involved exposing cells to serial dilutions of the active ingredient (PYRUS MALUS JUICE) at concentrations ranging from 0.01% to 1% v/v in serum-free medium. Glycerin (commonly used in cosmetic products as a standard hydrating agent) served as a positive control.

Tested substances:

• PYRUS MALUS JUICE, Glycerin, Sodium Benzoate, Potassium Sorbate
• Glycerin (positive control)

Viability and Metabolic Activity Assay

After 24 hours of incubation with the test substances, cellular metabolic activity was evaluated using the MTS assay (a colorimetric method for assessing mitochondrial function). Absorbance was measured at 490 nm, with values directly proportional to the number of viable, metabolically active cells.

Statistical Analysis

All experiments were conducted in triplicate. Statistical significance was assessed using unpaired two-tailed t-tests to compare treated samples to control (glycerin and untreated), with significance set at p < 0.05.

Part II: In Vivo Evaluation of Skin Hydration

Study Design and Ethics

The study tested a cosmetic formulation containing the active fruit-derived ingredient against a placebo (identical base formulation without the active). Applications were made to the volar forearms of each subject in a split-body design to eliminate inter-subject variability.

The study involved 10 healthy volunteers with an average age of 47.8 years. Informed consent was obtained from all participants in accordance with the Declaration of Helsinki. The study was designed as a controlled, comparative, randomized trial with double-arm treatment (active vs placebo on opposite forearms).

Formulation details for the in vivo efficacy test:

• Active product: Aqua, Pyrus Malus Juice, Glycerin, Carbomer, Sodium Hydroxide, Sodium Ethylparaben, Sodium Propylparaben
• Placebo product: Aqua, Carbomer, Sodium Hydroxide, Sodium Ethylparaben, Sodium Propylparaben

Inclusion and Exclusion Criteria

Inclusion Criteria:

• Caucasian subjects of either sex, aged 18–70.
• General good health and ability to comply with study procedures.
• Avoidance of UV exposure and tanning beds during the study.

Exclusion Criteria:

• Pregnancy, breastfeeding, known skin sensitivities.
• Ongoing treatment with systemic or topical agents affecting skin.
• Dermatological disorders (e.g., eczema, psoriasis).
• Participation in similar trials within the last 30 days.

Treatment and Application Protocol

A 3×3 cm area on each forearm was marked for treatment. A fixed amount of 2 mg/cm² of either the active product or placebo was applied post-baseline measurement. Applications were performed twice daily (morning and evening) for 30 days.

Instrumental Assessment

Surface Skin Hydration

Measured with Corneometer® CM 825 (Courage & Khazaka): Measures superficial stratum corneum hydration by quantifying electrical capacitance (expressed in arbitrary units from 0 to 120).

Deep Skin Hydration

Measured with MoistureMeterEpiD (Delfin Technologies): Non-invasive dielectric resonance-based probe that evaluates deep tissue hydration (up to 1mm) by converting the dielectric constant into percentage water content.

Conditions for Measurement Time Points

All measurements were performed in a climate-controlled room (24 ± 2°C; 50 ± 10% RH). Volunteers refrained from washing forearms for 2 hours and from applying any skincare product for at least 12 hours before testing.

• T0: Baseline (before application)
• T1: 30 minutes post-application (single dose)
• T2: Day 30 (after twice-daily application for one month)

All tested concentrations of PYRUS MALUS JUICE induced a dose-dependent increase in cell metabolic activity compared to both untreated and glycerin-treated controls.

• At 0.01%, viability increased to ~105.6% vs. ~102.9% for glycerin (p = 0.0198)
• At 0.05%, viability reached ~106.8% vs. ~103.7% (p = 0.0029)
• At 0.1%, although an upward trend was observed (~107.5%), the difference from glycerin (~104.6%) was not statistically significant (p = 0.126)
• At 0.5% and 1%, viability increased further (~107.9% and ~109.8%, respectively), with both comparisons showing statistical significance compared to glycerin (p = 0.0344 and p = 0.0374, respectively)

These results confirm that PYRUS MALUS JUICE significantly enhances mitochondrial activity, indicating improved cellular vitality and potential regenerative support.

Notably, the study was conducted in serum-free conditions, emphasising the intrinsic bioactivity of the test substance without interference from external growth factors. The use of glycerin as a comparator enabled us to distinguish the specific contribution of the apple-derived bioactives from the solvent vehicle, strengthening the conclusion that PYRUS MALUS JUICE displays biological activity.

The dose-response relationship observed further supports the hypothesis of receptor-mediated or concentration-dependent uptake mechanisms, potentially involving phytohormones or secondary metabolites known to interact with mammalian cells.

 In Vivo Hydration Measurements (7), (8), (9), (10), (11), (12), (13)

Superficial Hydration (Corneometer)

T1 (30 min post-application):

• • • • • Active Product: Increased from 43.7 CU to 50.6 CU (+15.8%), p < 0.05.
        • Placebo: Decreased from 43.2 CU to 39.7 CU (−8.1%), p < 0.05.
        • Comparison: Significant difference between active and placebo groups (p = 0.001).

A statistically significant increase (improvement) in the basal values of skin hydration was evidenced after 30 minutes from the single application of the PYRUS MALUS JUICE 5% product.

A statistically significant decrease (worsening) was registered in the area treated with the placebo

After Single Application (30 Minutes)

• Active Product: Increased from 43.2% to 45.0% (+1.8%), p < 0.05.
• Placebo: Decreased from 44.1% to 42.3% (−1.8%), p < 0.05.
• Comparison: Statistically significant (p < 0.01).

The statistical comparison between the variations recorded at 30 minutes from the single application evidenced a statistically significant difference between the product PYRUS MALUS JUICE 5% and the placebo.

• Active Product: Increased from 43.2% to 50.9% (+7.7%), p < 0.01.
• Placebo: Increased modestly from 44.1% to 46.9% (+2.8%), p > 0.05.
• Comparison: Statistically significant (p = 0.01).

A statistically significant increase (improvement) in the basal values of skin deep hydration was evidenced after 30 days of repeated application of the PYRUS MALUS JUICE 5% product.

No significant variation was registered in the area treated with the placebo