An upcycled fraction of Melaleuca alternifolia essential oil regenerates the skin through the skin melatonin pathway and improves sleep quality

Sleep is one of the most important factors affecting overall health. During the night, the skin repairs damage caused by daily stresses. Melatonin plays a key role in this process. Toxins are removed, and cellular repair and growth hormone production are increased. Inter alia, this also decreases signs of intrinsic aging.

or mortality. 2Lack of sleep can also degrade skin quality, such that sleep-deprived people are rated as less attractive and less healthy than fully rested individuals. 3After sleep deprivation, individuals were perceived as having darker circles under the eyes, paler skin, and more wrinkles. 3Attributes such as rough, dull, and dry skin are often observed.Importantly, sleep deprivation increases the production of glucocorticoids, which are thought to impair the integrity of the skin barrier. 4Other studies show that lack of sleep increases the secretion of stress hormones such as cortisol and the neuropeptide substance P. 5 Similarly, it has been observed that an increase of inflammatory cytokines and tumor necrosis factorα, observed after sleep deprivation, impedes skin barrier function recovery. 6Together, these effects are thought to induce skin dehydration, trans-epidermal water loss (TEWL), and a loss of skin elasticity and skin tone, thereby exacerbating the signs of intrinsic aging. 7Therefore, improving sleep might be a solution to improve skin condition.
Released by the pineal gland, melatonin is a critical regulator of the sleep cycle, through circadian clock modulation.The melatonin biosynthesis pathway is found in diverse tissues, including human skin, where it plays a key role in the repair process. 8A broad range of biological functions of melatonin has been identified, including antioxidancy, effecting decreases of ROS production and increases in the expression of antioxidant enzymes; and anti-inflammation, acting through NF-κB or cyclooxygenase-2. 9Therefore, the melatonin pathway may represent an interesting target to delay the skin aging process.
On this basis, we considered a dual approach to improving skin regeneration, by acting on both skin and sleep.To this end, we used an innovative upcycled fraction of Melaleuca alternifolia (FMA) essential oil, chosen for its volatile compound makeup.These molecules possess demonstrated efficacy in improving sleep quality and cognitive function. 10They are also present in skincare products, based on their reported health benefits 11 and antimicrobial properties. 12Tea tree oil (TTO) is well known for its topical antimicrobial, antifungal, and antiinflammatory effects. 13These are mainly attributed to monoterpenes and terpenoid alcohols, including terpinen-4-ol, γ-terpinene, and αterpinene.In order to minimize these antibacterial properties and instead valorize TTO for its anti-aging properties, a fraction of TTO devoid of terpenes but rich in sesquiterpenes was selected.The antiinflammatory and antioxidant properties of the latter family of molecules are well described. 14This fraction of M. Alternifolia essential oil showed good activity in the skin but also improved key parameters of sleep quality.The combination of these two effects results in a significant reduction in key indicators of skin fatigue in human participants.The identification of FMA and classical M. alternifolia molecules was achieved based on their mass spectra and an internal library of compounds.

| Measurement of reactive oxygen species
NHEKs were seeded at 1•10 4 cells/well in 96-well plates for 1 day before the medium was removed, and cells were washed with PBS.

| Measurement of Interleukin-8 (IL-8) secretion
NHDFs were seeded at 2•10 4 cells/well in a cell culture medium for 1 day before the medium was removed.Cells were then treated with IL1α (Sigma-Aldrich, #I2778) as an inducer of IL-8 production, in the presence of 0.001, 0.0001, 0.00001% FMA, or 0.05 μg/mL hydrocortisone (Sigma-Aldrich, #D1756) as a positive control, vs. untreated negative control, in a serum-free medium.After 24 h, the supernatant was collected, and IL-8 was quantified by ELISA (R&D System, #DY208) following the manufacturer's protocol.

| Ex vivo study
As described by the Bioethics group of the General Director Services of the French Research and Innovation Ministry (registered under no.DC-2008-542), the use of human skin from surgical waste as of May 5, 2010 is authorized.The skin samples were used with the consent of patients according to the Declaration of Helsinki.
Human skin explants from an abdominal skin biopsy from esthetic surgery procedures performed on a 46-year-old Caucasian woman with phototype II-III (according to the Fitzpatrick skin phototype classification) were used with the donor's consent.The explants were placed in a BEM (BIO-EC's Explants Medium) culture medium at 37°C under 5% CO 2 and 95% humidity.After 2 days in culture, the explant was stressed using a Vibert Lourmat RMX 3 W UV simulator, using a 13.5 J/cm 2 UVA dose and a 0.15 J/cm 2 UVB dose, vs. unirradiated control, before adding FMA at 2.5 × 10 −3 % in a Carbopol emulsion vs. vehicle control (Carbopol vehicle alone).UV radiation was used in this model to mimic skin stress by sleep deprivation, which is known to be characterized by high oxidative stress and inflammatory marker levels.After 24 h, explants were fixed in RNAlater for genomic study or in formalin solution for histological processing.
For genomic analysis, RNA was extracted from skin explants using Promega's ReliaPrep™ RNA Tissue Miniprep System (Promega, #Z6010) before reverse transcription (RT) into cDNA using iScript (Bio-Rad).Gene expressions were then evaluated.For quantification, the number of cycles was normalized based on reference gene B2M.A gene was considered induced if its expression showed a significant increase greater than or equal to 1.5 compared with the control (fold-change ≥1.5).Likewise, a gene was considered repressed if it showed a significant reduction of expression relative to the control (fold-change ≤0.65).For each gene of interest, values were calculated between treatment samples in triplicate compared to the matched explant control samples in triplicate for each kinetic time point.
For immunostaining analysis, samples were dehydrated and impregnated in paraffin using a Leica PEARL dehydration automat.After a 5μm-thick section was made and mounted on Superfrost® histological glass slides, the frozen samples were cut

| Clinical trial
Piper nigrum essential oil, not known to induce sleep, was used as a placebo control in the sleep study.FMA or Piper nigrum essential oil was incorporated at 2.5.10 −3 % into a simple face cream formulation, along with a corresponding unscented control (Table 1).At this low concentration, the formulations present no odor perceptible by volunteers.

| In vivo study-Measurement of sleep quality
Thirty-two healthy participants (16 males, 22-56 years old, median age 41.5 years) with self-reported mild sleep disturbances caused by lifestyle factors, but without a clinical diagnosis of a sleep disorder, were recruited for this study (Table 2).The Athens Insomnia Scale (AIS), a self-assessment psychometric instrument designed for quantifying sleep difficulties, was included to further allow us to screen out nondiagnosed clinical participants. 15 3).Participants applied the creams at bedtime in their natural sleep environment and were instructed to wash their faces with an unscented face wash that was provided to them before application.
The study lasted Sunday night through Friday morning for three consecutive weeks, excluding Friday and Saturday nights.After each night of the study, participants completed daily questionnaires and tracked their sleep using the SleepScore Max device (SleepScore Labs, Carlsbad, CA), a noncontact monitoring device that uses radio frequencies to track and stage sleep.The SSL technology platform has been validated against Polysomnography (PSG), the gold standard of sleep measurement. 16All self-reporting data were collected using Compusense®, a web-based data collection software.

Objective nightly sleep data were analyzed on JMP (version 15)
statistical software using multilevel regression analyses, considering the nested structure of the data (nights within subjects).An alpha level of 0.05 was used in a two-tailed test.The following regression model was used: SleepParameter ij = Const 0i + β*TestPeriod ij coded as 0 for the observations during baseline or control use and 1 for nights during active scent use.The primary comparisons were between the active and unscented control as well as between the active and placebo control.

| Measurement of skin quality
This study was realized in observance of Good Clinical Practices FRAP is based on the formation of the ferrous (FeII) form via the reduction of a ferric-tripyridyltriazine (FeIII-TPTZ) complex. 17

| Chemical composition of the fraction of Melaleuca alternifolia essential oil (FMA)
The chemical composition of the extract was elucidated by GC/MS and GC/FID.Thirty-six components representing 77.85% of the total extract were identified (Table 4).The analysis revealed that this fraction of FMA essential oil contains mainly sesquiterpenes, with 21.86% of delta-cadinene as a major component.Moreover, four other molecules were recorded: viridiflorene (17.3%), aromadendrene (8.4%), globulol (7.3%), and alloaromadendrene (3.5%), all also sesquiterpenes.Only 0.63% of 4-terpineol, the main component of classical TTO, was detected.
As can be noted, the molecular composition of this FMA is markedly different from the classical TTO analyzed before TA B L E 2 Demographic information for N = 32 participants included in the study.

| FMA essential oil decreases oxidative stress and inflammation
The antioxidant and anti-inflammatory properties of the FMA essential oil were evaluated in an in vitro model.
As can be observed in Figure 1A, a strong increase in ROS production was observed after pyocyanin induction in NHEK culture.
This shows FMA strongly reduced inflammation.

| FMA essential oil improves skin regeneration
Skin explants were cultured for 2 days before being stressed with UV and treated with FMA essential oil for 24 h.Gene expression was analyzed by RT-qPCR.
To study the potential effect of this essential oil on the melatonin pathway, four different markers were measured: ASMTL is an enzyme implicated in melatonin synthesis; RORα is a nuclear melatonin receptor and/or is modulated by melatonin 18 ; SIRT3 is a mitochondrial sirtuin that melatonin interacts with to neutralize oxidative stress 19 ; SLC15A1 (PEPT1), localized in the mitochondrial membrane, facilitates melatonin transportation into mitochondria.At the protein level, a significant increase in CAT and OGG1 levels (Figure 2C,D) was confirmed, with relative staining of +37% (***p < 0.001) and +17% (*p < 0.05), respectively.

As shown in
These results show the potential of the active to deliver regenerative properties after daily stress such as UV irradiation.

| FMA essential oil improves sleep quality
FMA significantly improved two parameters, sleep duration, and deep sleep duration compared to the unscented control (Table 5).All other parameters measured did not show significant changes versus controls.
Moreover, no significant changes were observed with the Piper nigrum essential oil (placebo control) compared with the unscented control.
FMA essential oil significantly increased total sleep duration (+2.5%, p < 0.05) compared with the unscented control.Participants on average slept for 6.7 h with FMA vs. 6.5 h with unscented control (Figure 3A).unscented control and placebo control, respectively (Figure 3B).

| FMA essential oil delivers skin antifatigue effect
FMA essential oil was tested in vivo on a panel of volunteers with a stressful lifestyle and signs of skin fatigue.The FRAP test was used to determine the skin's antioxidant potential, by measuring the total reducing power of the biological matrix, giving an indirect index of the skin's capacity to resist oxidative damage. 17Bearing out the data produced in vitro and ex vivo, the results showed that daily application of a cream containing FMA (2.5.10 −3 %) for 28 days significantly improved the skin's antioxidant potential (+17%, *p < 0.05) compared with the unscented placebo (Figure 4A).
An anti-aging effect was shown via a reduction of the Sa parameter by −5.7% (***p < 0.001) and −7.8% (***p < 0.001) in the crow's feet area after 7 days and 28 days, respectively (Figure 4B).This result reflects a reduction of wrinkle depth and an improvement in skin smoothness.In addition, an improvement in skin firmness was observed as an increase (+5.6% and +8.8% at D7 and D28, respectively, vs. unscented control) in the R0 parameter, indicating the capacity of the skin to resist deformation (Figure 4C).

| DISCUSS ION
Sleep disturbances have become a pervasive and prominent problem in modern 24/7 society.In addition to its effect on physical and

A
by-product of an Australian Melaleuca alternifolia essential oil (Geo Tag: −31.359, 152.823), obtained by standard fractional distillation of an FMA essential oil, was used.GC/MS analysis of this fraction's molecular composition was performed using an Agilent 6890 Series Gas Chromatograph coupled to an Agilent 5973 Mass Selective Detector (MSD, Agilent Technologies, Santa Clara, California, United States), equipped with an automatic liquid sampler and an HP-1MS capillary column (Agilent Technologies).The oven was heated to 60°C for 10 min, then the temperature was increased to 300°C at 2°C/min, and kept at 300°C for 10 min (total run time 140 min).Ion source temperature was 230°C; transfer-line temperature 260°C; ionization energy 70 eV; He (1.0 mL/min) carrier gas; injector temperature 250°C, with split mode injection (1:50).GC-FID analysis was performed with an Agilent 6890 Apparatus (Agilent Technologies) equipped with a flame-ionization detector (FID), an automatic liquid sampler, and the same column as above.The oven was heated at 60°C for 10 min, then temperature was increased to 300°C at 2°C/min, and finally held at 300°C for 10 min (total run time 140 min).Injector temperature was 250°C; detector temperature 300°C; H 2 (1.0 mL/min) carrier gas; split mode injection (1:50), injected volume 0.1 mL.The O 2 , H 2 , and makeup gas flows of the FID were 350, 35, and 20 mL/min, respectively.

into 7 -
μm-thick sections with a Leica CM 3050 cryostat before being mounted on Superfrost® plus silanized glass slides.A microscope (Leica DMLB, Olympus BX6) was used for microscopical observations, and images were recorded using a numeric DP74 Olympus camera with cellSens storing software.Immunostaining was performed on formalin-fixed, paraffin-embedded (FFPE) skin sections with a monoclonal anti-OGG-1 antibody (Invitrogen, #PA1-31402, 1:600) or catalase antibody (Novus biologicals, ref.NBP2-00492, 1:200).After 1 h of incubation at room temperature with a Vectastain Kit Vector avidin/biotin amplifier system, the section was revealed by VIP, a substrate of peroxidase giving a violet signal when oxidized (Vector laboratories, #SK-4600).Staining intensity in the whole epidermidis and in the stratum corneum (as Hunter L*, a*, b* values) was determined by image analysis.Each marker was quantified based on the stain intensity for each treatment, compared with the stressed untreated condition.Image processing was performed using the Fiji software.
Only individuals with an AIS score of less than 8 were included in this study.All study procedures including participant recruitment were reviewed and approved by the Western Institutional Review Board (WIRB).All materials were reviewed and approved by the Global Regulatory Affairs (GRA) department at International Flavors and Fragrances Ltd. (IFF), for safe use with human subjects in face cream application at the specified dosages.Participants used a different sample each week, following an incontext, randomized, counterbalanced design where the unscented control sample was always used during the second week and the two scented samples were counterbalanced across participants (Table

(
International Recommendations ICH Topic E6, CPMP/ICH/135/95 of May 1, 1996, European Parliament and Council Guideline 2001/20/CE-DOCE of May 1, 2001).Forty-four healthy volunteers, distinct from those in the sleep study, were selected, aged 30-65, presenting redness-prone skin and dark circles, and complaining of sleep disturbances.Volunteers were distributed into two groups of 22 individuals in a randomized manner, and given one of two separate face cream samples: one active product containing FMA and the unscented placebo.Volunteers applied the products on the face every day before going to bed, for 28 days.Skin wrinkles were quantified using a Primos 3D device (GFMesstechnik Canfield, Parsippany, NJ), measuring the Sa parameter.Skin slackening was evaluated with a Cutometer® MPA 580 (Courage-Khazaka, Köln, Germany), measuring the R0 parameter.The color of dark circles was assessed by image analysis (expressed as ITA°, calculated from Hunter L*, a*, b* values) of pictures acquired by VISIA-CR (Canfield, Parsippany, NJ) before (D0) and after 7 days (D7) and 28 days (D28) of topical product application.To measure skin antioxidant potential, a ferric-reducing ability (FRAP) assay was performed at Day 0 (D0) and Day 28 (D28) of topical product application on skin stripping samples taken with Corneofix® (Courage-Khazaka, Köln, Germany).

1
FMA essential oil decreases oxidative stress and inflammation on NHEKs and NHDFs, respectively.(A) FMA essential oil decreases the release of reactive oxygen species (ROS) after pyocyanin induction.(B) FMA essential oil decreases IL-8 production after IL-1α induction.Data are presented as mean ± SEM values (n = 3).Statistical analysis was performed using ANOVA (*p < 0.05, **p < 0.01, and ***p < 0.001).F I G U R E 2 Effect of FMA essential oil (2.5.10 −3 %) on stress markers versus UV-stressed condition in skin explants.(A) Expression of mRNA of melatonin-associated gene expression.(B) Expression of mRNA of stress markers from melatonin pathway.(C) Protein staining and (D) relative intensity for Catalase (CAT) and OGG1.Data are presented as mean ± SEM values.Statistical analysis was performed using t-test ( # p ≤ 0.1, *p ≤ 0.05, **p ≤ 0.01, and ***p ≤ 0.001).

Furthermore, participants spent
more time in the deep sleep stage with FMA compared with both unscented control (+7.3%, p < 0.05) and placebo control (+10.5%,p < 0.05).Participants spent 79 min in deep sleep with FMA compared to 73 min and 71 min with mental well-being, poor sleep also impacts skin quality.Sleep is an important physiological mechanism to fight skin aging through the regulation of systemic inflammation, oxidative stress, or DNA damage.In the work presented here, we explored the capacity of a unique upcycled FMA essential oil formulated into a cream with no perceptible smell to improve sleep quality via the inhalation of volatile compounds.We also measured the effect of the same product on the skin.To the best of our knowledge, these effects have not been investigated in conjunction to date, with researchers generally focusing separately on sleep or on skin benefits.Our in vitro and ex vivo results indicate anti-inflammatory andantioxidant properties of the FMA essential oil.These properties can be attributed to its rich sesquiterpene composition, these molecules having been shown to decrease inflammation and oxidative stress.14Specifically, our ex vivo study showed the regenerative effect of FMA following UV stress.A decrease in pro-inflammatory F I G U R E 3 In vivo efficacy of FMA essential oil (2.5.10 −3 %) on sleep quality.Measurement of total sleep duration (A), measurement of deep sleep duration (B).Statistical analysis was performed using ANOVA (*p ≤ 0.05, **p ≤ 0.01, and ***p ≤ 0.001, ****p ≤ 0.0001).F I G U R E 4 In vivo efficacy of FMA essential oil (2.5 × 10 −3 %).Skin antioxidant potential using FRAP test after skin stripping (A), skin smoothness as Sa parameter (B), skin firmness as R0 parameter (C), dark circles lightening as ITA° parameter (D).Statistical analysis was performed using ANOVA (*p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, and ****p ≤ 0.0001).