Cosmetic composition

Abstract

Cosmetic compositions comprising gardenia fruit extract are provided.

Description

Technical Field

[0001] The present invention relates to a cosmetic composition containing gardenia fruit extract.

Background Art

[0002] In modern society, digital technology has become more common than ever, leading to an increase in exposure to blue light. Blue light is a type of high-energy light and is part of the visible light spectrum, having wavelengths between 400 and 495 nm. Typical blue light sources include mobile phones, computers, tablets, TVs, and lights. Much of the exposure results from light-emitting diodes (LEDs). People are increasingly being exposed to blue light through everyday technology. A 2015 Pew Research Center study found that 68% of U.S. adults own a smartphone and 45% own a tablet. The study also found that technology ownership levels vary by age; 86% of Americans ages 18 - 29 and 83% of those ages 30 - 49 own a smartphone. In contrast, computer ownership rates are lower for older Americans.

[0003] Blue light is thought to be able to affect the skin and damage cells. Moreover, Dong et al. demonstrated that blue light disrupts the circadian rhythm and affects people's sleep quality. Melatonin is a well-known sleep-related hormone that is naturally secreted from our organs (brain and skin) on a daily cycle. The peak of its production occurs at night and plays an important role in our ability to fall asleep and the quality of our sleep. In addition, melatonin also acts as a powerful anti-aging agent due to its strong antioxidant properties and by triggering a biological defense cascade through binding to the melatonin membrane receptor MT1R. Exposure to blue light disrupts the melatonin production rhythm, leading to skin aging (loss of antioxidant defenses and damage to mitochondria) and perturbations to sleep-related functions (difficulty falling asleep, frequent awakenings during the night, and morning fatigue). This results in premature skin aging, making the skin more vulnerable to external attacks and unable to recover during the body's rest phase. [Overview of the Initiative]

[0004] Therefore, an object of the present invention is to provide a cosmetic means for preventing negative effects caused by exposure to blue light. This problem is solved by the cosmetic surfactant and cosmetic composition of the present invention.

[0005] In a first aspect, the present invention provides a cosmetic surfactant comprising gardenia fruit extract. Gardenia (also known as Cape jasmine or Dandan) is an evergreen flowering plant belonging to the Rubiaceae family. It originates in Asia and is most commonly found growing wild in Vietnam, southern China, South Korea, Taiwan, Japan, Myanmar, India, and Bangladesh. There are approximately 250 species of gardenia worldwide. Throughout this disclosure, the term “Gardenia” means to cover all species of the genus Gardenia, including Gardenia jasminoides, Gardenia angustifolia, Gardenia augusta, Gardenia florida, Gardenia grandiflora, Gardenia radicans, Gardenia longisepala, Gardenia maruba, and Gardenia pictorum, and in particular Gardenia jasminoides J. Ellis.

[0006] Traditionally, gardenia fruit has been used orally in folk remedies to treat inflammation, headaches, edema, fever, liver damage, and hypertension. The fragrant flowers are also used to flavor tea and are sometimes eaten raw as a delicacy, pickled, or even preserved in honey. Today, the gardenia plant is mainly used as a food additive, dye, and ornamental plant. Gardenia fruits have a red color due to natural carotenoid pigments. In particular, they contain crocetin, crocin, and other crocetin esters. As used throughout this disclosure, the term "crocin" encompasses aglycone crocetin, its digenthiobioside derivative crocin, as well as other crocetin monoglucosides or diglucosides, and any other crocetin derivatives having an 8,8'-diapocarotenoic acid core.

[0007] Crocetin is a naturally occurring apocarotenoid dicarboxylic acid found in the flowers of Crocus sativus L. and the fruits of Gardenia jasminoides. It has the following structure: [ka] Crocin is a diester formed from the disaccharide genthiobiose and the dicarboxylic acid crocetin. It has the following structure: [ka]

[0008] Crocin is one of the main components in gardenia fruit extract. Crocin is particularly interesting because it is one of the few water-soluble carotenoids, which makes it suitable as a coloring agent for food applications. One of the main drawbacks of crocin is its notorious instability, particularly its decomposition under the influence of light, decreased pH, and increasing temperature. This fact makes the cosmetic use of gardenia fruit extract and crocin in general difficult, if not impossible. Therefore, it is important to stabilize the gardenia fruit extract within the cosmetic surfactant.

[0009] As a result, the cosmetic surfactant of the present invention further contains a solvent, which enables the stabilization of the extract, and in particular the crocin contained therein. Surprisingly, it has been found that the stability of crocin can be improved by adding a eutectic solvent with a pH of at least 5. This finding has been confirmed by long-term stability studies (see Example 2 below). Therefore, the solvent used in the cosmetic surfactant of the present invention is a eutectic solvent having a pH of at least 5. The eutectic solvent also enables good solubilization and stabilization of the gardenia fruit extract, particularly the crocin contained therein.

[0010] Eutectic solvents are generally known. They consist of multiple components, typically each within a specific concentration range, and each component has a lower melting point than the individual components. The cosmetic surfactant of this invention is intended for topical application. The cosmetic surfactants of the present invention have been found to possess several advantageous effects, which have been demonstrated in both in vitro and clinical studies (see Example 2 below). In particular, it was found to have anti-aging properties and to improve sleep quality, especially in people who are frequently exposed to blue light.

[0011] Without being bound by theory, the cosmetic surfactant of the present invention is thought to protect melatonin production in the skin when the skin is exposed to digital stress, i.e., blue light, thereby preventing premature skin aging. It has been found that crocin present in the cosmetic surfactant of the present invention not only protects the natural skin melatonin cycle but can also be converted to crocetin by the skin microbiome. Based on microbiome studies (see Example 3 below), crocin is completely converted to crocetin within approximately 210 hours during culture.

[0012] Without being bound by theory, the applicant hypothesizes that crocetin can bind to the major melatonin receptor MT1R, while crocin cannot. This hypothesis is supported by molecular modeling studies based on the crystal structures of MT1R, crocin, crocetin, and melatonin obtained from the Protein Databank (www.rcsb.org). Calculations of molecular interactions between each of the other molecules and MT1R showed that crocetin has a positive affinity score of 3.6232, which is very close to that of melatonin (5.6553), while crocin has a negative affinity score of -36.4141. Therefore, it is believed that crocetin can trigger the cutaneous melatonin receptor (MT1). Consequently, thanks to its activation by the skin microbiome, the cosmetic surfactant of the present invention can actively participate in the skin's defense and repair mechanisms.

[0013] Clinical trials (see Example 8 below) have shown that the cosmetic surfactant of the present invention reverses the visible signs of aging (21% reduction in wrinkle count compared to placebo). Therefore, it is optimal for preventing and treating early skin aging. This finding was also confirmed by panelists' self-assessments, during which 80% of volunteers reported increased skin moisture (compared to 40% for placebo), and 75% of volunteers reported smoother skin (compared to 45% for placebo).

[0014] More specifically, the cosmetic active agent of the present invention makes it possible to protect the mitochondrial network and cell diffusion in vitro (see Examples 5 and 6 below). Exposure to blue light has been shown to damage the mitochondrial network. However, in the presence of the cosmetic active agent of the present invention, the network is less fragmented. Cell diffusion is also significantly affected by blue light. However, as before, the cosmetic active agent of the present invention makes it possible to prevent this effect.

[0015] The cosmetic active agent of the present invention makes it possible to reduce the content of oxidized proteins ex vivo (see Example 7 below). After exposure to blue light, the content of oxidized proteins in human skin explants increases significantly. This effect can be reversed by the cosmetic active agent of the present invention. Moreover, the cosmetic active agent of the present invention has been found to preserve the melatonin cycle in vitro (see Example 4 below), which ensures nocturnal melatonin release and, thus, protects the skin from digital stress.

[0016] The cosmetic active agent of the present invention has also been found to have a positive effect on the quality of sleep, thereby contributing to overall health. In particular, topical application of the cosmetic active agent has been found to reduce the number of awakenings during the night and increase the ease of falling asleep (see the clinical study in Example 8 below). The cosmetic active agent is thought to act as a phytomelatonin-like molecule and activate biological mechanisms related to the circadian rhythm.

[0017] In one aspect of the present invention, the eutectic solvent has a pH of at least 5.5, more preferably at least 6, and most preferably at least 7. Thanks to the higher pH, the stabilization of crocin in the gardenia fruit extract is improved. However, for skin care applications, it is desirable that the pH is not too high, for example, 10 or less, more preferably 9 or less, and most preferably 8 or less.

[0018] In one aspect of the present invention, the constituents of the eutectic solvent are of natural origin. Eutectic solvents formed from constituents of natural origin are generally known and are typically described as "natural deep eutectic solvents", or NaDES. More preferably, all of the constituents of the eutectic solvent are 100% of natural origin according to ISO 16128.

[0019] In one aspect of the present invention, the eutectic solvent comprises betaine, glycerol and water. This eutectic solvent has been found to be particularly well-suited for the solubilization and stabilization of gardenia fruit extracts. Betaine can also act as a humectant in skin care applications. Eutectic solvents based on glycerol, betaine and water are generally known (for example, from WO2016 / 162703). Apart from avoiding the drawbacks of common synthetic organic solvents such as inherent toxicity, high volatility, or lack of recyclability, they also have the advantage of being of natural origin. For this reason, they are also described as "natural deep eutectic solvents", or NaDES.

[0020] In one aspect of the present invention, the eutectic solvent comprises from about 30 to about 40 wt% betaine, from about 35 to about 45 wt% glycerol, and from about 20 to about 30 wt% water, more preferably about 35 wt% betaine, about 40 wt% glycerol, and about 25 wt% water. In a particularly preferred aspect, the eutectic solvent consists of 34.6 wt% betaine, 40.4 wt% glycerol, and 25.0 wt% water. This eutectic solvent has a pH of about 7.45.

[0021] Without being bound by theory, the applicant believes that in the presence of a eutectic solvent based on betaine, glycerol and water, transesterification occurs by replacing at least one of the gentiobioside moieties of crocin with a glycerol-betaine moiety, forming the following adducts: [ka] This hypothesis is supported by 2D-NMR analysis (NOESY and HSQC). This derivative is also intended to be included within the term "crosin" as defined above.

[0022] In an alternative embodiment, the eutectic solvent is a mixture of pentylene glycol, betaine, and water. In particular, the eutectic solvent may contain about 35 to about 50 wt% pentylene glycol, about 25 to about 35 wt% betaine, and about 20 to about 35 wt% water, more preferably about 44 wt% pentylene glycol, about 29 wt% betaine, and about 28 wt% water. In a particularly preferred embodiment, the eutectic solvent consists of 43.7 wt% pentylene glycol, 28.8 wt% betaine, and 27.5 wt% water. This eutectic solvent has a pH of about 6.85. The concentration of gardenia fruit extract in the cosmetic surfactant of the present invention should be selected so as to achieve the above-mentioned advantageous effects.

[0023] In one aspect of the present invention, the concentration of gardenia fruit extract in the cosmetic surfactant is about 0.01 to about 10 wt%, more preferably about 0.05 to about 1 wt%, and most preferably about 0.1 wt%. To ensure the activity of the cosmetic surfactant of the present invention, a relatively high content of crocin in the gardenia fruit extract is preferred. As can be seen from the analysis of commercially available gardenia fruit extracts (see Example 1 below), the crocin content can vary from supplier to supplier.

[0024] In one aspect of the present invention, the gardenia fruit extract contains at least 1 wt% crocin, more preferably at least 10 wt% crocin, and most preferably at least 25 wt% crocin. In a further aspect, the present invention provides a cosmetic composition comprising the cosmetic surfactant and cosmetically acceptable excipients of the present invention. The cosmetic composition of the present invention is intended for topical application.

[0025] Any excipients commonly used in the preparation of cosmetic preparations for use on human skin can be used in this invention. Suitable excipients include, but are not limited to, components that may affect the functional properties, skin penetration, and bioavailability of the gardenia fruit extract. More specifically, they include liquids such as water, oil, or surfactants, which are of petroleum, animal, plant, or synthetic origin, such as, but are not limited to, peanut oil, soybean oil, mineral oil, sesame oil, castor oil, polysorbate, sorbitan ester, ether sulfate, sulfate, betaine, glycoside, maltoside, fatty alcohol, nonoquinol, poloxamer, polyoxyethylene, polyethylene glycol, dextrose, glycerol, digitonin, and the like.

[0026] Formulations for topical application to the skin may take any physical form. For example, cosmetic compositions, and in particular skincare compositions, may take the form of liposome compositions, mixed liposomes, oleosomes, niosomes, etosomes, milliparticles, microparticles, nanoparticles and solid-lipid nanoparticles, vesicles, micelles, surfactant mixed micelles, surfactant-phospholipid mixed micelles, myrispheres, microspheres and nanospheres, lipospheres, millicapsules, microcapsules and nanocapsules, as well as microemulsions and nanoemulsions, which can be added to achieve higher penetration of gardenia fruit extract.

[0027] Cosmetic compositions, and in particular skincare compositions, may be manufactured in any solid, liquid, or semi-solid form useful for topical or transdermal application. Thus, these preparations for topical or transdermal application include, but are not limited to, creams, numerous emulsions, for example, oil-in-water and / or silicone emulsions, water-in-oil and / or silicone emulsions, water / oil / water or water / silicone / water emulsions, and oil / water / oil or silicone / water / silicone emulsions, micro-emulsions, emulsions and / or solutions, liquid crystals, anhydrous compositions, aqueous dispersions, oils, milks, balsams, foams, aqueous or oily lotions, aqueous or oily gels, creams, hydro-alcohol solutions, hydro-glycol solutions, hydrogels, liniments, ceramics, soaps, face masks, serums, polysaccharide films, ointments, mousses, pomades, pastes, powders, bars, pencils, sprays or aerosols (sprays), which include leave-on and rinse-off formulations.

[0028] Therefore, the present invention also provides skincare compositions, and in particular anti-aging skincare compositions. The advantageous effects of the cosmetic surfactant of the present invention and the cosmetic composition thereof are described in detail above. Certain embodiments of cosmetic surfactants are also advantageously applicable to cosmetic compositions.

[0029] In a further aspect, the present invention also provides a method for reducing signs of aging in the skin, which comprises the step of topically applying the cosmetic surfactant or cosmetic composition of the present invention to the skin, particularly the skin of the face. The advantageous effects are described in detail above and are further supported by the examples below. In a further aspect, the present invention also provides a method for protecting skin from oxidative stress, which comprises the step of topically applying the cosmetic surfactant or cosmetic composition of the present invention to skin, particularly facial skin. The advantageous effects are described in detail above and are further supported by the examples below.

[0030] In a further aspect, the present invention also provides a method for protecting skin from the effects of blue light, which comprises the step of topically applying the cosmetic surfactant or cosmetic composition of the present invention to skin, particularly facial skin. The advantageous effects are described in detail above and are further supported by the examples below. In a further aspect, the present invention also provides a non-therapeutic method for protecting an individual's melatonin cycle, which comprises the step of topically applying the cosmetic activator or cosmetic composition of the present invention to the skin, particularly the skin of the face. The advantageous effects are described in detail above and are further supported by the examples below.

[0031] In a further aspect, the present invention also provides a non-therapeutic method for improving an individual's sleep, which comprises the step of topically applying the cosmetic surfactant or cosmetic composition of the present invention to the skin, particularly the skin of the face. The advantageous effects are described in detail above and are further supported by the examples below. In a further aspect, the present invention also relates to the use of gardenia fruit extract to improve an individual's sleep. In particular, the present invention also relates to the use of the present invention's cosmetic surfactants or cosmetic compositions for improving an individual's sleep. [Brief explanation of the drawing]

[0032] [Figure 1] Figure 1 shows melatonin release at different time points, with the x-axis indicating the time elapsed since the last FCS shock. [Figure 2] Figure 2 shows the results of the network segmentation analysis. [Modes for carrying out the invention]

[0033] The present invention can be further described by the following non-limiting examples: Example 1: Gardenia fruit extract Gardenia fruit extract is commercially available from multiple suppliers. For this study, commercially available extracts were obtained from Indfrag Bioscience Private Limited ("Gardenia Florida Extract," batch numbers GFP-ROE-14002 and GFP-ROE-19001; plants cultivated in Anhui Province, China; according to the supplier's specifications, the extract was obtained from Gardenia Florida fruit by water extraction, fractionation, concentration, drying, and powdering) and Yunnan Rainbow Bio-tech. Corp., Ltd ("Gardenia Yellow Powder," batch number ZT180201, received July 6, 2018; plants cultivated in Guangxi Province, China; according to the supplier's specifications, the extract was obtained from gardenia fruit (Gardenia Jasminoides Ellis) by extraction, filtration, purification, concentration, and spray drying). Both products are in powder form and range in color from yellowish to reddish or brownish.

[0034] The authenticity of the products was confirmed by macroscopic and microscopic identification of dried fruit samples obtained from each supplier, HPTLC, DNA analysis (provided by an external provider: Tru-ID), and HPLC. The composition of gardenia fruit extracts from both suppliers was determined by further analysis using HPLC-UV and LC-TOF. It was found that the sample from Indfraq contained approximately 10 wt% crocin, while that from Yunnan Rainbow contained approximately 30 wt%.

[0035] Commercially available gardenia fruit extract was further processed as follows: - Crushing - Extract with water (Yunnan Rainbow) or methanol / water 75:25 (Indfrag). - filtration - Adsorption and desorption with ethanol (70%) - Concentrate - Spray drying, with maltodextrin (for Yunnan Rainbow only) The solid material obtained in this way was then dissolved in a desired solvent(s).

[0036] Example 2: Stability study The stability of gardenia fruit extract in different solvents was tested under different conditions. long term stability Long-term stability was tested at room temperature and 40°C in the following two different eutectic solvents: (a) a mixture of betaine, lactic acid, and water, and (b) a mixture of betaine, glycerol, and water. The pH of the solvents alone was 3.27 for (a) and 7.45 for (b), respectively. Gardenia fruit extracts from both Yunnan Rainbow (a1) and (b1) and Indfrag (a2) and (b2) were tested. The eutectic solvent was prepared by mixing the components at 50°C. 0.1 wt% of gardenia fruit extract (in powder form) was then added, and the resulting composition was heated to 80°C for 1 hour.

[0037] The samples were stored at room temperature and 40°C for 3 months each. pH, Gardner index, and λ were measured. max The stability of the sample was evaluated by measuring the optical density (in the range of 250-600 nm). The results are presented in the table below: [Table 1] As can be seen from the above, the gardenia fruit extract was clearly more stable in the betaine / glycerol / water mixture compared to the betaine / lactic acid / water mixture.

[0038] Samples for the study of sunlight ("Suntest") and elevated temperatures Gardenia fruit extract (in powder form; derived from Yunnan Rainbow) was dissolved in (c) water and (d) a eutectic solvent (35:40:25 w / w) formed from betaine, glycerol, and water. The eutectic solvent (d) was prepared by mixing the constituent components at 50°C until they were completely homogeneous. 0.1 wt% of gardenia fruit extract was added to each solvent, and the resulting compositions were heated to 80°C for 1 hour.

[0039] At T0, the color of the sample was identified by CIELAB: [Table 2]

[0040] Sunlight ("Suntest") The sample was subjected to 450 W / m². 2 Irradiation was performed over a 24-hour period with light at 300-800 nm. pH, color change (dE2000), Gardner index, and optical density at 440 nm (λ) were measured at 8, 12, 18, and 24 hours. max The stability of the sample was evaluated by measuring ). The results are presented in the table below: [Table 3] As can be seen from the above, gardenia fruit extract is completely stable in a eutectic solvent formed of betaine / glycerol / water, while the optical density of the sample in water alone decreases, accompanied by a significant change in color.

[0041] Rising temperature The samples were heated to 100°C and 120°C for 24 hours each. After 8, 12, 18, and 24 hours, pH, color change (dE2000), Gardner index, and optical density at 440 nm (λ) were measured. max The stability of the sample was evaluated by measuring ). The results are presented in the table below: [Table 4]

[0042] Similar to the previous study, the samples in water were less stable than those in the eutectic solvent of betaine, glycerol, and water. In particular, the samples in water became turbid, and at later time points, even precipitates formed. For both solvents, the color visibly changed over time, but the change was more pronounced in the samples in water.

[0043] Example 3: Conversion of crocin to crocetin by the skin microbiome (in vitro) Skin microbiome samples were collected from seven volunteers using sterile gauze impregnated with NaCl in five areas: forehead, cheeks, nose, neck, and forearms. By combining all samples, a representative skin microbiome composition was obtained. The microbiome thus obtained was cultured in liquid medium (buffered HT medium) at 30°C in the presence of crocin. The supernatant was collected periodically and analyzed by HPLC-MS to detect crocin and crocetin.

[0044] The results are shown in the table below: [Table 5] As can be seen from the above, the microbiome present on the skin surface converts crocin to crocetin within a few days.

[0045] Example 4: Evaluation of melatonin release (in vitro) The effect of blue light on melatonin release was investigated in in vitro co-culture models of human sensory neurons and primary keratinocytes.

[0046] cell culture The sensory neurons originated from hiPS (human induced pluripotent stem cell) cells themselves, which were derived from human fibroblasts. hiPS cells were seeded at a density of 250,000 cells per well into 6-well plates coated with a thin layer of Matrigel® (Corning, reference: 354277, batch: 72005017) in a differentiation medium consisting of DMEM-F12 (Panbiotech, reference: P04-41450, batch: 2730618) supplemented with 10% Knockout Serum Replacement (KSR, Life Technologies, reference: 10828028, batch: 1896527), 0.1 μM retinoic acid (Sigma, reference: R4643, batch: SLBF3638V), a cocktail of central differentiation pathway inhibitors, and 1% penicillin-streptomycin antibiotic (PS, Panbiotech, reference: P06-07100, batch: 7631018). The cells were maintained in culture at 37°C and 5% CO2 for 6 days. The culture medium was changed every 2 days.

[0047] After 6 days of culture, cells were cleaved at 37°C for 10 minutes using Accutase (Sigma Aldrich, reference: A6964, batch: SLBT9789V). The reaction was stopped by adding culture medium. The cell suspension was centrifuged at 1200 rpm for 5 minutes. Cell viability was determined by cell counting using trypan blue, and the cells were seeded at a density of 100,000 cells per well into 24-well plates coated with a thin layer of Matrigel® in the same differentiation medium used previously.

[0048] After 9 days of culture, the culture medium was replaced with a maturation medium for sensory neurons. This maturation medium consisted of DMEM-F12 supplemented with 1% N2 (Life Technologies, ci: 11520536, batch: 2004543), 10 ng / mL BDNF (PanBiotech, ci: CB-1115002, batch: 051861), 10 ng / mL GDNF (PeproTech, ci: 450-10, batch: H170806), 10 ng / mL NT3 (PeproTech, ci: 450-03, batch: H171010), 10 ng / mL NGF (Sigma, ci: N1408, batch: SLBW7063), and 1% antibiotic PS. Cells were maintained in culture at 37°C and 5% CO2. The culture medium was changed every 2-3 days.

[0049] After 14 days of culture, keratinocytes were added to a plate on top of a layer of differentiated hiPS cells. Keratinocytes derived from skin explants from a 30-year-old donor were previously amplified on a cycle in keratinocyte growth medium (Lonza, reference: 192152, batch 723883) before being dissociated by trypsin treatment and freezing. These keratinocytes were thawed, and cell viability was determined by cell counting. The keratinocytes were seeded at 30,000 cells per well in a culture medium consisting of 2 / 3 sensory nerve medium and 1 / 3 keratinocyte growth medium. The cells were maintained in culture at 37°C and 5% CO2. The culture medium was changed every 2-3 days.

[0050] A cyclical protocol was developed based on the use of glutamate and temperature increase ("daytime" phase). In addition, shocks with a medium containing 50% FCS were performed to synchronize the cell cycle (Ramanathan et al., Monitoring Cell-autonomous Circadian Clock Rhythms of Gene Expression Using Luciferase Bioluminescence Reporters, Journal of Visualized Experiments, 2012, 67: p.1-9; Buhr et al., Temperature as a universal resetting cue for mammalian circadian oscillators, Science, 2010, 330(6002): 379-385; Balsalobre et al., A Serum Shock Induces Circadian Gene Expression in Mammalian Tissue Culture Cells, Cell, 1998, 93: 929-937).

[0051] From this day forward, the cultures were placed under conditions simulating the daytime phase (10 nM glutamate and a temperature of 39.5°C) for 8 hours per day. On day 15 of co-culture, FCS-rich medium (50% FCS and 50% medium, 2 / 3 maturation medium for sensory nerves and 1 / 3 growth medium for keratinocytes) was incubated in the presence of cells for the first two hours of the "daytime" phase. This FCS shock was applied to all cells (those alternated between daytime and nighttime, and controls without alternation).

[0052] On day 17 of co-culture, further FCS shock was applied to the cells. Gardenia fruit extract (from Indfrag) was diluted to 0.004% (w / v) in the culture medium and applied to the cells during the "daytime" phase. On the same day, 30 minutes before the "nighttime" phase, the co-cultured cells were exposed to blue light. From this day forward, 0.004% (w / v) gardenia fruit extract was incubated with each change of culture medium. The cultures were maintained under the same conditions and treated with blue light 30 minutes before the "nighttime" phase each day.

[0053] For analysis, culture supernatant samples were taken 30 minutes before the “night” phase and 2, 5, and 8 hours after the shift to the “night” phase, and stored at -80°C. This procedure was performed on days 17, 18 (i.e., 24 hours after the last FCS shock), and 19 (i.e., 48 hours after the last FCS shock). These culture supernatant samples were thawed, and ELISA assays were performed to dose the amount of melatonin released (BlueGene, see ABIN511419). After 20 days of culture, the cells were washed once with PBS, and an MTT test was performed to verify cell viability.

[0054] statistical analysis The results were statistically analyzed using the Kruskal-Wallis ANOVA followed by the Mann-Whitney U nonparametric test. Significance of the results is indicated as * for p<0.05, ** for p<0.01, and *** for p<0.001.

[0055] result Figure 1 shows melatonin release at different time points, with the x-axis indicating the time elapsed since the last FCS shock. In the untreated control group ("Untreated" in Figure 1), cellular synchronization induced a melatonin release cycle after 24 hours. Melatonin levels were significantly increased at 2, 5, and 8 hours compared to the level 30 minutes prior to the "daytime" phase.

[0056] In parallel, to replicate pre-sleep digital device exposure, blue light stress was induced at the end of each “daytime” phase in a separate co-culture. This condition (“Control Blue Light” in Figure 1) was found to exhibit a different response: on day 18, i.e., 24 hours after the final FCS shock, there was no increase in melatonin release. This difference was statistically significant. These results suggest that blue light stress disrupted or delayed the melatonin release cycle. Under the same culture conditions, a third co-culture was treated with 0.004% gardenia fruit extract and blue light ("0.004% activity" in Figure 1). This treatment was found to result in melatonin release very similar to that in the untreated control.

[0057] The results of the melatonin release study are summarized in the table below: [Table 6]

[0058] As is clearly observed, both the untreated condition ("untreated") and those treated with 0.004% gardenia fruit extract and blue light ("0.004% activity") showed significant increases at 2, 5, and 8 hours into the night, i.e., 26, 29, and 32 hours after the last FCS shock, respectively. On day 2 after treatment (day 19), there was a smaller but still noticeable increase in melatonin. Samples treated with blue light but not with the extract ("control blue light") had significantly lower melatonin release than both of the other conditions. In conclusion, it was found that a 0.004% gardenia fruit extract can protect cells from the effects of blue light exposure. In particular, it allows for the preservation of melatonin release levels and their cycle.

[0059] Example 5: Mitochondrial network analysis and cell diffusion (in vitro) - Study 1 Mitochondrial networks and cell diffusion are both biomarkers for cellular senescence. Cell culture and treatment Human dermal primary fibroblasts from a 57-year-old female donor were thawed and amplified in flasks for several days in CnT-Prime culture medium specifically for epithelial cell culture (CellnTEC). 24 hours before starting the assay, the cells were divided into three groups: - Group 1 was left untreated; - Group 2 was treated with 0.002% (w / v) gardenia fruit extract (from Yunnan rainbow; diluted in culture medium); and - Group 3 was treated with 0.004% (w / v) gardenia fruit extract (from Yunnan rainbow; diluted in culture medium).

[0060] Next, the cells were loaded with Mitotracker Green dye for 15 minutes. The cells were washed with PBS, detached, and seeded at 2000 cells / well in 10% serum medium into CYTOO plates with an oversized Y micropattern. After 1.5 hours, once the cells had attached to and spread along the micropattern, the culture medium was replaced with a medium containing less serum and further containing the same concentration of gardenia fruit extract previously applied to each group. The cells were then incubated at 37°C with 5% CO2 for 2 hours.

[0061] Two hours after the procedure, the cells were given a dose of 20 J / cm², corresponding to a 1-month (28-day) exposure to a screen at a distance of 10 cm. 2 The device was irradiated with 447nm LEDS (reference Kingbright KA-3529AQB25Z4S) for 1 hour. Hoechst was added to each well for 15 minutes to stain the nuclei. The medium was replaced with fresh medium to wash off the Hoechst, and the cells were incubated with activity in CnT-prime culture medium.

[0062] Image acquisition Live imaging was performed using a Leica microscope. At the end of the live imaging, the cells were fixed and F-actin was stained with Phalloidin 555. Images were acquired on the Operetta HCS platform from Perkin Elmer.

[0063] Network Analysis Once a mitochondrial network was detected, the total length of all fibers in the single-cell network was calculated and averaged across all single cells from the same well to determine the "total network length." The mitochondrial network can be divided into groups of continuously connected fibers: this basic unit is called a "tree." The number of trees per network, as well as their total lengths, were averaged across all single cells detected in each well. Each tree is divided into “branches” separated at each end by either a branching point or an endpoint. These branches were characterized by measuring their average and maximum lengths across the entire network of single cells (“average branch length”).

[0064] Cell diffusion analysis Dedicated image analysis was performed to detect single cells on micropatterns and measure their area. 1800 μm 2 We counted properly spread cells that had a larger surface area than the specified area. statistical analysis The results were statistically analyzed using standard ANOVA with multiple comparisons. Significance of the results is indicated as * for p<0.05, ** for p<0.01, and *** for p<0.001.

[0065] Results: Mitochondrial network Literature has indicated that blue light exposure may lead to cellular oxidation and ultimately affect the mitochondrial network (Rascalou et al., Mitochondrial damage and cytoskeleton reorganization in human dermal fibroblasts exposed to artificial visible light similar to screen-emitted light, Journal of Dermatological Science, 2018, 91: 195-205). The more fragmented the network becomes, the more disrupted the cell becomes.

[0066] In the first part of the study, the mitochondrial network was analyzed through network segmentation analysis. The results are shown in Figure 2. The untreated condition was found to show a clear and distinct mitochondrial network (Figure 2a). After exposure to blue light, the network became fragmented and scattered as a result of oxidative stress (Figure 2b). In the presence of 0.002% gardenia fruit extract, the network appeared to be less fragmented (Figure 2c). This protective effect was even more pronounced in the presence of 0.004% gardenia fruit extract (Figure 2d). This network was then numerically segmented for quantitative analysis. Various parameters, including the total network length, number of trees, number of branches, and their average length, were evaluated.

[0067] The results are summarized in the table below: [Table 7] As can be seen from the above, blue light stress led to a significant reduction in the total length of the mitochondrial network. This result was confirmed by data from the literature. However, in the presence of gardenia fruit extract, significant protection of the network was observed, at +68% (p<0.001) and +98% (p<0.001), respectively.

[0068] The total length of the network is naturally tied to the number of trees and branches: as the network shrinks, the number of trees and branches increases, and the average length of each decreases. As expected, blue light stress was found to induce a significant increase in the number of trees and branches, as well as a significant decrease in the mean tree length and branch length. Similar to before, gardenia fruit extract provided significant protection, reducing the number of trees by 58% and 73% (p<0.001), and the number of branches by 60% and 68% (p<0.001), respectively.

[0069] Results: Cell diffusion In the second part of the study, cell diffusion and their area were analyzed. This diffusion is closely related to the stress that occurs in cells: when cells are stressed, their cytoplasm contracts. The results are summarized in the table below: [Table 8] It was found that the percentage of enlarged cells was significantly reduced after exposure to blue light. The same was true for mean cell area. However, in the presence of gardenia fruit extract, significant protection was observed, increasing the percentage of enlarged cells by +165% (p<0.001) and +149% (p<0.001) at 0.002% and 0.004% concentrations, respectively. Cell area was improved by +183% and +175% at 0.002% and 0.004% extract concentrations, respectively (p<0.001).

[0070] Example 6: Mitochondrial network analysis (in vitro) - Study 2 The mitochondrial network analysis described in Example 5 was repeated for gardenia fruit extract at 0.0015% in a natural deep eutectic solvent (NaDES) consisting of 35 wt% betaine, 40 wt% glycerol, and 25 wt% water, versus NaDES alone. The results are summarized in the table below: [Table 9]

[0071] As can be seen from the above, gardenia fruit extract in NaDES enables significant protection of the network: it allowed for a -38% reduction in the number of trees and a +66% increase in average tree length compared to untreated light-stress conditions. The same trend was observed for branch parameters: gardenia fruit extract in NaDES allowed for a -14% reduction in the number of branches and a 19% increase in average branch length. On the other hand, natural deep eutectic solvents alone do not provide protection against blue light stress.

[0072] Example 7: Protein oxidation analysis (ex vivo) Protein oxidation is another biomarker for cellular senescence. Culture and treatment Twelve human skin explants with an average diameter of 12 mm (±1 mm) were obtained from a 35-year-old Caucasian woman (reference: P2159-AB35, phototype III) and prepared via abdominal reconstruction. The explants were kept viable in BEM culture medium (BIO-EC Explants Medium) in a humid 5% CO2 environment at 37°C.

[0073] The explants were assigned to four groups as follows (3 explants each): - Untreated control: Explants exposed to light rhythms - Blue light control: Explants exposed to photorhythmics and blue light stress - 0.002% gardenia fruit extract (from Indfrag): Explants exposed to photorhythmic and blue light stress, with topical application of 0.002% gardenia fruit extract. - 0.004% Gardenia fruit extract: Explants exposed to photorhythmic and blue light stress, with topical application of 0.004% Gardenia fruit extract. Gardenia fruit extract was prepared by diluting commercially available raw material (Yunnan Rainbow) in phosphate-buffered saline (PBS) at the respective concentrations (w / v).

[0074] From day 0 to day 4 of the study, skin explants were exposed to photocycling and blue light irradiation according to the following pattern, in order to mimic circadian rhythms: - 7 PM to 7 AM (12 hours): Exposure of explants to daylight using a SlimStyle W021 / 02 lamp (Dayvia) that exhibits an emission spectrum close to that of solar radiation. Skin explants were kept in BEM culture medium during daylight exposure. - 7:00 AM to 10:00 AM (3 hours): 63.75 J / cm³ in 1 mL of HBSS medium using a Solarbox® device (BioEC). 2 Exposure of explants to a dose of blue light. Untreated control explants were kept in 1 ml of HBSS in the dark for the entire duration of blue light exposure. At the end of exposure, all explants were returned to 2 ml of BEM medium. - 10:00 AM to 7:00 PM (9 hours): Skin explants were kept in BEM culture medium in the dark.

[0075] For the last two groups, gardenia fruit extract was used on explants (2 mg / cm³). 2 The solution was applied topically based on 2 μl per 100°C and spread using a small spatula on days 1, 2, 3, and 4 (before exposure to blue light). Untreated control explants received no treatment other than replacement of the culture medium. Half of the culture medium (1.2 ml per well) was replaced at the end of each dark phase of the photocycle on days 1, 2, and 3. On the fourth day, immediately after the final blue light irradiation, three explants were collected from each condition and cut into two parts. One part was fixed in buffered formalin, and the other part was frozen at -80°C.

[0076] Immunostaining of oxidized proteins Oxidized proteins were stained on frozen sections after pre-incubation with DNPH (2,4-dinitrophenylhydrazine, Millipore, cf. 90448) and incubation at 37°C for 1 hour with anti-DNP antibody (Millipore, cf. 90451) diluted 1:250 in PBS, 0.3% BSA, using a biotin / streptavidin amplification system, and identified with VIP (Vector, cf. SK-4600), a purple substrate for peroxidase. Immunostaining was performed manually and evaluated by microscopic observation.

[0077] Image analysis: Quantification of color indicators The staining intensity of oxidized proteins was quantified using two open-source optical imaging software programs. Microscope images (in JPEG format) were opened in GIMP - GNU Image Manipulation Program. Strong to light pink signals corresponding to the stains were selected, copied, pasted into a new image, and saved as a JPEG file, which contained only the selected stains. This image was then opened using the ImageJ program. A region within the dermis was selected for analysis. A histogram of the section was then created, and the total number of pixels in the image was separated into 255 color categories spanning the visible spectrum. Peaks corresponding to strong to light pink were determined by cropping and summing the appropriate counts from each microscope image. Alternatively, it was also possible to paste the numbers corresponding to the peaks into an Excel spreadsheet and sum them. The pigmentation index was then divided by the surface area (expressed in the arbitrary unit AU).

[0078] statistical analysis The results were statistically analyzed using the Kruskal-Wallis ANOVA followed by the Mann-Whitney U nonparametric test. Significance of the results is indicated as * for p<0.05, ** for p<0.01, and *** for p<0.001.

[0079] result The results are summarized in the table below: [Table 10] As can be seen from the above, exposure to blue light led to a significant increase in oxidized proteins (+93%, p<0.01). On the other hand, in the presence of gardenia fruit extract, clear protection from blue light was observed, demonstrated by reductions in oxidized proteins of -81% (p<0.05) and -86% (p<0.01) with 0.002% and 0.004% of the extract, respectively.

[0080] Example 8: Clinical research prescription For the following clinical studies, cosmetic formulations with the following INCI formulations were used: AQUA / Water, Cetyl Alcohol, Glyceryl Stearate, PEG-75 Stearate, CETETH-20, STEARETH-20, Isodecyl Neopentanoate, Gardenia Fruit Extract, Phenoxyethanol, Methylparaben, Propylparaben, Ethylparaben, Dimethicone, Fragrance, Benzyl Salicylate, Linalool, D-Limonene. In the placebo composition, gardenia fruit extract was omitted.

[0081] More details are as follows: [Table 11]

[0082] panel The clinical study was conducted with 40 female volunteers aged 18 to 50 years, with an average age of 35 ± 9 years. Participation criteria required volunteers to have facial wrinkles and to spend at least four hours per day in front of a screen (digital device), with two consecutive hours in the evening at 100% brightness. Volunteers were informed about the possible side effects of using the product and the technical conditions under which the evaluation would be conducted. They willingly signed consent forms written in accordance with the Declaration of Helsinki and the Code de la Sante Public of December 20, 1988. During the study, volunteers applied either a facial cream containing 0.002% gardenia fruit extract (from Indfrag) or a placebo twice daily (morning and evening) for 56 days. The anti-aging properties of the product were analyzed by quantifying the number of wrinkles using VISIA® (Canfield) analysis, and the quality of sleep cycles was analyzed by daily logging.

[0083] Analysis of wrinkle count by VISIA (registered trademark) Digital photographs of faces were obtained at D0, D28, and D56 using VISIA® (6th generation). Repositioning control was performed directly on the data processing screen using image overlay visualization with each acquisition. VISIA® allows for photographing under different types of lighting and extremely rapid image acquisition. A series of photographs taken under multispectral imaging and analysis allows for the capture of visual information that influences the appearance of the skin. In this study, we analyzed wrinkles around the eyes.

[0084] Analysis of sleep quality through daily logs Volunteers kept daily logs to collect data on their sources of blue light exposure, duration of blue light exposure, fatigue levels, ease of falling asleep, number of nighttime awakenings, type of skin reaction to the product, and intensity of skin reaction. self-evaluation Product feel, efficacy, and cosmetic quality were evaluated by volunteers after 27 and 55 days of product application during the study, through online questionnaires completed on Eval&Go (https: / / www.evalandgo.com / ).

[0085] statistical analysis First, Gauss's law was examined using the Shapiro-Wilk test (α=0.05). The data on wrinkle reduction did not follow Gauss's law; therefore, nonparametric statistical analysis was performed. For comparison with D0, a paired nonparametric Wilcoxon test was used (a significant result was obtained if p<0.05). For comparisons between the two products (active and placebo), an unpaired nonparametric analysis was performed along with the Mann-Whitney U test (a significant result was obtained if p<0.05). A chi-squared test (a binary analysis consisting of comparing the number of relevant responses) was performed to analyze the results of the self-assessment questionnaire and daily logs.

[0086] Result: Reduction in the number of wrinkles Treatment with a facial cream containing 0.002% gardenia fruit extract was found to result in a statistically significant reduction in the number of wrinkles in the crow's feet area, by -26% compared to D0. Furthermore, it was demonstrated that even after 56 days of application, there was a statistically significant difference of -21% between the facial cream containing 0.002% gardenia fruit extract and the placebo. In fact, the placebo cream had no effect on wrinkles after 56 days of application.

[0087] Results: Improved sleep cycle During the study, daily logs were used to track the number of times volunteers woke up during the night and their ease of falling asleep. To that end, the questionnaire included the following three questions, which volunteers were instructed to answer daily over a 56-day period: - Did you wake up in the middle of the night? → Yes or No - How many times did you wake up? - Did you fall asleep easily? → Yes or No

[0088] After 28 days of application, it was found that only 31.1% of volunteers who received a facial cream containing 0.002% gardenia fruit extract woke up at least once during the night, while 68.9% did not wake up at all. In the placebo group, 49.5% of volunteers woke up at least once during the night, while only 50.5% did not wake up at all. This difference is statistically significant. After 56 days of application, it was found that between day 29 and day 56, only 29% of volunteers who received the facial cream containing 0.002% gardenia fruit extract woke up at least once during the night, while 71% did not wake up at all. In the placebo group, 49.6% of volunteers woke up at least once during the night, while only 50.4% did not wake up at all. This difference, as previously noted, is statistically significant. Therefore, it was shown that gardenia fruit extract can significantly reduce the frequency of waking up in the middle of the night compared to a placebo.

[0089] The second question allowed us to quantify the number of nightly awakenings over the duration of the study. Volunteers who received a placebo were found to have woken up an average of 23 times in the first 28 days and 41.1 times over the entire 56 days. On the other hand, volunteers who received a facial cream containing 0.002% gardenia fruit extract woke up an average of only 3 times in the first 28 days and 7.5 times over the entire 56 days. Thus, gardenia fruit extract led to a significant reduction in the number of awakenings after 28 and 56 days, of -87% and -82% compared to placebo, respectively. These results demonstrate that gardenia fruit extract can reduce the number of times one wakes up during the night, thereby improving sleep quality.

[0090] A third question assessed ease of falling asleep. Using a dichotomy analysis, it was shown that, on average, 90.6% of volunteers who applied the facial cream containing 0.002% gardenia fruit extract fell asleep easily after one month of application, compared to only 84.8% of volunteers who received a placebo. After two months, the percentages were 89.8% and 85.8%, respectively. All of these differences were statistically significant, as before.

[0091] Data analysis based on volunteer age The results were then subjected to further analysis based on the age of the tested volunteers. For this purpose, the younger group (18-35 years old) and the older group (35-50 years old) were evaluated separately. Regarding wrinkle reduction, the gardenia fruit extract (0.002%) of the present invention was found to be more effective in older volunteers than in younger volunteers: in the older group, a significant reduction in the number of wrinkles of -25% was observed after 2 months of application. This effect was also significant compared to placebo, which resulted in only a -5% reduction.

[0092] The results for the frequency of nighttime awakenings (Question 1) are shown in the table below: [Table 12] As can be seen from the above, the gardenia fruit extract (0.002%) of the present invention resulted in a significantly smaller number of young volunteers waking up during the night compared to the placebo group. The elderly group showed a similar trend, but with slightly lower efficacy.

[0093] Regarding the average number of times participants woke up during the night (second question), the younger group showed a significant and dramatic decrease after 28 and 56 days, with reductions of -83% and -82% compared to placebo, respectively. The same trend was observed in the older group; however, the difference between gardenia fruit extract and placebo was not statistically significant. Regarding ease of falling asleep, it was found that the gardenia fruit extract of the present invention was more effective in younger groups than in older groups.

Claims

1. A cosmetic surfactant comprising gardenia fruit extract and a solvent, wherein the solvent is a eutectic solvent having a pH of at least 5, and wherein the eutectic solvent contains 30-40.4 wt% betaine, 34.6-45 wt% glycerol, and 20-30 wt% water.

2. The cosmetic surfactant according to claim 1, wherein the eutectic solvent has a pH of at least 5.

5.

3. The cosmetic surfactant according to claim 2, wherein the eutectic solvent has a pH of at least 6.

4. A cosmetic surfactant according to any one of claims 1 to 3, wherein the components of the eutectic solvent are of natural origin.

5. The cosmetic surfactant according to any one of claims 1 to 4, wherein the eutectic solvent comprises 35 wt% betaine, 40 wt% glycerol, and 25 wt% water.

6. A cosmetic surfactant according to any one of claims 1 to 5, wherein the concentration of gardenia fruit extract in the cosmetic surfactant is 0.01 to 10 wt%.

7. The cosmetic surfactant according to any one of claims 1 to 6, wherein the gardenia fruit extract contains at least 1 wt% crocin.

8. A cosmetic composition comprising a cosmetic surfactant and a cosmetically acceptable excipient according to any one of claims 1 to 7.

9. The cosmetic composition according to claim 8, which is a skincare composition.

10. A method for reducing signs of aging in the skin, comprising the step of topically applying to the skin a cosmetic surfactant according to any one of claims 1 to 7 or a cosmetic composition according to claim 8 or 9.

11. A method for protecting skin from oxidative stress, comprising the step of topically applying to the skin a cosmetic surfactant according to any one of claims 1 to 7 or a cosmetic composition according to claim 8 or 9.

12. A method for protecting skin from the effects of blue light, comprising the step of topically applying to the skin a cosmetic surfactant according to any one of claims 1 to 7 or a cosmetic composition according to claim 8 or 9.

13. A non-therapeutic method for protecting an individual's melatonin cycle, comprising the step of topically applying to the skin a cosmetic surfactant according to any one of claims 1 to 7 or a cosmetic composition according to claim 8 or 9.

14. A non-therapeutic method for improving an individual's sleep, comprising the step of topically applying to the skin a cosmetic surfactant according to any one of claims 1 to 7 or a cosmetic composition according to claim 8 or 9.

15. The method according to any one of claims 10 to 14, wherein a cosmetic surfactant or cosmetic composition is applied to the skin of the face.

16. A cosmetic surfactant according to any one of claims 1 to 7, or a cosmetic composition according to claim 8 or 9, for improving an individual's sleep.

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