COSMETIC COMPOSITION

Abstract

Cosmetic compositions comprising a Gardenia fruit extract are provided.

Claims

1. Cosmetic active agent comprising a Gardenia fruit extract and a solvent, wherein the solvent is a eutectic solvent having a pH of at least 5.

2. Cosmetic active agent according to claim 1, wherein the eutectic solvent has a pH of at least 5.5, more preferably of at least 6, and most preferably of at least 7.

3. Cosmetic active agent according to claim 1, wherein the components of the eutectic solvent are of natural origin.

4. Cosmetic active agent according to claim 1, wherein the eutectic solvent comprises betaine, glycerol and water.

5. Cosmetic active agent according to claim 4, wherein the eutectic solvent comprises from about 30 to about 40 wt % of betaine, from about 35 to about 45 wt % of glycerol, and from about 20 to about 30 wt % of water.

6. Cosmetic active agent according to claim 1, wherein the concentration of the Gardenia fruit extract in the cosmetic active agent is from about 0.01 to about 10 wt %, more preferably from about 0.05 to about 1 wt %, and most preferably about 0.1 wt %.

7. Cosmetic active agent according to claim 1, wherein the Gardenia fruit extract comprises at least 1 wt % of Crocins, more preferably at least 10 wt % of Crocins, and most preferably at least 25 wt % of Crocins.

8. Cosmetic composition comprising the cosmetic active agent according to claim 1 and a cosmetically acceptable excipient.

9. Cosmetic composition according to claim 8, which is a skin care composition, and preferably an anti-ageing skin care composition.

10. Method of reducing the signs of ageing in skin, comprising the step of topically applying the cosmetic active agent according to claim 1, preferably to facial skin.

11. Method of protecting the skin against oxidative stress, comprising the step of topically applying the cosmetic active agent according to claim 1, preferably to facial skin.

12. Method of protecting the skin against the effects of blue light, comprising the step of topically applying the cosmetic active agent according to claim 1, preferably to facial skin.

13. Non-therapeutic method of protecting an individual's melatonin cycle, comprising the step of topically applying the cosmetic active agent according to claim 1, preferably to facial skin.

14. Non-therapeutic method of improving an individual's sleep, comprising the step of topically applying the cosmetic active agent according to claim 1, preferably to facial skin.

15. A method of improving an individual's sleep which method comprises the use of Gardenia fruit extract.

16. Cosmetic active agent according to claim 5, wherein the wherein the eutectic solvent comprises about 35 wt % of betaine, about 40 wt % of glycerol, and about 25 wt % of water.

Description

[0119] FIG. 1 shows the melatonin release at different time points, with the x-axis indicating the time lapsed after the last FCS shock.

[0120] In the untreated control (“Untreated” in FIG. 1), the synchronisation of the cells induced a cyclization of the release of melatonin after 24 hours. The quantity of melatonin was significantly increased after 2, 5 and 8 hours in comparison to the level 30 minutes before the “day” phase.

[0121] In parallel, a blue light stress was induced to another co-culture at the end of each “day” phase in order to reproduce the exposition to digital instruments before sleep. It was found that this condition (“Control Blue Light” in FIG. 1) showed a different response: On day 18, i.e. 24 h after the last FCS shock, there was no increase in melatonin release. This difference was statistically significant. These results indicate that the blue light stress disturbed or delayed the cycle of melatonin release.

[0122] In the same culture conditions, a third co-culture was treated with Gardenia fruit extract at 0.004% and blue light (“Active 0.004%” in FIG. 1). It was found that this treatment led to a very similar melatonin release cycle as in the untreated control.

[0123] The results of the melatonin release studies are summarized in the following table:

TABLE-US-00006 Untreated Control Blue Light Active 0.004% Time Average Average Average (h) (pg/ml) SEM (pg/ml) SEM (pg/ml) SEM −0.5 10.60 0.0000 10.60 0.0000 10.60 0.0000 2 10.67 0.0333 10.70 0.0000 10.70 0.0577 5 10.67 0.0333 10.67 0.0667 10.63 0.0333 8 10.60 0.0000 10.67 0.0333 10.70 0.0000 23.5 10.70 0.0000 10.90 0.2082 10.63 0.0333 26 11.43 0.2603 10.43 0.1333 11.50 0.1155 29 11.80 0.1155 10.43 0.0882 11.80 0.2082 32 11.50 0.1000 10.47 0.0667 11.47 0.3180 47.5 10.57 0.3180 10.57 0.0333 10.93 0.0882 50 11.03 0.1856 10.53 0.1453 11.13 0.1202 53 10.80 0.1528 10.60 0.0577 11.33 0.0333 56 11.00 0.1732 10.87 0.0882 11.17 0.0333

[0124] As can be clearly seen, both the untreated condition (“Untreated”) and that treated with Gardenia fruit extract at 0.004% and blue light (Active 0.004%″) showed a significant increase at 2, 5 and 8 hours after the night, i.e. 26, 29 and 32 hours after the last FCS shock, respectively. On the second day after the treatment (day 19), there is a smaller but still noticeable increase in melatonin.

[0125] The samples treated with blue light but not the extract (“Control Blue Light”) had a significantly lower melatonin release than both other conditions.

[0126] In conclusions, it was found that Gardenia fruit extract at 0.004% is able to protect the cells against the effects of blue light exposure. In particular, it allows for preserving the level melatonin release, as well as its cycle.

EXAMPLE 5: MITOCHONDRIAL NETWORK ANALYSIS AND CELL SPREADING (IN VITRO)—STUDY 1

[0127] The mitochondrial network and cell spreading are both biomarkers for cell ageing.

[0128] Cell Culture and Treatment

[0129] Human dermal primary fibroblasts from a 57 years old female donor were thawed and amplified in flasks for a few days in CnT-Prime culture medium dedicated to epithelial cell culture (CelInTEC). 24 h before starting the assay, the cells were divided into 3 groups: [0130] Group 1 was left untreated; [0131] Group 2 was treated with 0.002% (w/v) of Gardenia fruit extract (from Yunnan rainbow; diluted in culture medium); and [0132] Group 3 was treated with 0.004% (w/v) of Gardenia fruit extract (from Yunnan rainbow; diluted in culture medium).

[0133] Then, the cells were loaded with the Mitotracker Green dye for 15 min. Cells were washed with PBS, detached and seeded into a CYTOOplate with extra-large Y micropatterns at 2000 cells/well in a 10% serum medium.

[0134] 1.5 h later, once the cells attached and spread on micropatterns, the medium was replaced by a medium containing less serum, further containing the same concentrations of Gardenia fruit extract as previously applied to each of the groups. The cells were then incubated during 2 h at 37° C. with 5% CO.sub.2.

[0135] After 2 h of treatment, cells were irradiated with LEDS (reference Kingbright KA-3529AQB25Z4S) at 447 nm for 1 h at 20 J/cm.sup.2 corresponding to the dose of 1 month (28 days) of screen exposition at 10 cm distance.

[0136] Hoechst was added for 15 minutes in each well to stain nuclei. The medium was renewed to wash off Hoechst and cells were incubated in CnT-prime culture medium with the active.

[0137] Image Acquisition

[0138] Live imaging was performed on the Leica microscope. At the end of the live imaging, cells were fixed and F-actin was stained with Phalloidin 555. Images were acquired on the Operetta HCS platform from Perkin Elmer.

[0139] Network Analysis

[0140] Once the mitochondrial network was detected, the sum of the length of all the filaments of a single cell network was calculated in order to determine the “Network total length”, which is averaged between all single cells from the same well. The mitochondrial network can be divided into groups of filaments that are continuously linked: This basal unit is called a “tree”. The number of trees per network, as well as their total length were averaged between all single cells detected in each well.

[0141] Each tree is divided into “branches” that are delimited at each end by either a junction or an endpoint. These branches were characterized by measuring their average and maximum lengths in the whole network of each single cell (“average branch length”).

[0142] Cell Spreading Analysis

[0143] A dedicated image analysis was run in order to detect single cells on micropatterns and to measure their area. Correctly spread cells with an area above 1800 μm.sup.2 were counted.

[0144] Statistical Analysis

[0145] The results were statistically analyzed by ordinary ANOVA with multi-comparative. Significance of results is indicated as p<0.05 with *, p<0.01 with ** and p<0.001 with ***.

[0146] Results: Mitochondrial Network

[0147] In the literature, it has been described that blue light exposure can lead to an oxidation of the cells and finally impact 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 is, the more disturbed the cell.

[0148] In a first part of the study, the mitochondrial network was analyzed through a network segmentation analysis, the results of which are shown in FIG. 2. It was found that the untreated condition showed a clear and defined network of mitochondria (FIG. 2a). After exposure to blue light, the network became fragmented and diffused as a result of oxidative stress (FIG. 2b). In the presence of Gardenia fruit extract at 0.002%, the network seemed to be less fragmented (FIG. 2c). This protective effect was even more pronounced in the presence of Gardenia fruit extract at 0.004% (FIG. 2d).

[0149] This network was then numerically segmented to perform a quantitative analysis. Various parameters were evaluated, including the total length of the network, the number of trees, the number of branches, and their average lengths.

[0150] The results are summarized in the following table:

TABLE-US-00007 Untreated + Gardenia fruit extract Gardenia fruit extract blue light at 0.002% + blue at 0.004% + blue Untreated (20 J/cm.sup.2) light (20 J/cm.sup.2) light (20 J/cm.sup.2) Network total length 454.1 ± 21.1  259.7 ± 22.0  391.9 ± 22.0  450.4 ± 20.4  Number of trees 0.127 ± 0.007 0.356 ± 0.015 0.223 ± 0.009 0.190 ± 0.006 Number of branches 0.464 ± 0.005 0.678 ± 0.012 0.549 ± 0.010 0.533 ± 0.007 Average tree length 8.787 ± 0.438 2.788 ± 0.114 4.476 ± 0.136 5.397 ± 0.192 Average branch length  2.021 ± 0. 025 1.334 ± 0.029 1.720 ± 0.037 1.747 ± 0.025

[0151] As can be seen from the above, blue light stress led to a significant reduction of the total length of the mitochondrial network. This result confirmed the data of the literature. In presence of the Gardenia fruit extract, however, a significant protection of the network by +68% (p<0.001) and +98% (p<0.001), respectively, was observed.

[0152] The network total length is automatically linked to the number of trees and branches: if the network is reduced, the number of trees and branches is increased and the average length of each one is decreased.

[0153] As expected, blue light stress was found to induce a significant increase of the number of trees and branches, and a significant decrease of the average of tree length and branch length. Again, the Gardenia fruit extracts exhibited a 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.

[0154] Results: Cell Spreading

[0155] In second part of the study, the spreading of the cells and their area was analyzed. This spreading is intimately linked to the stress incurred on the cells: when the cell is stressed, its cytoplasm is retracted.

[0156] The results are summarized in the following table:

TABLE-US-00008 Untreated + Gardenia fruit extract Gardenia fruit extract blue light at 0.002% + blue at 0.004% + blue Untreated (20 J/cm.sup.2) light (20 J/cm.sup.2) light (20 J/cm.sup.2) % of spread cells 69.96 ± 1.83 52.10 ± 1.59 81.56 ± 1.20 78.77 ± 1.03 Average cell area   2089 ± 20.83   1887 ± 20.93   2257 ± 20.49   2239 ± 21.23

[0157] It was found that after exposure to blue light, the percentage of spread cells was significantly reduced. The same applies to the average cell area. In presence of Gardenia fruit extract, however, a significant protection was observed, increasing the percentage of spread cells by +165% (p<0.001) and by +149% (p<0.001) at 0.002% and at 0.004%, respectively. The cell area was improved by +183% and by +175% at 0.002% and 0.004% of the extract, respectively (p<0.001).

EXAMPLE 6: MITOCHONDRIAL NETWORK ANALYSIS (IN VITRO)—STUDY 2

[0158] The mitochondrial network analysis described in Example 5 was repeated for Gardenia fruit extract at 0.0015% in a natural deep eutectic solvent consisting of 35 wt % of betaine, 40 wt % of glycerol, and 25 wt % of water (NaDES), vs. NaDES alone. The results are summarized in the following table:

TABLE-US-00009 Untreated + Gardenia fruit extract NaDES blue light at 0.0015% in NaDES + blue at 0.0015% + blue Untreated (20 J/cm.sup.2) light (20 J/cm.sup.2) light (20 J/cm.sup.2) Network total length 455.31 ± 19.62  243.29 ± 12.08  297.38 ± 19.15  198.88 ± 14.03  Number of trees  0.15 ± 0.004 0.37 ± 0.01 0.23 ± 0.02 0.37 ± 0.02 Number of branches 0.48 ± 0.01  0.7 ± 0.01 0.60 ± 0.01 0.69 ± 0.01 Average tree length 6.69 ± 0.25 2.70 ± 0.08 4.48 ± 0.46 2.80 ± 0.19 Average branch length 1.92 ± 0.03 1.25 ± 0.02 1.49 ± 0.06 1.27 ± 0.04

[0159] As can be seen from the above, Gardenia fruit extract in NaDES is able to provide a significant protection of the network: it was able to decrease the tree number by −38% compared to the untreated light stress condition and to increase the average tree length by +66%. The same tendency was observed for the branch parameters: Gardenia fruit extract in NaDES was able to reduce the number of branches by −14% and to increase the average length of the branches by 19%.

[0160] The natural deep eutectic solvent alone, on the other hand, has not protective effect against blue light stress.

EXAMPLE 7: PROTEIN OXIDATION ANALYSIS (EX VIVO)

[0161] Protein oxidation is another biomarker for cell ageing.

[0162] Culture and Treatments

[0163] 12 human skin explants of an average diameter of 12 mm (±1 mm) were prepared on an abdominal plasty coming from a 35-year-old Caucasian woman (reference: P2159-AB35, phototype III). The explants were kept in survival in BEM culture medium (BIO-EC's Explants Medium) at 37° C. in a humid, 5%-CO.sub.2 atmosphere.

[0164] The explants were assigned to 4 groups as follows (3 explants each): [0165] Untreated control: explants exposed to light rhythm [0166] Blue light control: explants exposed to light rhythm and blue light stress [0167] Gardenia fruit extract (from Indfrag) at 0.002%: explants exposed to light rhythm and blue light stress, with topical application of Gardenia fruit extract at 0.002% [0168] Gardenia fruit extract at 0.004%: explants exposed to light rhythms and blue light stress, with topical application of Gardenia fruit extract at 0.004%

[0169] The Gardenia fruit extracts were prepared by diluting the commercial material (Yunnan Rainbow) in phosphate buffered saline solution (PBS) at the respective concentrations (w/v).

[0170] From day 0 to day 4 of the study, skin explants were exposed to a light cycle with the purpose to mimic the circadian rhythm, and to blue light irradiations, according to the following pattern: [0171] from 7 pm to 7 am (12 hours): exposure of explants to day light using the SlimStyle W021/02 lamp (Dayvia) which presents an emission spectrum close to solar radiation. Skin explants were kept in BEM culture medium during day light exposure. [0172] from 7 am to 10 am (3 hours): exposure of explants to a dose of 63.75 J/cm.sup.2 of blue light, in 1 mL of HBSS medium, using the Solarbox® device (BioEC). The untreated control explants were kept in 1 ml of HBSS, in the dark, during the whole time of blue light exposure. At the end of the exposure, all the explants were put back in 2 mL of BEM medium. [0173] from 10 am to 7 pm (9 hours): skin explants were kept in the dark, in BEM culture medium.

[0174] For the last two groups, Gardenia fruit extract was applied topically on the basis of 2 μl per explant (2 mg/cm.sup.2) and spread using a small spatula on days 1, 2, 3 and 4 (before blue light exposure). The untreated control explants did not receive any treatment except the renewal of culture medium.

[0175] Half of the culture medium (1.2 ml per well) was renewed on days 1, 2 and 3, at the end of each dark phase of the light cycle.

[0176] On day 4, immediately after the last blue light irradiations, 3 explants from each condition were collected and cut into two parts. One part was fixed in buffered formalin and the other part was frozen at −80° C.

[0177] Immunostaining of Oxidized Proteins

[0178] Oxidized proteins were stained on frozen sections after a pre-incubation with DNPH (2,4-dinitrophenylhydrazine, Millipore, ref. 90448) and an incubation with anti-DNP antibody (Millipore, ref. 90451) diluted at 1:250 in PBS, BSA 0.3% for 1 h at 37° C., with a biotin/streptavidin amplifying system and revealed with VIP, a violet substrate of peroxidase (Vector, ref. SK-4600).

[0179] The immunostaining was performed manually and was assessed by microscopic observation.

[0180] Image Analysis: Color Index Quantification

[0181] The staining intensities of the oxidized proteins were quantified using two open source optical imaging software programs. Photomicrographs (jpeg format) were opened in GIMP-GNU Image Manipulation Program. The strong-to-light-pink color signals corresponding to the staining were selected, copied and pasted into a new image and saved as a jpeg file, this jpeg file consisting solely of the staining selected. This image was subsequently opened using the ImageJ program. An area within the dermis was selected for analysis. Then, a histogram of the section was created, separating the total number of pixels in the image into 255 color categories spanning the visible spectrum. The peak corresponding to the strong-to-light-pink color was determined by cutting and summing the appropriate counts from each photomicrographs. Alternatively, the numbers corresponding to the peak could be pasted into an Excel spreadsheet and summed.

[0182] The pigmentation index was then divided by the surface area (expressed in arbitrary units, A.U.).

[0183] Statistical Analysis

[0184] The results were statistically analyzed by Kruskal-Wallis ANOVA followed by Mann Whitney U non-parametric test. Significance of results is indicated as p<0.05 with *, p<0.01 with ** and p<0.001 with ***.

[0185] Results

[0186] The results are summarized in the following table:

TABLE-US-00010 Untreated + Gardenia fruit extract Gardenia fruit extract blue light at 0.002% + blue at 0.004% + blue Untreated (20 J/cm.sup.2) light (20 J/cm.sup.2) light (20 J/cm.sup.2) Oxidized proteins (color 0.010 ± 0.003 0.138 ± 0.042 0.026 ± 0.003 0.019 ± 0.007 index/surface (A.U.))

[0187] As can be seen from the above, exposure to blue light led to a significant increase of oxidized proteins (+93%, p<0.01). In the presence of Gardenia fruit extract, on the other hand, a clear protection from the blue light was observed, demonstrated by a reduction of oxidized proteins by −81% (p<0.05) and by −86% (p<0.01) with the extract at 0.002% and 0.004%, respectively.

EXAMPLE 8: CLINICAL STUDIES

[0188] Formulation

[0189] For the clinical studies described below, a cosmetic formulation having the following INCI formula was used:

[0190] AQUA/WATER, CETYL ALCOHOL, GLYCERYL STEARATE, PEG-75 STERATE, CETEH-20, STEARETH-20, ISODECYL NEOPENTANOATE, GARDENIA FRUIT EXTRACT, PHENOXYETHANOL, METHYL PARABEN, PROPYL PARABEN, ETHYL PARABEN, DIMETHICONE, FRAGRANCE, BENZYL SALICYLATE, LINALOOL, D-LIMONENE.

[0191] In the placebo composition, the Gardenia fruit extract was omitted.

[0192] In more detail:

TABLE-US-00011 INCI Active Placebo AQUA/WATER 89.698% (w/v) 89.700% (w/v) CETYL ALCOHOL, GLYCERYL 5.0% (w/v) 5.0% (w/v) STEARATE, PEG-75 STEARATE, CETETH-20, STEARETH-20 ISODECYL NEOPENTANOATE 4.5% (w/v) 4.5% (w/v) PHENOXYETHANOL, METHYL 0.4% (w/v) 0.4% (w/v) PARABEN, PROPYL PARABEN, ETHYL PARABEN DIMETHICONE 0.3% (w/v) 0.3% (w/v) FRAGRANCE, LINALOOL, 0.1% (w/v) 0.1% (w/v) D-LIMONENE GARDENIA FRUIT EXTRACT 0.002% (w/v) — (from Indfrag)

[0193] Panel

[0194] The clinical studies were carried out on 40 female volunteers, aged between 18 and 50 with an average age of 35±9 years. Inclusion criteria required the volunteers to have wrinkles on their face and to be in front of a screen (digital devices) at least 4 hours per day, of which 2 consecutive hours during the evening at 100% of the digital devices' luminosity. The volunteers were informed of the possible adverse effects from using the product and the technical conditions, under which the assessment was performed. They willingly signed the consent form which was written in compliance with the Declaration of Helsinki and the Dec. 20, 1988 act of the Code de la Santé Publique.

[0195] During the study, volunteers applied a facial cream containing 0.002% of Gardenia fruit extract (from Indfrag) or a placebo twice daily (morning and evening) for 56 days. The anti-ageing properties of the product was analyzed by the quantification of the number of wrinkles using VISIA® (Canfield) analysis, and the quality of the sleeping cycle was analyzed by a daily log.

[0196] Wrinkle Number Analysis by VISIA®

[0197] Using VISIA® (6th generation), digital photographs of the face were obtained on D0, D28, and D56. The control of the repositioning took place directly on the data-processing screen, using an overlay visualization of the images at each time of acquisition. VISIA® allows taking pictures with different types of illuminations and a very rapid capture of images. A series of photos taken under multi-spectral imaging and analysis allows capturing visual information affecting appearance of the skin.

[0198] In this study, the crow's feet wrinkles were analyzed.

[0199] Analysis of Sleep Quality by Daily Log

[0200] Volunteers filled in a daily log to collect data on the source of their blue light exposure, the duration of the blue light exposure, tiredness state, easiness to fall asleep, number of nocturnal awakenings, type of skin reactions to the product, and intensity of skin reactions.

[0201] Self-Assessment

[0202] The assessment of the sensation felt, efficacy and cosmetic quality of the product was performed through an online questionnaire completed on Eval&Go (https://www.evalandgo.com/) by the volunteers after 27 and 55 days of product application during the study.

[0203] Statistical Analysis

[0204] First, the Gaussian law by a Shapiro-Wilk test (α=0.05) was verified. The data on wrinkles reduction did not follow the Gaussian law; consequently, a non-parametric statistical analysis was done. For the comparison with D0, a paired and non-parametric Wilcoxon test was used (significant result if p<0.05). Regarding the comparison between the two products (Active and placebo), an unpaired and non-parametric analysis with Mann Whitney test was performed (significant result if p<0.05).

[0205] For the analysis of the self-assessment questionnaire and the daily log results, a Chi-square test was done (dichotomous analysis which consists in comparing the number of associated answers).

[0206] Results: Reduction of Number of Wrinkles

[0207] It was found that treatment with the facial cream containing 0.002% of Gardenia fruit extract led to a statistically significant reduction of the number of wrinkles in the crow's feet area by −26% compared to D0.

[0208] In addition, it was demonstrated that there was also a statistically significant difference between the facial cream containing 0.002% of Gardenia fruit extract and the placebo of −21% after 56 days of application. In fact, the placebo cream did not have any effect on the wrinkles after 56 days of application.

[0209] Results: Improved Sleep Cycle

[0210] During the study, the daily log was used to follow up the number of awakenings during the night and on how easy volunteers fell asleep.

[0211] To this end, the questionnaire contained the following three questions, which the volunteers had to answer every day during 56 days: [0212] Did you wake up during the night?->YES or NO [0213] How many times did you wake up? [0214] Did you fall asleep easily->YES or NO

[0215] After 28 days of application, it was found that only 31.1% of volunteers applying the facial cream containing 0.002% of Gardenia fruit extract had woken up during the night at least once, while 68.9% had never woken up during the night. For the placebo, 49.5% of the volunteers had woken up during the night at least once, while only 50.5% had never woken up during the night. This difference is statistically significant.

[0216] After 56 days of application, it was found that, from day 29 to day 56, only 29% of volunteers applying the facial cream containing 0.002% of Gardenia fruit extract had woken up during the night at least once, while 71% had never woken up during the night. For the placebo, 49.6% of the volunteers had woken up during the night at least once, while only 50.4% had never woken up during the night. This difference is again statistically significant.

[0217] Thus, it was shown that the Gardenia fruit extract is able to significantly reduce the frequency of awakenings during the night in comparison to placebo.

[0218] The second question allowed quantifying the number of nightly awakenings over the study period. It was found that volunteers applying the placebo had, on average, woken up 23 times in the first 28 days and 41.1 times in the total 56 days. Volunteers applying the facial cream containing 0.002% of Gardenia fruit extract, on the other hand, had only woken up 3 times in the first 28 days and 7.5 times in the total 56 days, on average. Thus, the Gardenia fruit extract led to a significant reduction in the number of awakenings by −87% and −82% after 28 and 56 days, respectively, compared to the placebo.

[0219] These results demonstrated that the Gardenia fruit extract is able to reduce the number of awakenings during the night, thereby improving the sleep quality.

[0220] With the third question, the ease of falling asleep was assessed. Using dichotomy analysis, it was shown that, on average, 90.6% of the volunteers applying the facial cream containing 0.002% of Gardenia fruit extract easily fell asleep, while only 84.8% of the volunteers using the placebo said so after 1 month of application. After 2 months, the respective percentages were 89.8% and 85.8%. All these differences were again significant.

[0221] Data Analysis Based on Age of Volunteers

[0222] Results were further analysis based on the age of the volunteers tested. To this end, a Younger Group (age 18-35) and an Older Group (age 35-50) were evaluated separately.

[0223] Regarding the reduction of wrinkles, it was found that the Gardenia fruit extract (0.002%) of the present invention was more effective for volunteers of the Older Group than for those of the Younger Group: For the Older Group, a significant reduction by −25% in the number of wrinkles was observed after 2 months of application. This effect was also significant compared to the placebo, which only led to a reduction by −5%.

[0224] For the frequency of awakenings per night (first question), the results are shown in the following table:

TABLE-US-00012 Days 1-28 Days 29-56 Woke up at Did not Woke up at Did not least once wake up least once wake up Younger Gardenia 22% 78% 19% 81% Group fruit extract Placebo 60% 40% 58% 42% Older Gardenia 27% 63% 29% 61% Group fruit extract Placebo 48% 52% 51% 49%

[0225] As can be seen from the above, the Gardenia fruit extract (0.002%) of the present invention led to a significantly smaller number of volunteers of the Younger Group waking up during the night than the placebo. The Older Group displayed the same tendency, but with a slightly lower efficacy.

[0226] For the average number of awakenings during the night (second question) a significant and drastic reduction was observed for the Younger Group, with a reduction of −83% and −82% in comparison to the placebo after 28 and 56 days, respectively. For the Older Group, the same tendency was observed; however, the difference between Gardenia fruit extract and placebo was not significant.

[0227] Also with regard to the ease of falling asleep, the Gardenia fruit extract of the present invention was found to be more efficient for the Younger Group than for the Older Group.