Improvements in or related to organic compounds

Abstract

This invention provides extracts and cosmetic compositions that exhibit whitening and anti-aging activity on the skin. [Solution] An extract of Himanthalia elongata, a carrier, and a cosmetic composition containing the extract are provided.

Description

[Technical Field]

[0001] This invention is directed toward the Himanthalia elongata extract and its use for skin treatment. Furthermore, it is directed toward cosmetic compositions containing the Himanthalia elongata extract. [Background technology]

[0002] Himanthalia elongata is an edible brown alga also known as "sea spaghetti." It is a biennial alga with two morphological stages: a vegetative form and a reproductive form. The reproductive form has long, hair-like threads.

[0003] H. elongata is found in the Baltic Sea, the North Sea, and the northeastern Atlantic Ocean from southern Scandinavia to Portugal. For example, this alga can be harvested between May and August (but preferentially in July or August) in the northern part of Brittany, France, for example, near Roscoff or Landrellec, preferably in the area between Ile Grande and Pleubian.

[0004] Himanthalia elongata contains many original molecules that may be of interest to dermatological cosmetics. For example, this brown alga contains two polyols. The first, D-mannitol, is common in brown algae, but the second, D-althritol or D-talitol, was first found in nature in Himanthalia elongata and is considered a rare sugar. This polyol is involved in regulating intracellular osmotic pressure, energy storage, and the protection of biomacromolecules. Himanthalia elongata is also rich in peptides and amino acids and contains polysaccharides (alginates, laminarins, fucans) and polyphenols. [Overview of the project]

[0005] Surprisingly, it has been found that Himanthalia elongata extract, and in particular certain fractions thereof, can possess impressive skincare properties.

[0006] In a first aspect, the present invention provides Himanthalia elongata extract, which is useful in compositions and methods for skin treatment, particularly cosmetics, non-therapeutic compositions and methods.

[0007] Himanthalia elongata extract is an aqueous extract obtained by treating frozen algae under acidic conditions. In particular, the extract is obtained by treatment with aqueous acids.

[0008] The Himanthalia elongata extract of the present invention is a concentrated extract containing polyphenols and, in particular, phlorotannins. Phlorotannins are a group of complex polymers of phloroglucinol (1,3,5-trihydroxybenzene) that are unique to macroalgae. These phenolic compounds are not only essential structural components of the cell walls of brown algae but also play many secondary ecological roles, such as protection from ultraviolet irradiation and defense against grazing. The extract contains fatty acids, citric acid, D-mannitol, D-althritol, and phlorotannins in the form of oligomers and phloroglucinol polymers with various degrees of polymerization (DP).

[0009] Some examples of phlorotannins found in Himanthalia elongata are shown in Figure 1. In the first in-tubo screening of Himanthalia elongata extract, anti-tyrosinase, anti-hyaluronidase, anti-collagenase, and anti-elastase activity was observed (Examples 3-6), which are of interest in the skincare field.

[0010] Anti-tyrosinase activity is associated with the whitening properties desired in cosmetics. Himanthalia elongata extract can interact with tyrosinase, an enzyme that controls melanin production. Upon contact with its natural substrate, the enzyme tyrosinase catalyzes the oxidation of L-DOPA to the reactive intermediate dopaquinone, which further reacts to ultimately form melanin oligomers. The presence of an inhibitor of this enzyme allows for a reduction in the rate of dopaquinone formation.

[0011] The anti-hyaluronidase, anti-collagenase, and anti-elastase activities of Himanthalia elongata extract are related to anti-aging properties such as reduction of fine lines and wrinkles, and increased skin elasticity, firmness, and moisture, as well as other properties desired in cosmetics and skincare.

[0012] Hyaluronic acid is a compound naturally produced in the dermis of the skin and is constantly broken down by an enzyme called hyaluronidase. Therefore, with age, the ratio of hyaluronic acid synthesized by cells to hyaluronic acid broken down by hyaluronidase decreases. This leads to a decrease in the moisturizing capacity of the dermis and the progression of sagging, which results in the appearance of wrinkles. Inhibiting hyaluronidase (anti-hyaluronidase activity) will reduce the amount of broken-down hyaluronic acid in the dermis and thus limit the appearance of wrinkles.

[0013] Collagenase enzymes have the ability to break down molecules such as elastin, fibronectin, and collagen. These proteins, present in the dermis, play essential roles in the extracellular matrix (ECM), and their breakdown leads to premature skin aging. Therefore, agents that inhibit collagenase activity (exhibiting anti-collagenase activity) may have beneficial effects in maintaining healthy skin by preventing the breakdown of the dermal matrix. Collagenase has the ability to cleave the "X-gly" bond of synthetic peptides with blocked amino terminals.

[0014] Elastin is a dermal protein that is a component of the extracellular matrix (ECM) and is primarily responsible for skin firmness and elasticity. Over time, the metabolism of ECM proteins slows down. In parallel, the activity of enzymes, including elastases that break down elastin, increases. One way to prevent this loss of elasticity is to use active ingredients with anti-collagenase activity that can inhibit these enzymes. Therefore, the observed anti-tyrosinase, anti-hyaluronidase, anti-collagenase, and anti-elastase activities of the Himanthalia elongata extract suggest the whitening and anti-aging properties of the extract. It was concluded that cosmetic compositions containing Himanthalia elongata extract can reduce fine lines and wrinkles and increase skin elasticity, firmness, and moisture. It was also concluded that cosmetic compositions containing Himanthalia elongata extract can whiten and / or brighten the skin.

[0015] A comparison between Himanthalia elongata extract and phenylethyl resorcinol (a commercially available whitening compound) showed that it has an equivalent or superior whitening effect. In addition, Himanthalia elongata extract exhibits anti-aging properties. Furthermore, Himanthalia elongata extract is a natural product that can be obtained from renewable sources.

[0016] The whitening activity of Himanthalia elongata extract was investigated in more detail. First, inhibition of melanin synthesis by Himanthalia elongata extract was observed in normal human melanocytes, confirming its whitening activity at the in vitro level.

[0017] It has been revealed in in tubo tests that the extract of Himanthalia elongata has strong anti-tyrosinase activity. Therefore, it can be extremely effective against hyper pigmented disorders such as pigmented senile lentigines. Indeed, tyrosinase is an important enzyme involved in the biosynthesis of melanin, which causes skin pigmentation. Excessive activity of this enzyme leads to dermatological disorders, including senile lentigines and solar lentigines, which can be classified into two types of age spots.

[0018] Therefore, in the next step, ex vivo tests of age spots related to senile lentigines (Example 7) and solar lentigines (Example 8) were carried out. It was found that the extract of Himanthalia elongata can reduce melanin content, reduce oxidized proteins, control inflammation related to solar lentigines, and improve the skin barrier.

[0019] Senile lentigines are related to cell aging, and particularly to dermal fibroblasts that are densely based across the dermal-epidermal junction. These play an active role in the control of skin pigmentation through the release of SDF-1 (stromal cell-derived factor Ⅰ). These cells play an active role in the control of skin pigmentation through the release of SDF-1 (stromal cell-derived factor Ⅰ). Unfortunately, with aging, a dramatic reduction in SDF-1 expression is observed, which leads to hyperpigmentation and age spot formation.

[0020] Cell aging is caused, and quite surprisingly, solar lentigines are mediated by ultraviolet exposure, which subsequently leads to a reduction in SDF-1 expression related to the loss of pigmentation control. Furthermore, ultraviolet exposure also leads to an increase in oxidized proteins due to a reduction in proteasome activity that correlates with an increase in the thickness of the stratum corneum. As a result, a significant increase in lipofuscin, which is well known to be involved in age spot formation, is observed. In parallel, an increase in ultraviolet-induced inflammation related to excessive stimulation of melanogenesis is observed. All these disrupted mechanisms lead to hyperpigmentation and the formation of pigmented age spots.

[0021] SDF-1 has been described in the literature as being associated with the aging of dermal fibroblasts. Indeed, in the aged state, these fibroblasts have lost the ability to express SDF-1 (Yoon et al, 2018). On the other hand, ultraviolet light is known to induce cellular aging of fibroblasts and keratinocytes from the skin (Debacq-Chainiaux et al, 2012 ; Amaro-Ortiz et al, 2014). Therefore, it was speculated that ultraviolet light involved in the formation of solar lentigines could induce skin aging. In such a case, although not described in the literature, an impact of ultraviolet light on SDF-1 expression in the dermal papilla was expected.

[0022] Surprisingly, it has been shown that SDF-1 is involved in hyperpigmentation related to ultraviolet exposure that results in solar lentigines. Furthermore, it has been shown that this effect can be affected by treatment with an extract of Himanthalia elongata.

[0023] An extract of Himanthalia elongata has been found to affect senile lentigines and solar lentigines (see Examples 7 and 8). It can be concluded that a cosmetic composition containing an extract of Himanthalia elongata can increase SDF1 expression, thereby activating the control of pigmentation. It can also be concluded that a cosmetic composition containing an extract of Himanthalia elongata can reduce melanin biosynthesis, as observed by the reduction of melanin content.

[0024] Subsequently, ex vivo studies were conducted on skin explants from different ethnic groups, including Caucasians, Asians, and Africans (Example 9). Himanthalia elongata extract was shown to possess whitening activity, resulting in an increase in non-pigmented cells and a decrease in highly pigmented cells. Himanthalia elongata extract has a whitening effect on the skin of various ethnic groups. Its effect is comparable to, or even greater than, that of phenylethyl resorcinol.

[0025] Finally, the whitening activity of Himanthalia elongata extract was tested in clinical studies. The first trial investigated the reduction of blemishes on the hands of Caucasian volunteers (Example 10). The second trial studied the reduction of blemishes on the faces of Asian volunteers (Example 11). The third trial investigated the reduction of blemishes on the faces of African volunteers (Example 12). Overall, Himanthalia elongata extract was demonstrated to reduce blemishes on the skin, particularly on the hands and face.

[0026] It has been demonstrated that the application of Himanthalia elongata extract to the skin can achieve one or more of the following effects: inhibition of tyrosinase, inhibition of hyaluronidase, inhibition of elastase, inhibition of collagenase, increase in SDF1 expression, reduction of melanin biosynthesis, reduction of melanin content, reduction of oxidized proteins after UV exposure, reduction of lipofuscin accumulation after UV exposure, reduction of inflammation after UV exposure, and reduction of stratum corneum thickness after UV exposure.

[0027] It has been demonstrated that applying Himanthalia elongata extract to the skin can produce one or more of the following effects: skin whitening, activation of pigment control, reduction of pigmentation or hyperpigmentation, reduction of skin redness, reduction of visible spots, reduction of age spots, and reduction of skin aging.

[0028] In a further aspect, the present invention provides a cosmetic composition comprising a carrier and a Himanthalia elongata extract as a first cosmetic active ingredient. The Himanthalia elongata extract should be present in an amount of 2 to 5% by weight, preferably 2.5 to 3.5% by weight, and more preferably 3% by weight of the cosmetic composition. The cosmetic composition may contain further cosmetic active ingredients. The carrier in the cosmetic composition should be a dermatologically acceptable carrier.

[0029] In a further aspect of the present invention, cosmetic compositions that are skincare compositions are provided. Typical skincare compositions are, for example, creams, serums, essences, masks, or lotions. In a further aspect of the present invention, a method of skin treatment is provided, comprising the step of applying a cosmetic composition containing an extract of Himanthalia elongata to the skin. In particular, the cosmetic composition to be applied is a skin care composition.

[0030] In a further aspect of the present invention, a method for skin treatment for skin whitening is provided. Furthermore, the provided method for skin treatment is for anti-aging treatment. In a further aspect of the present invention, the use of Himanthalia elongata extract in skincare is provided. In a further aspect, the present invention relates to a method for preparing a cosmetic active ingredient, comprising the step of extracting Himanthalia elongata.

[0031] To extract Himanthalia elongata, the frozen algae are crushed, suspended in water, and heated and acid-treated at high temperature. After acid hydrolysis, the reaction mixture is cooled and the pH is set to 2.2. This solution is centrifuged and filtered to give the first extract. This extract is then purified by adsorption onto a resin, washing with water, and desorption with ethanol. The ethanol is then evaporated and water is added. The pH is adjusted to 3 and then filtered through a 0.2 μm sieve. Glycerin is added at the end of filtration. The resulting extract contains 1 to 30 g / L of polyphenols (quantified in terms of gallic acid anhydride), preferably 3 to 25 g / L of polyphenols, more preferably 5 to 20 g / L of polyphenols, and even more preferably 9 to 18 g / L of polyphenols. [Brief explanation of the drawing]

[0032] [Figure 1] Figure 1 shows some examples of phlorotannins found in Himanthalia elongata. [Figure 2] Figure 2 shows a flowchart of the extraction process. [Figure 3a] Figure 3a shows the results of an anti-tyrosinase test on purified Himanthalia elongata extract. [Figure 3-b] Figure 3b shows the results of anti-tyrosinase tests on unpurified Himanthalia elongata extract and purified extract HEX phenylethyl resorcinol. [Figure 4] Figure 4 shows the results of anti-hyaluronidase tests for unpurified Himanthalia elongata extract and purified extract HEX phenylethyl resorcinol. [Figure 5] Figure 5 shows the results of anti-elastase tests on unpurified Himanthalia elongata extract and purified extract HEX phenylethyl resorcinol. [Figure 6] Figure 6 shows the results of anticollagenase tests for unpurified Himanthalia elongata extract and purified extract HEX phenylethyl resorcinol.

[0033] [Figure 7-a] Figure 7a shows the results of SDF1 immunostaining. [Figure 7-b] Figure 7b shows the scoring results for SDF1-positive cells. [Figure 8-a] Figure 8a shows the results of image analysis using Fontana's Masson staining. [Figure 8-b] Figure 8b shows the calculation of the pigmentation index using image analysis. [Figure 8-c] Figure 8c shows the results of calculating the pigmentation index using image analysis. [Figure 9-a] Figure 9a shows the results of SDF1 immunostaining. [Figure 9-b] Figure 9b shows the scoring results for SDF1-positive cells. [Figure 10-a] Figure 10a shows the results of image analysis using Fontana's Masson staining. [Figure 10-b] Figure 10b shows the calculation of the pigmentation index using image analysis. [Figure 10-c] Figure 10c shows the calculation of the pigmentation index using image analysis.

[0034] [Figure 11-a] Figure 11a shows a fluorescence image. [Figure 11-b] Figure 11b shows the results of quantifying oxidized proteins. [Figure 12-a] Figure 12a shows a micrograph of a Sudan Black B tissue section. [Figure 12-b] Figure 12b shows the results of quantifying lipofuscin accumulation. [Figure 13] Figure 13 shows the quantification results for pro-inflammatory cytokines (IL-8). [Figure 14-a] Figure 14a shows the results of filaggrin immunostaining. [Figure 14-b]Figure 14b shows the results of calculating the stratum corneum thickness.

[0035] [Figure 15-a] Figure 15a shows the results of whitening in Caucasian skin explants. [Figure 15-b] Figure 15b shows the results of whitening in Caucasian skin explants. [Figure 16-a] Figure 16a shows the results of whitening in Asian skin exgrafts. [Figure 16-b] Figure 16b shows the results of whitening in Asian skin exgrafts. [Figure 17-a] Figure 17a shows the results of whitening in African skin explants. [Figure 17-b] Figure 17b shows the results of whitening in African skin exgrafts.

[0036] [Figure 18] Figure 18 shows the results of melanin content in clinical trials. [Figure 19] Figure 19 shows the effect of reducing skin redness. [Figure 20] Figure 20 shows the effect of reducing visible facial blemishes. [Figure 21] Figure 21 shows the effect of reducing brown spots on the face. [Figure 22] Figure 22 shows the reduction effect of the b* parameter in the facial blemish area. [Figure 23] Figure 23 shows the effect of reducing melanin content.

[0037] The present invention will be further illustrated by the following non-limiting examples: General: All raw data, from in vitro to in vivo, were analyzed by the Shapiro-Wilk test to determine whether the data followed Gaussian law. If so, the data were analyzed with parametric paired or unpaired Student's t-test. Otherwise, the data were analyzed using the nonparametric Wilcoxon test or nonparametric Mann-Whitney U test. p<0.1#, p<0.05 * p<0.01 ** and p<0.001 *** In this case, the results are considered statistically significant.

[0038] Example 1: Preparation of Himanthalia elongata extract (HEX) Figure 2 shows a flowchart of the extraction process, which is performed in two steps, a) and b). a) Unpurified extract A 4% suspension of dried Himanthalia elongata (harvested in northern Brittany) in water was prepared. First, the seaweed was finely chopped and immersed for 10 minutes under stirring, then coarsely ground on an Ultratrax (12000 rpm) for 3 minutes. The solution was then stirred and heated. When the temperature reached 65°C, citric acid was added (the amount of added citric acid corresponds to 7.5% of the dried material in the suspension). Then, 1N sulfuric acid was added to set the pH to 2.2. Acid hydrolysis continued at 70°C for 2 hours. After 2 hours, the solution was cooled and filtered through a 170 μm sieve. The unpurified extract could be centrifuged (12000 g, 18°C, 15 minutes) and then contacted with a macroporous adsorption resin (e.g., Diaion HP20).

[0039] b) Purified extract (HEX) The unpurified extract (approximately 4000g) is brought into contact with the resin (approximately 1600g, wet) in a batch under stirring for 1 hour. After the adsorption step, the resin is washed three times with water for 5 minutes each (3 × 4.6 L) to remove salts and polar compounds (e.g., sugars and proteins). The adsorbed chemicals are then desorbed from the resin with 90% ethanol (maximum density 0.835, 1 × 4.6 L and 3 × 3.8 L for 30 minutes). Finally, the resin is washed again with 3 L of water for 5 minutes. The ethanol fraction and the final aqueous eluate are pooled and evaporated in a vacuum rotary evaporator until the ethanol content is less than 1% and the mass of the concentrated solution is 1 / 85 of the mass of the initial extract. The pH is adjusted to 3.0 with sodium hydroxide, and the product is filtered to 0.2 μm. The final product (purified extract) is obtained by diluting it 2-fold with glycerol (w / w). It contains polyphenols between 9 and 18 g / L (quantified as gallic acid anhydride).

[0040] Example 2: Analysis of phlorotannins present in Himanthalia elongata extract a) Concentration of phlorotannins An aqueous extract of Himanthalia elongata (purified extract obtained in Example 1) was diluted with distilled water and mixed with n-heptane + acetonitrile + methyl-tert-butyl ether in a separation funnel. After mixing and decantation, the resulting three immiscible liquid phases (upper phase 1UP1, middle phase MP1, and lower phase LP1) were separated. The lower phase LP1 was diluted with distilled water and mixed with acetonitrile + methyl-tert-butyl ether. The resulting two liquid phases (upper phase UP2 and lower phase LP2) were separated. The upper phase UP2 was finally mixed with MP1. The solvent was evaporated under vacuum, resulting in a fraction rich in phlorotannins, denoted as E-Phlor.

[0041] b) Fraction The phlorotannin-rich extract E-Phlor was fractionated by centrifugation and partition chromatography (CPC) using a Rousselet-Robatel-Kromaton FCPE300® instrument under the following conditions: - Injection mass of 1.5g of E-Phlor - Two-phase solvent system: methyl-tert-butyl ether / acetonitrile / water (4 / 1 / 5, v / v) - Stationary phase: Lower phase of a two-phase solvent system (heating mode) - Mobile phase: Upper phase of a two-phase solvent system - Column rotation speed: 1200 rpm - Flow rate: 20mL / min The mobile phase was passed through the stationary phase and delivered for 60 minutes. During this elution step, organic compounds with affinity for the mobile phase were collected at the outlet of the column. The stationary phase was then extruded. During this extrusion step, all compounds remaining trapped in the column were recovered.

[0042] c) Analysis of fractions Eleven consecutive CPC fractions F01-F11 were obtained. All fractions were analyzed using a Bruker Avance AVIII-600 spectrometer (Karlsruhe, Germany) equipped with a cryoprobe. 13 Analysis was performed by 13C NMR. All CPC fractions contained significant amounts of phlorotannins, as summarized in Table 1. * This is due to the presence of lower amounts of phlorotannin with the indicated degree of polymerization, and ** This indicates the presence of higher amounts. LC / MS analysis was also performed to tentatively identify the various chemical structures of phlorotannins contained in the CPC fraction.

[0043] [Table 1] Higher-degree-of-polymerization phlorotannins were also present in the CPC fraction, but were not sufficiently detected by MS.

[0044] Example 3: In-tubo evaluation of anti-tyrosinase activity of Himanthalia elongata extract The unpurified and purified extracts of Himanthalia elongata obtained in Example 1 were tested for their ability to inhibit the enzyme tyrosinase in order to confirm their whitening activity, which is of interest for cosmetic applications.

[0045] Tyrosinase will dissolve L-DOPA in its natural substrate. The presence of an enzyme inhibitor reduces the hydrolysis rate. This activity is detected by measuring the absorbance (OD) of dopaquinone released into the culture medium after substrate hydrolysis. If the enzyme is not inhibited, the concentration of released dopaquinone increases, and a brown coloration is observed.

[0046] The results obtained are expressed in terms of IC50 (mg / ml). IC50 is an indicator of the effectiveness of a substance that inhibits a specific biological or biochemical function. A lower IC50 indicates stronger biological activity at lower concentrations of the extract.

[0047] This assay is adapted for a 96-well microplate. Mix 40 μL of sample with 60 μL of phosphate buffer pH 6.8. Then add 40 μL of tyrosinase. Shake the reaction mixture and pre-incubate at 37°C for 15 minutes. Then add 40 μL of L-DOPA solution. Incubate the microplate at 37°C for 15 minutes, reading at a wavelength of 475 nm every 5 minutes. Use kojic acid as a positive standard. The purified extracts were tested at different concentrations. The results are shown in Figure 3a. The results are expressed as a percentage of tyrosinase inhibition.

[0048] Figure 3b shows the results obtained for Himanthalia elongata extract (obtained in Example 1a), purified extract HEX (obtained in Example 1b, which has a higher polyphenol content), and phenylethyl resorcinol. The purified extract was 2.6 times more active than phenylethyl resorcinol (benchmark) and 160 times more active than the unpurified extract. This demonstrates that Himanthalia elongata extract can inhibit tyrosinase.

[0049] Example 4: In-tubo evaluation of anti-hyaluronidase activity of Himanthalia elongata extract The evaluation of anti-hyaluronidase activity by turbidimetry is based on the formation of a precipitate obtained by contacting hyaluronic acid polymer with the surfactant CTAB (cetyltrimethylammonium bromide).

[0050] During the enzymatic reaction, hyaluronic acid (formed from 20 to 12,500 units) is broken down by hyaluronidase, giving smaller polymers. Therefore, the resulting disaccharides and polymers smaller than 8 kDA do not precipitate in the presence of CTAB, while the undegraded hyaluronic acid polymers and polymers larger than 8 kDA precipitate upon contact with CTAB.

[0051] The assay was performed in a 96-well microplate. 20 μl of sample was first mixed with 10 μl of Mac Ilvaine's buffer pH 4.6. Then, 20 μl of hyaluronidase and 20 μl of hyaluronic acid were added. The mixture was shaken and incubated at 37°C for 40 minutes. Finally, 180 μl of CTAB was added to all wells to precipitate the unhydrolyzed hyaluronic acid polymer. This mixture was incubated at room temperature for 20 minutes, and the absorbance at 600 nm was measured using a spectrophotometer. DSCG (disodium cromoglycate) was used as a positive control.

[0052] Figure 4 shows the results obtained from unpurified and purified phlorotannins of Himanthalia elongata extract (obtained in Examples 1a and 1b, respectively) versus phenylethyl resorcinol. The purified extract of Himanthalia elongata exhibited the best anti-hyaluronidase activity, and the global extract of Himanthalia elongata also demonstrated significant anti-hyaluronidase activity. This demonstrates that Himanthalia elongata extract can inhibit hyaluronidase.

[0053] Example 5: In-tubo evaluation of anti-elastase activity of Himanthalia elongata extract Upon contact with its natural substrate, elastase dissolves N-succinyl-(Ala)3-p-nitroanilide. The presence of inhibitors for this enzyme allows for a reduction in the rate of hydrolysis. The expression of this activity corresponds to the measurement of the absorbance (OD) of p-nitroaniline released into the culture medium after substrate hydrolysis. If the enzyme is not inhibited, the concentration of released p-nitroaniline increases and appears yellow.

[0054] The anti-elastase assay used was spectrophotometric and performed in a 96-well microplate. Porcine pancreatic elastase was used as the enzyme and N-succinyl-(Ala)3-p-nitroanilide as the substrate, and the release of p-nitroanilide was monitored. The reaction mixture contained 25 μL of sample, 25 μL of 0.2 M Tris-HCl buffer (pH 8), and 25 μL of the enzyme elastase. The mixture was pre-incubated at 37°C for 15 minutes. Then, 100 μL of N-succinyl-(Ala)3-p-nitroanilide was added as the substrate. After incubation at 37°C for 30 minutes, the absorbance was monitored at 405 nm. Elastatinal was used as a positive control.

[0055] Figure 5 shows the results obtained for unpurified and purified phlorotannins (obtained in Examples 1a and 1b, respectively) of different Himanthalia elongata extracts versus phenylethyl resorcinol. It was shown that the purified Himanthalia elongata extract had the best anti-elastase activity, and the unpurified Himanthalia elongata extract had significant anti-elastase activity. It was demonstrated that Himanthalia elongata extracts can inhibit elastase.

[0056] Example 6: In-tubo evaluation of anticollagenase activity of Himanthalia elongata extract The anti-aging activity is estimated by determining the ability of the extract to delay the enzymatic reaction between the collagenase of the bacterium Clostridium histolyticum (ChC) and the fluorescent peptide substrate (MMP-2) by spectrofluorescence at 320 nm (excitation) and 405 nm (emission). PADS (phosphoramidone disodium sulfate) is used as the positive standard.

[0057] The results obtained for Himanthalia elongata extract (obtained in Examples 1a and 1b, respectively) versus phenylethyl resorcinol are shown in Figure 6. Both purified and unpurified extracts of Himanthalia elongata were shown to have significant anti-collagenase activity. Himanthalia elongata extract was shown to be able to inhibit collagenase.

[0058] Example 7: Ex vivo study for senile lentigines (age spots). i) Treatment of skin explants Dermal fibroblast senescence was induced in fresh human skin explants (derived from a 35-year-old female donor) by 2 hours of chemical systemic treatment with 200 μM H2O2. To evaluate the effects of topical application of Himanthalia elongata extract, the explants were treated as follows: After induction of aging, the skin culture medium was refreshed with fresh medium, and either a) skin explants were topically treated with 3% (v / v) Himanthalia elongata extract (purified extract), or b) skin explants in the untreated aging state were not treated. Other skin explants for the untreated basal control were left untreated, and their medium was simply refreshed daily. These conditions were repeated daily for 5 days. After sampling, the skin explants were fixed with formalin, dehydrated, and embedded in paraffin.

[0059] ii) Hyperpigmentation (SDF1) Method: SDF1 immunostaining The paraffin-embedded excision was cut into 4 μm thick slices and degreased. Antigen retrieval was performed in EDTA (ethylenediaminetetraacetic acid) pH 8 buffer, and then nonspecific areas of the skin sample were saturated with bovine serum albumin. The sample was then incubated overnight at 4°C with anti-SDF1 antibody, rinsed, and incubated again for 45 minutes at room temperature in the dark with a secondary antibody conjugated to Alexa Fluor 488. The sample was rinsed and assembled in a coverslip and mounting medium containing DAPI (4',6-diamino-2-phenylindole). SDF1-positive cells were scored.

[0060] Results: Regulation of hyperpigmentation by SDF1 expression Comparison with untreated skin (Figure 7a, left) confirmed that aging induction significantly reduced the number of fibroblasts expressing SDF1 (Figure 7a, center), which signifies a loss of pigmentation. A significant increase in SDF1 expression was observed after 5 days of aging explant treatment with 3% HEX (Figure 7a, right), which indicates activation of latent pigmentation control. Furthermore, the number of fibroblasts expressing SDF1 near the dermal-epidermal junction under each condition was scored (Figure 7b) to confirm the above results. Himanthalia elongata extract has been shown to increase SDF1 expression, thereby activating the regulation of pigmentation, particularly in relation to senile lentigines.

[0061] iii) Hyperpigmentation (Fontana's Masson staining) Methods: Melanin quantification using Fontana's Masson staining and image analysis. The formalin-fixed and paraffin-embedded excised specimens were cut into 4 μm thick sections, degreased, and then stained for melanin using the Fontana Masson silver method. Images were acquired in bright-field mode using an Axio Observer Inverted fluorescence microscope (Zeiss).

[0062] Melanin was quantified using two open-source optical imaging software programs. Micrographs (JPEG format) of Fontana-Masson tissue sections were opened in GIMP - GNU Image Manipulation Program. The brown-black color signals corresponding to melanin granules in the stained sections were selected, copied and pasted into a new image, and saved as a JPEG file; this JPEG file consists only of black / brown (melanin granules) on a white background. This image was then opened using the ImageJ program. A histogram of the image was created, separating the total number of pixels in the image into 255 color categories across the visible spectrum. The peaks corresponding to brown-black (melanin) were determined by cutting and summing the appropriate counts from each channel of the melanin peak. Alternatively, the numbers corresponding to the pigment peaks could be pasted into an Excel spreadsheet and summed. The pigment index was taken as the absolute number of representative 20× field black pixels determined in ImageJ, and 10 -3 It was obtained by multiplying by .

[0063] Results: Control of hyperpigmentation using Fontana's Masson staining and image analysis Fontana's Masson staining was performed on the same skin explants to visualize the melanin content before and after application (Figure 8a). It was revealed that H2O2 induced a significant increase in melanin content compared to the untreated condition by inhibiting SDF1 expression. This result indicates that this model mimics the hyperpigmentation seen in senile lentigo. Topical application of 3% HEX for 5 days reduced melanin content to the same level as we observed in the untreated condition, which is likely due to the reactivation of SDF1 expression by senescent fibroblasts. The calculation of the pigmentation index using image analysis (Figure 8b) demonstrated significant hyperpigmentation associated with aging induction, as a +145% result was observed in the presence of H2O2. Local application of 3% HEX for 5 days led to a significant reduction in melanin biosynthesis, as observed by the reduction in melanin content (Figure 8c).

[0064] Example 8: Ex vivo study of senile blemishes - solar lentigines i) Treatment of skin explants Fresh human skin (from a 35-year-old female donor) was irradiated daily for 5 days, a total of 5 single irradiations, with UV-A (9 J / cm 2 ) and UV-B (0.33 J / cm 2 , UV-A / UV-B ratio = 27). After each single irradiation, 3 explants were topically treated with 3% (v / v) Himanthalia elongata extract (purified extract), and the other explants were not treated due to the untreated UV irradiation conditions. The explants in the control group were neither irradiated nor treated. After sampling, the explants were cut into two, one was stored in OCT and used for carbonyl score imaging, and the other was fixed in formalin, dehydrated, and embedded in paraffin for Fontana Masson staining and complementary immunochemistry. To measure IL-8 production, the supernatant was collected 8 hours after the final day of irradiation.

[0065] ii) Hyperpigmentation (SDF1) For the method, refer to Example 7 ii) Results: Control of hyperpigmentation via SDF1 expression Daily UV exposure has been documented in the literature to cause premature skin aging through the induction of skin cell aging. By performing SDF1 immunostaining, the effect of UV-induced aging on SDF1 expression was demonstrated. It was also demonstrated how the Himanthalia elongata extract can change this phenomenon.

[0066] Compared to untreated skin (Figure 9a, left), UV irradiation significantly reduced the number of fibroblasts expressing SDF1 (Figure 9a, center), indicating a loss of control over pigmentation. After treating UV-irradiated explants with 3% HEX for 5 days, a significant increase in SDF1 expression was observed (Figure 9a, right), suggesting activation of latent pigmentation control. Furthermore, the number of fibroblasts expressing SDF1 near the dermal-epidermal junction was scored under each condition (Figure 9b), and the results were confirmed. Himanthalia elongata extract was shown to increase SDF1 expression, thereby activating the regulation of pigmentation, particularly related to solar lentigines.

[0067] iii) Hyperpigmentation (Fontana's Masson staining) See Example 7iii) for details on the method. Results: Control of pigmentation using Fontana's Masson staining and image analysis As observed using Fontana's Masson staining compared to the untreated state, UV irradiation was shown to induce an increase in melanin content in skin explants (see Figure 10a, top and middle). Topical application of 3% HEX for 5 days reduced UV-induced hyperpigmentation (Figure 10a, bottom). Image analysis-based calculation of the pigmentation index (Figures 10b, 10c) demonstrated that significant hyperpigmentation induced by UV exposure was significantly reduced 5 days after topical application of HEX 3%. This indicates that Himanthalia elongata extract can reduce UV exposure-induced pigmentation.

[0068] iv) Reduction of oxidized proteins Method: Quantification of oxidized proteins by carbonyl score measurement. Explant sections with a thickness of 4 μm were obtained using a cryostat (Leica) and fixed with a solution containing 95% ethanol and 5% acetic acid. Carbonylated proteins were labeled using a specific fluorescent probe that reacts with the carbonyl moiety (Ex = 480 nm / Em = 530 nm) and DAPI (4',6-diamino-2-phenylindole) for nuclear labeling. Fluorescence images were acquired using an epifluorescence microscope (EVOS M5000 Imaging System; Thermo Fisher Scientific) and analyzed with ImageJ software (Schneider, 2012). Image comparisons under different conditions were achieved using identical acquisition conditions with the same magnification (10× or 60× objective lens).

[0069] Results: Reduction of oxidized proteins after UV irradiation. As observed by the detection intensity of red fluorescence, UV irradiation was found to induce an increase in oxidized proteins. This effect correlates with a decrease in proteasome activity, which is responsible for the detection and removal of oxidized proteins. Five days after topical application of 3% HEX, a significant reduction in oxidized proteins was observed. Therefore, it was demonstrated that HEX can reactivate proteasome activity, which is dramatically affected in solar lentigines (Figure 11a). Signal quantification confirmed the observations, showing that a significant increase of +24% in oxidized proteins after UV irradiation was significantly reduced by -40% with local application of 3% HEX (Figure 11b). Himanthalia elongata extract has been shown to reduce the presence of oxidized proteins after UV irradiation.

[0070] v) Reduction of lipofuscin accumulation after UV irradiation Method: Sudan Black B staining Frozen sections with a thickness of 8 μm were obtained using a cryostat and fixed in 10% formalin for 10 minutes. After fixation, the sections were treated with a continuous bath of anhydrous propylene glycol for 12 minutes, followed by a 15-minute bath at 60°C in a 0.7% Sudan Black B solution prepared in anhydrous propylene glycol. Subsequently, the sections were rinsed three times in propylene glycol at 85°C and distilled water. Coverslips were mounted on the frozen sections in aqueous mounting medium, and images were acquired in bright-field mode using an Axio Observer Inverted fluorescence microscope (Zeiss).

[0071] Quantification of lipofuscin accumulation: Lipofuscin was quantified using two open-source optical imaging software programs. Micrographs (in Tif format) of Sudan Black B tissue sections were opened in ImageJ software, and the color channels were split. The green channel, which more representative of the Sudan Black B stain, was selected and saved before opening in GIMP - GNU Image Manipulation Program. The dark color signal corresponding to lipofuscin on the stained section was selected, copied and pasted into a new image, and saved as a JPEG file; this JPEG file consisted only of dark lipofuscin on a white background. This image was then opened using the ImageJ program and inverted to black background and white lipofuscin stain corresponding to the signal to be measured. Stain intensity was measured in the epidermal region and gave a value corresponding to the amount of lipofuscin.

[0072] Results: Reduction in lipofuscin accumulation after UV irradiation. We evaluated whether the accumulation of oxidized proteins could lead to the formation of lipofuscins in the epidermis. To visualize lipofuscin accumulation after UV irradiation, we used Sudan Black B staining, which is specific for detecting lipids (Evangelou et al. 2017). Sudan Black B staining revealed a significant increase of +69% in lipofuscin accumulation after UV irradiation. This is clearly visible in the increased blue staining after UV exposure compared to the untreated state (Figure 12a top and middle panels, Figure 12b)). Topical application of HEX 3% significantly reduced UV-induced lipofuscin accumulation by -39% after 5 days, which was also observed by a reduction in blue staining in photographs (Figure 12a bottom panel, 12b). These results indicate that HEX has a protective effect against lipid oxidation and reduces the accumulation of lipofuscins in the epidermis, which is a marker of skin aging. Himanthalia elongata extract was shown to reduce lipofuscin accumulation after UV exposure.

[0073] vi) Pro-inflammatory cytokine (IL-8) Method: Quantification of pro-inflammatory cytokine (IL-8) The supernatant from skin explants was diluted 1:5 for non-irradiated explants and 1:20 for irradiated explants. IL-8 released from the explants was quantified by ELISA using the Human IL-8 Quantikine ELISA Kit (Biotechne). ELISA was performed according to the supplier's instructions. The optical density obtained from this colorimetric assay was read using a Tecan microplate reader (Spark).

[0074] Results: Reduction of inflammation after UV irradiation. The increase in the inflammatory response was observed, particularly through the quantification of the pro-inflammatory cytokine IL-8, which showed a significant increase of +121% with UV irradiation. Topical application of 3% HEX significantly and dramatically reduced UV-induced IL-8 overproduction, demonstrating that HEX can control inflammation related to solar lentigines (Figure 13). Himanthalia elongata extract has been shown to reduce inflammation after UV exposure.

[0075] vii) Reduction of stratum corneum thickness after UV irradiation Method: Filaggrin immunohistochemistry Explants embedded in paraffin were cut into 4 μm thick sections and degreased. Antigen retrieval was performed with EDTA pH8 buffer, and then non-specific areas of the skin sample were saturated with bovine serum albumin. The sample was then incubated with anti-filaggrin antibody at room temperature for 2 hours, followed by rinsing, and incubated again with a secondary antibody conjugated to Alexa Fluor 488 at room temperature in the dark for 1 hour. After rinsing, the sample was assembled in a coverslip and mounting medium containing DAPI (4',6-diamino-2-phenylindole). Stratum corneum thickness was measured using ImageJ software, following specific filaggrin staining that allows visualization of the stratum corneum.

[0076] Results: UV irradiation was shown to affect the skin barrier by increasing stratum corneum thickness, which is equivalent to a loss of intercellular bonding between keratinocytes. Filaggrin immunostaining revealed that topical application of 3% HEX significantly reduced stratum corneum thickness, followed by filaggrin expression (green fluorescence, Figure 14a). Indeed, it was shown that the stratum corneum thickness increased by +26%, from 11 μm to 15 μm, after UV irradiation. Topical application of 3% HEX was observed to restore the stratum corneum thickness to near the unirradiated state of 11 μm, by -27% (Figure 14b). Himanthalia elongata extract was shown to reduce the stratum corneum thickness after UV irradiation.

[0077] Example 9: Ex vivo study of whitening effect i) Treatment of skin explants Skin fragments from 3 Asian, 3 African, and 3 Caucasian subjects were measured in 1 cm². 2 The area was cut and washed three times with antibiotics. Subcutaneous fat and subdermis were mechanically removed using a surgical scalpel under a stereomicroscope. Skin biopsies were placed epithelial side up at the air / liquid interface on a culture insert (filter pore size 12 μm; Costar, VWR International, Fontenay-sous-Bois, France). These inserts were set in a 12-well plate and cultured in 5% CO2 at 37°C for 14 days in a humidified incubator. The culture medium was added to the wells so that the surface of the medium was horizontal with the filter. Organ cultures were maintained in Dulbecco's Minimum Essential Medium (Invitrogen Corporation, Paisley, UK), which contains antibiotics, L-glutamine, bovine pituitary extract, growth factors, and fetal bovine serum (DAP, Neuf-Brisach, France). Compared to untreated skin, 3% Himanthalia elongata extract (unpurified extract), 0.5% phenylethyl resorcinol, and 2% hydroquinone (SIGMA H9003) as a positive control were applied topically to the epidermis of skin fragments daily for 14 days.

[0078] We compared four conditions (which occurred three times): - Untreated skin - Skin treated with 3% (v / v) Himanthalia elongata extract - Skin treated with 0.5% (w / v) phenylethyl resorcinol - Skin treated with 2% hydroquinone (w / v).

[0079] ii) Method: Histological analysis using Fontana's Masson staining and whitening scoring by experts. To test for depigmentation products, melanin pigment present in melanocytes and in continuous keratinocytes of the epidermal basal layer is evaluated from treated skin compared to untreated skin. The specimens were fixed in formalin on day 14 and embedded in paraffin. Sections (4 μm) were then stained for melanin using the Fontana Masson silver method (see Example 4iii). Quantitative counting of melanin-containing cells was performed on approximately 300 basal cells of the epidermis under a light microscope at a magnification of ×40.

[0080] I counted two types of cells. - Score 1: Cells that are not pigmented or cells that display a small amount of isolated melanin pigment in the cytoplasm. - Score 3: Cells that display important melanin pigment throughout the cytoplasm. Epidermal melanin content was calculated as the percentage of epidermal cells in each scoring system.

[0081] result: a) Whitening in Caucasian skin explants (Figure 15a,b) Topical application of 3% unpurified Himanthalia elongata extract over 14 days significantly reduced the percentage of highly pigmented cells (-21.6%) and significantly increased the percentage of non-pigmented cells (+36.7%) in Caucasian skin explants, demonstrating the whitening effect of 3% Himanthalia elongata extract in Caucasian skin. In contrast, phenylethyl resorcinol only significantly increased the proportion of non-pigmented cells (+38.8%), and had no effect on highly pigmented cells (-4.2%). Therefore, we can conclude that 3% Himanthalia elongata extract is more effective than phenylethyl resorcinol in Caucasian skin explants. Compared to 2% hydroquinone, which showed no significant effect on either non-pigmented or heavily pigmented cells in Caucasian skin, 3% Himanthalia elongata extract was also significantly better.

[0082] b) Whitening in Asian skin exgrafts (Figure 16a,b) Topical application of 3% unpurified Himanthalia elongata extract over 14 days significantly reduced the percentage of highly pigmented cells (-18.9%) and significantly increased the percentage of non-pigmented cells (+75.9%) in Asian skin explants, demonstrating the whitening effect of 3% Himanthalia elongata extract on Asian skin. Topical application of phenylethyl resorcinol significantly increased the percentage of non-pigmented cells (+110.7%) and significantly decreased the percentage of pigmented cells (-29.2%). Therefore, 3% Himanthalia elongata extract is as effective as phenylethyl resorcinol in Asian skin explants. Himanthalia elongata extract 3% is also as effective in whitening activity as hydroquinone 2% in Asian skin, causing a significant increase in non-pigmented cells (+54.1%) and a significant decrease in highly pigmented cells (-25.8%).

[0083] c) Whitening of African skin exgrafts (Figure 17a, b) Topical application of 3% unpurified Himanthalia elongata extract over 14 days significantly reduced the percentage of highly pigmented cells (-23.6%) and significantly increased the percentage of non-pigmented cells (+88.4%) in African skin explants, demonstrating the whitening effect of 3% Himanthalia elongata extract in African skin. Treatment with phenylethyl resorcinol also significantly increased the percentage of non-pigmented cells (+91.7%) and significantly decreased the percentage of pigmented cells (-22.7%), indicating that 3% Himanthalia elongata extract was as effective as phenylethyl resorcinol in African skin explants. However, 3% Himanthalia elongata extract is more effective in whitening activity than 2% hydroquinone, which showed no significant results in non-pigmented cells or highly pigmented cells after topical application to African skin. Himanthalia elongata extract has been shown to have a whitening effect on different skin types.

[0084] Example 10: Clinical Trial - Reduction of Hand Spots in Caucasian Volunteers i) INCI formulation used [Table 2]

[0085] ii) Panel description This study was a double-blind, randomized, and placebo-controlled study. Assessment was based on within-subject comparisons between results obtained from creams containing 3% HEX (active product), 0.3% phenylethyl resorcinol, and placebo. Measurements were taken in a controlled atmosphere at a temperature of 21±1°C and a humidity of 50±10%. This study was conducted on 39 volunteers (healthy Caucasian women aged 50 to 75 years, mean age: 61.7±5.6 years) with pigmented senescence spots on their hands.

[0086] iii) Treatment The study was conducted in two groups of approximately 20 volunteers each (a total of 39 volunteers aged 50 to 70). The cream was applied to the hands twice daily. Evaluations were performed before treatment and at 28 and 56 days later. Group 1 received a placebo on one hand and HEX 3% on the other, while Group 2 tested HEX 3% on one hand against phenylethyl resorcinol 0.3% on the other hand.

[0087] iv) Method: Mexameter® analysis The measurement is based on the principle of absorption. The MEXAMETER MX 18 probe emits radiation at three defined wavelengths. The receiver measures the reflection of light reflected by the skin. The positions of the transmitter and receiver ensure that only diffuse light is measured. Using the defined amount of emitted light, the amount of light absorbed by the skin can be calculated. Melanin is measured at two wavelengths. The wavelengths are selected according to the different absorption peaks of the melanin pigment. For measuring erythema (redness of the skin), two different wavelengths are used to measure the absorption capacity of the epidermis. One of these corresponds to the absorption spectrum of hemoglobin. The other wavelength is selected because it has other pigments that affect skin color.

[0088] v) Results: a) Reduction of melanin content (Figure 18) HEX3% significantly reduced skin melanin content at 28 and 56 days compared to D0 by -13.8% and -21.3%, respectively. While the placebo cream produced a slight whitening effect during the study, HEX 3% significantly reduced melanin content compared to the placebo and showed 2.2 times more effective whitening activity after 28 days of HEX application. Phenylethyl resorcinol 0.3% showed the same melanin-reducing efficacy as HEX 3%, and no significant difference was observed between the two products. Himanthalia elongata extract has been shown to reduce the melanin content of the skin, thereby reducing blemishes on the skin, particularly on the hands of Caucasian volunteers.

[0089] b) Reduction of skin redness (Figure 19) HEX3% significantly reduced skin redness at 28 and 56 days compared to D0 by -8.9% and -14.8%, respectively. While the placebo caused a slight reduction in redness during the study, HEX 3% was significantly more effective than the placebo in reducing redness, showing a 1.6 times greater reduction in redness after 56 days compared to the placebo. Phenylate resorcinol showed the same efficacy as the placebo. This result demonstrated that HEX is more effective than phenylethyl resorcinol in reducing skin redness in pigmented age spots on the hands. Himanthalia elongata extract has been shown to reduce skin redness in pigmented senescence, particularly in the hands of Caucasian volunteers.

[0090] vi) Example photos using DermaScop® (registered trademark) Exemplary photographs of specific pigmented age spots on the hands before and after product application were taken using Dermascop®. These photographs were used solely for qualitative evaluation of the effect. A clear positive effect of HEX 3% in reducing age spots was visualized, similar to that of phenylethyl resorcinol 0.3%. Placebo did not show a positive effect in comparison.

[0091] Example 11: Clinical trial - Reduction of blemishes on the faces of Asian volunteers i) INCI formulation used The formulation described in Example 10i) was used. ii) Panel description This study was a double-blind, randomized, and placebo-controlled study. Assessment was based on within-subject comparisons between results obtained from creams containing 3% HEX (active product), 0.3% phenylethyl resorcinol, and placebo. Measurements were taken in a controlled atmosphere at a temperature of 21±1°C and a humidity of 50±10%. This study was conducted on 72 volunteers (healthy Asian women aged 45 to 65 years) with pigmented age spots on their faces.

[0092] iii) Treatment The study was conducted in two groups of 36 volunteers each (72 volunteers in total). The cream was applied to the face twice daily. Evaluations were performed before treatment and at 28 and 56 days later. Group 1 received a placebo on one side of their face and HEX 3% on the other side. Group 2 tested HEX 3% on one side of their face against phenylethyl resorcinol 0.3% on the other side.

[0093] iv) Method: Analysis of aging spots using VISIA CR2.0 The VISIA® device from CANFIELD® was used. It allows for taking photographs under different types of lighting and extremely rapid image acquisition. Repositioning control can be performed directly on the data processing screen using image overlay visualization at each acquisition.

[0094] A series of photographs taken under multispectral imaging and analysis (white light, ultraviolet, or polarized light—parallel or crossed) allows us to capture visual information that influences the health and appearance of the complexion: Visible plaques can be evaluated by visualizing brown or red skin lesions, including freckles, acne scars, hyperpigmentation, and vascular lesions, under standard lighting. The combination of cross-polarization and Canfield's RBX® Technology allows for the evaluation of brown plaques, particularly by analyzing the state of melanin beneath the surface. Canfield's RBX® Technology isolates the unique color signs of red and brown skin components for unparalleled visualization of conditions that result in color concentration, such as spider veins, hyperpigmentation, inflammation, and other conditions. Results are given in terms of lesion count, which is the total number of lesions detected by the device, regardless of size or intensity.

[0095] v) Results: a) Reduction of visible facial blemishes (Figure 20) We analyzed the number of visible spots using VISIA photographs. HEX3% significantly reduced the number of visible plaques by -5.6% and -3.8% compared to D0 at 28 and 56 days after application, respectively. The placebo cream slightly reduced the number of visible spots, but was clearly less effective than the cream containing HEX 3%. Furthermore, HEX 3% caused a significant reduction in visible spots, with 2.9 times higher efficacy compared to placebo after 56 days of HEX cream application. A cream containing 0.3% phenylethyl resorcinol showed efficacy very close to that obtained with 3% HEX, but there was no significant difference between the two products, indicating that HEX is as effective as phenylethyl resorcinol in reducing visible blemishes on the face. Himanthalia elongata extract has been shown to reduce visible blemishes on the face, particularly in Asian volunteers.

[0096] b) Reduction of brown spots on the face (Figure 21) Brown spots related to pigmented aging on the face were evaluated using VISIA analysis. Cream containing HEX 3% significantly reduced brown spots by -3.4% and -2.7% compared to D0 after 28 and 56 days of application, respectively. In addition, HEX 3% significantly reduced brown spots by 4.9 times after 28 days compared to placebo, demonstrating significantly greater effectiveness than placebo. Compared to phenylethyl resorcinol 0.3%, it showed slightly better activity than HEX in reducing facial brown spots, but there was no significant difference between the two products, indicating that HEX 3% was as effective as phenylethyl resorcinol in reducing facial brown spots. It was shown that Himanthalia elongata extract can reduce facial brown spots, particularly in Asian volunteers.

[0097] vi) Method: Spectrophotometer measurement Skin colorimetric measurements were performed using a MINOLTA CM700-d Spectrophotometer® with an 8mm diameter head. The Spectrophotometer® measures human-perceived color using three parameters: L * : In relation to brightness (from dark to bright), a * For the spectrum from green to red, b * For the blue to yellow spectrum, a * and b * L is the chrominance parameter. * This is the luminance parameter. It is converted into a digital code composed of these elements.

[0098] It is possible to represent subtle differences between two skin zones that appear to be the same color. After a calibration phase, measurements are taken directly on the skin using a pulsed xenon light source and a dual-beam system designed to measure transmitted light and correct for minor errors. b * The parameter (yellow melanin in the skin) has been studied in research on pigmented spots. * The parameter is skin brightness (L * ) and melanin parameters (b * Considering this, the following formula:

number

[0099] vii) Result: b of the facial blemish area * Parameter reduction (Figure 22) Using a spectrophotometer, especially b * Focusing on the parameters, we performed a complementary analysis of the color from the pigmented aging spot area. The cream containing 3% HEX is a yellow pigment related to pheomelanin content (a natural yellow pigment). * The parameter was significantly reduced. HEX3% was also significantly better than placebo, showing a significant difference between both 28-day and 56-day application periods. Regarding phenylethyl resorcinol, after application on day 28 and day 56, b * A significant reduction in the parameter resulted in a whitening effect similar to that of HEX3%, which means a reduction in skin yellowness. In other words, HEX3% is effective in reducing pigmented aging spots on the face of Asian volunteers. * In reducing parameters, it is as effective as phenylethyl resorcinol 0.3%. Himanthalia elongata extract is effective in reducing pigmented aging spots on the face, particularly on the faces of Asian volunteers. * It was shown that the parameters could be reduced.

[0100] Example 12: Clinical trial - Reduction of hyperpigmentation spots on the faces of African volunteers. i) INCI formulation used The formulation described in Example 10i) was used. ii) Panel description This study was a double-blind, randomized, and placebo-controlled study. Assessment was based on within-subject comparisons between results obtained from creams containing 3% HEX (active product), 0.3% phenylethyl resorcinol, and placebo. Measurements were taken in a controlled atmosphere at 20°C and 40% humidity. The study was conducted on 70 volunteers (aged 19 to 57 years) with hyperpigmentation on their faces.

[0101] iii) Treatment The study was conducted on three groups. The cream was applied to the entire face twice daily. Evaluations were performed before treatment and at 28 and 56 days later. Groups 1 and 2, each with 25 volunteers, received placebo and HEX 3%, respectively, while group 3, with 20 volunteers, received phenylethyl resorcinol 0.3%.

[0102] iv) Method: Mexameter® analysis See Example 10 (iv) v) Results: Reduction in melanin content (Figure 23) HEX3% significantly reduced melanin content by -3.4% compared to D0 after 56 days. This effect was also statistically significant, showing a -4.9% reduction compared to placebo after 56 days of application (Figure 23 left). Phenylethyl resorcinol was used as a benchmark and caused a slight reduction in melanin content in pigmented areas, showing a significant effect of -2.1% only after 28 days, which was not maintained after 56 days. This effect was significantly -4.3% greater than placebo only after 28 days of application (Figure 23 right). These results reveal that phenylethyl resorcinol appears to be less efficient than Himanthalia elongata extract in reducing hyperpigmentation from African skin. The efficacy of both products was compared, and no relevant difference was observed, demonstrating that the Himanthalia elongata extract is as effective as the benchmark in reducing hyperpigmented spots on African skin compared to D0 and placebo. It was shown that the Himanthalia elongata extract can reduce hyperpigmented spots on the skin, particularly in African volunteers.

Claims

1. A cosmetic skincare composition containing acidic Himantaria elongata extract.

2. The composition according to claim 1 for providing skin whitening.

3. The composition according to claim 1 for providing a reduction in oxidized proteins after UV irradiation.

4. The composition according to claim 1, for providing a reduction in lipofuscin accumulation after UV irradiation.

5. The composition according to claim 1 for providing a reduction in inflammation after UV irradiation.

6. The composition according to claim 1, for providing a reduction in the thickness of the stratum corneum after UV irradiation.

7. The composition according to claim 1, wherein the acidic Himantaria elongata extract is an aqueous extract obtained by treating frozen algae under acidic conditions.

8. The composition according to claim 7, wherein the acidic Himantaria elongata extract is purified.

9. The composition according to claim 1, wherein the acidic Himantaria elongata extract has a pH of 3.

0.

10. A cosmetic skincare method that includes the step of topically applying an acidic Himantaria elongata extract to the skin.

11. The method according to claim 10, which provides skin whitening.

12. The method according to claim 10, which provides a reduction in oxidized proteins after UV irradiation.

13. The method according to claim 10, which provides a reduction in lipofuscin accumulation after UV irradiation.

14. The method according to claim 10, which provides a reduction in inflammation after UV irradiation.

15. The method according to claim 1, which provides a reduction in the thickness of the stratum corneum after UV irradiation.

16. The method according to claim 10, wherein the acidic Himantaria elongata extract is an aqueous extract obtained by treating frozen algae under acidic conditions.

17. The method according to claim 16, wherein an acidic Himantaria elongata extract is purified.

18. The method according to claim 1, wherein the acidic Himantaria elongata extract has a pH of 3.

0.

19. The use of acidic Himantaria elongata extract in cosmetic skincare compositions to provide a skin whitening effect.

Images