Patent Application
20220071891
The invention relates to a peptide extract of the microalga Chlamydomonas acidophila and to a cosmetic, dermatological or pharmaceutical composition comprising such an extract. The invention also relates to a process for extracting a peptide extract of Chlamydomonas acidophila, and to the extract obtainable by said process. The invention also relates to a composition or such an extract for use in the prevention or treatment of disorders or pathologies of the skin, mucous membranes or skin appendages, for use in the prevention or treatment of vascular disorders, or for use in the prevention or treatment of alterations of the adipose tissue. Finally, the invention relates to a process for cosmetic care of the skin, mucous membranes or skin appendages, with a view to improving their condition or their appearance, consisting in administering such a composition or such an extract.
Microalgae are unicellular, eukaryotic organisms which are endowed with photosynthesis and therefore able, like higher plants, to use CO2 from the air for their metabolism in addition to other nutrients such as phosphorus, nitrates, etc. They were among the first species to colonize the earth. There are about 30 000 described species but there are believed to be many more. Microalgae are found in their natural state, in fresh, brackish and salt water throughout the world.
Microalgae can be cultivated according to processes known to the person skilled in the art, such as in photoreactors in light-, pH- and nutrient-controlled environments, and they have many outlets. They, like higher organisms, are able to synthesize proteins, carbohydrates and lipids. Some lipids are particular, such as complex fatty acids or pigments with particular biological properties (xanthophylls). They have become extremely popular for the possible production of biofuel and their production in bioreactors has expanded. Other outlets are diverse: fish feed (aquarium and fish farms), food and human health (astaxanthin extracted from Haematococcus pluvialis, spirulina proteins) and some outlets in the cosmetic industry.
Chlamydomonas acidophila of the class Chlorophyceae (Family: Chlamydomonadaceae) is a green freshwater microalga that proliferates in very acidic waters (pH 2.3 to 3.4) and is adapted to environments laden with heavy metals. In particular, it was first identified and collected in volcanic lakes in Argentina. It is said to be rich in phytochelatins, particular structures able to chelate metals, and in carotenoids (beta-carotene, lutein). Apart from the publications concerning its possible culture and development conditions (tolerance to extreme pH and to heavy metals), there is little concerning its composition and use. Its “cousin” Chlamydomonas reinhardtii is used as a model organism in different scientific sectors such as genetics.
The Applicant has discovered that peptide extracts of the microalga Chlamydomonas acidophila exhibit cosmetic, pharmacological and dermatological properties never described before. In particular, it is the first time that such Chlamydomonas acidophila extracts are used as such, for their specific properties.
The invention relates to a peptide extract of the microalgae Chlamydomonas acidophila.
In the sense of the present invention, “peptide extract” means an extract comprising mainly peptides.
In the sense of the present invention, “peptide” means a polymer of amino acids linked together by peptide bonds. A peptide is characterized in particular by a molecular weight comprised between 200 and 10 000 Daltons (Da), inclusive.
Advantageously, the Chlamydomonas acidophila extract according to the invention comprises at least 20% by weight of peptides, the percentages being expressed with respect to the total weight of said extract. In particular, the extract according to the invention comprises from 20% to 90%, advantageously from 20% to 75%, more advantageously from 30% to 70%, typically 65%, by weight of peptides, the percentages being expressed with respect to the total weight of said extract.
Advantageously, the Chlamydomonas acidophila extract according to the invention is substantially free of any protein, in particular of any residual native protein. Among other things, this avoids allergic reactions and improves the solubility and bioavailability of the extract according to the invention.
In the sense of the present invention, “protein” means biological macromolecules formed of one or more polypeptide chains. Each of these chains consists of a sequence of amino acid residues linked together by peptide bonds. A protein is characterized in particular by a molecular weight greater than 10 000 Daltons (Da).
Advantageously, the Chlamydomonas acidophila extract according to the invention is substantially free of free amino acids. The free amino acids have a molecular weight of less than 200 Da.
In the peptide extract of Chlamydomonas acidophila according to the invention, the peptides advantageously have a molecular weight of less than 3500 Daltons (Da). Advantageously, these peptides cover all the amino acid-based compounds initially present in the extract.
Advantageously, in the extract according to the invention, at least 80%, more advantageously at least 90%, of the peptides have a molecular weight of less than 1000 Da.
Advantageously, in the extract according to the invention, at least 30% of the peptides, more advantageously at least 35%, more advantageously at least 40% of the peptides, have a molecular weight of less than 500 Da.
The molecular weight distribution of the peptides is expressed as a percentage of the total peptide concentration.
In the context of the present invention, the peptide extract of Chlamydomonas acidophila is advantageously obtained by enzymatic hydrolysis, more advantageously in the presence of at least one protease. The extract according to the invention is more advantageously obtainable by the process described below in the description.
The invention also relates to a process for preparing a peptide extract of the microalga Chlamydomonas acidophila, comprising at least one enzymatic hydrolysis step. This step is advantageously carried out under the optimal pH and temperature conditions, known to the skilled person, in particular under the optimal pH and temperature conditions related to the enzyme used.
Advantageously, said enzymatic hydrolysis step is carried out in the presence of at least one protease. Said protease can advantageously be an alkaline protease or an acid protease, advantageously it is an alkaline protease.
Advantageously according to the invention, the process for preparing a peptide extract of Chlamydomonas acidophila comprises at least the following steps:
a) aqueous phase dispersion of the microalga Chlamydomonas acidophila;
b) enzymatic hydrolysis of the aqueous dispersion obtained in step a);
c) heat treatment of the mixture obtained in step b); and
d) recovery of the peptide extract at the end of step c).
In step a), the aqueous phase is advantageously water. Furthermore, the content of the microalgae Chlamydomonas acidophila in the aqueous dispersion is advantageously comprised between 0.1% and 20%, more advantageously 1% and 10%, dry extract equivalent of the microalgae.
The enzymatic treatment (step b) is advantageously carried out by adding at least one protease, advantageously under the optimal pH and temperature conditions known to the skilled person, for example at a pH comprised between 3.0 and 9.0 and typically at a temperature comprised between 20° C. and 90° C. In particular, the enzymatic treatment comprises the addition of an alkaline or acid protease, advantageously an alkaline protease.
The enzymatic hydrolysis step of the process according to the invention is very important, since it transforms or “cuts” the native proteins present in Chlamydomonas acidophila to obtain peptides.
In the context of the present invention, the enzymatic hydrolysis step is advantageously followed by a heat treatment step to denature the enzymes. This heat treatment step is advantageously carried out at a temperature greater than 40° C., typically between 80° C. and 100° C.
In step d), the peptide extract is advantageously recovered by extraction of the dispersion obtained at the end of step c), advantageously with stirring, and advantageously at a pH comprised between 3.0 and 9.0 and at a temperature comprised between 20° C. and 90° C.
Advantageously, the process comprises an additional filtration or centrifugation step, located between steps c) and d), optionally followed by ultrafiltration, diafiltration, or nanofiltration.
The filtration or centrifugation steps, optionally followed by membrane ultrafiltration or diafiltration, are used to remove residual proteins. The nanofiltration steps are used to remove mineral salts or free amino acids, for example.
The process according to the invention advantageously comprises a step of ultrafiltration at 15 kDa, advantageously between 10 and 15 kDa, carried out between steps c) and d), which is used to remove any potentially-allergenic residual protein.
Advantageously, the process according to the invention further comprises a nanofiltration step with, for example, a cut-off threshold comprised between 100 Daltons and 300 Daltons, advantageously between 130 and 300 Daltons, typically between 200 Daltons and 300 Daltons, in order to remove some of the amino acids or mineral salts, following the ultrafiltration step. Advantageously, said nanofiltration step is carried out on a 200 Da membrane.
The aqueous hydrolysate obtained, i.e., the peptide extract according to the invention, can then be physically and microbiologically stabilized by addition of solvent such as glycerol or glycols like 1,3-propanediol in different proportions suitable for such stabilization. Advantageously, glycerol will be present alone or in combination with water or a glycol, advantageously in a proportion comprised between 40% and 95% and preferentially between 50% and 90%, by weight with respect to the total weight of the peptide extract and the solvent. Similarly, glycol and preferentially 1,3-propanediol, will advantageously be present alone or in combination with water or glycerol, advantageously in a proportion comprised between 40% and 95% and preferentially between 50% and 90%, by weight with respect to the total weight of the peptide extract and the solvent. Thus, the present invention further relates to a composition comprising the peptide extract of Chlamydomonas acidophila according to the invention, a solvent selected from glycerol, glycols and mixtures thereof in an effective amount for physical and microbiological stabilizing action, and optionally water. The effective amounts for physical and microbiological stabilizing action are as described above.
There is an alternative in which the peptide extract can be stabilized by drying, by processes known to the skilled person, in the presence or absence of a carrier such as, for example, maltodextrins or acacia fibers (Fibregum® from the company CNI). The carrier content typically varies according to a ratio ranging from 0% to 80% of carrier in relation to the percentage of dry matter obtained in the liquid form of the extract. The extract can be dried by atomization, freeze-drying or any process known to the skilled person and is preferentially dried by freeze-drying to obtain a final powder. The final powder advantageously comprises 30% to 70% by weight of dry matter of the extract, the balance to 100% being the freeze-drying carrier. More advantageously, the final powder comprises 50% dry matter from the extract and 50% freeze-drying carrier, said freeze-drying carrier preferably being of the maltodextrin or acacia fiber type.
Preferentially, by way of example, the peptide extract can be obtained according to the following process:
a) solution of the microalgae Chlamydomonas acidophila in water at a content of about 10% dry extract equivalent of the microalgae;
b) enzymatic hydrolysis by an alkaline protease (Alcalase from the company Lyven);
c) heat treatment to denature the enzymes;
c′) centrifugation, ultrafiltration and diafiltration on 15 kDa membranes in order to eliminate potentially-allergenic residual proteins;
c″) 200 Da membrane nanofiltration to remove mineral salts or free amino acids for example; and
d) recovery of the peptide extract obtained at the end of step c″).
In the context of the process according to the invention, the Chlamydomonas acidophila microalga used as raw material can be derived from a culture in an open environment, for example in “raceways” (oval track-shaped tank used for hatchery rearing), or from a culture in a closed environment, in photobioreactors. Advantageously, said microalgae used as raw material is derived from a culture in a photobioreactor, in particular in a stirred-tank photobioreactor. More advantageously, said microalgae used as raw material is derived from a culture in horizontal tubular wave—ventilated stirred—tank photobioreactors, such as, for example, those developed by the company Microphyt and described in particular in the patent application FR 2 943 685 and the international application WO 2011/058267.
The present invention also relates to a Chlamydomonas acidophila extract obtainable by the above-mentioned process. Such an extract meets the specifications defined above.
The invention also relates to a cosmetic, dermatological or pharmaceutical composition comprising a peptide extract of Chlamydomonas acidophila as active principle and, if need be, a suitable excipient.
Advantageously, in the composition according to the invention, the peptide extract of Chlamydomonas acidophila is as defined above or is obtainable by the above-mentioned process. Thus, said extract is advantageously as defined in the above paragraphs concerning the extract according to the invention as such or those concerning the extract obtainable by the process according to the invention.
Said composition is advantageously formulated to be administered external topically, vaginally or orally.
Advantageously, the composition according to the invention comprises from 0.001% to 10%, advantageously 0.01% to 5%, of said peptide extract of Chlamydomonas acidophila, by weight expressed as dry extract, based on the total weight of the composition.
The composition according to the invention may further comprise one or more other active principles.
According to a first alternative, the various preparations are suitable for topical administration and include in particular creams, emulsions, milks, ointments, lotions, oils, aqueous or hydroalcoholic or glycolic solutions, powders, patches, sprays, shampoos, varnishes or any other product for external application. And according to the following alternatives, the various preparations include in particular intimate hygiene care, oral care, such as for example, toothpastes, oral solutions, gingival gels.
Depending on its nature (cosmetic, pharmaceutical or dermatological), the composition according to the invention may further comprise at least one cosmetically, pharmaceutically or dermatologically acceptable excipient. In particular, the composition according to the present invention may further comprise at least one cosmetically, pharmaceutically or dermatologically acceptable adjuvant known to the skilled person, selected from surfactants, thickeners, preservatives, fragrances, dyes, chemical or mineral filters, moisturizing agents, geothermal waters, etc. The skilled person knows how to adapt the formulation of the composition according to the invention by using his or her general knowledge.
The optimal dosages and galenic forms of the compositions according to the invention can be determined according to the criteria generally taken into account in the establishment of a pharmacological, dermatological or cosmetic treatment adapted to a patient or an animal, such as, for example, the age or body weight of the patient or animal, the severity of his or her general condition, the tolerance to the treatment, the side effects observed, and the skin type.
The invention also relates to an extract according to the invention or an extract obtainable by the process according to the invention or a composition according to the invention, for use in preventing and/or treating:
The invention also relates to the use of an extract according to the invention or an extract obtainable by the process according to the invention or a composition according to the invention, for the manufacture of a cosmetic, pharmaceutical or dermatological composition for preventing and/or treating:
The invention further relates to a method for preventing and/or treating:
In particular, the extract according to the invention or the extract obtainable by the process according to the invention or the composition according to the invention is intended for the prevention and/or treatment of allergic, inflammatory, irritative reactions or pathologies or disorders of the barrier or homeostasis of the skin, immature, normal, or mature/aged skin appendages (hair and nails) and/or mucous membranes (gums, periodontium, genital mucosa).
Advantageously, the composition or extract according to the invention can be used for the prevention and/or treatment of reactions, disorders or pathologies of:
the skin, such as acne, rosacea or erythrocouperosis, psoriasis, vascular disorders, diaper rash, atopic dermatitis, eczema, contact dermatitis, irritant dermatitis, allergic dermatitis, seborrheic dermatitis (cradle cap), psoriasis, sensitive skin, reactive skin, dry skin (xerosis), dehydrated skin, skin with redness, skin erythema, aged or photoaged skin, photosensitized skin, pigmented skin (melasma, post-inflammatory pigmentation, etc.), depigmented skin (vitiligo), skin with cellulite, sagging skin, skin with stretch marks, scabs, chapped skin, punctures, cracks, in particular of the breasts, sunburn, inflammation due to all kinds of rays, irritation by chemical, physical (for example stress for pregnant women), bacteriological, fungal or viral, parasitic (lice, scabies, ringworm, mites, dermatophytes) or radiological agents or by deficiency of innate (antimicrobial peptides) or acquired (cellular, humoral, cytokines) immunity, and/or
the mucous membranes such as gums and periodontium that may present gingivitis (sensitive gums of newborns, hygiene problems, due to smoking or others), periodontal disease, or genital mucosa that may present irritation of the external or internal male or female genital areas and/or
the skin appendages such as immature, normal or mature nails (brittle, fragile nails, etc.) and hair (alopecia, dandruff, hirsutism, seborrheic dermatitis, folliculitis) presenting in particular disorders of the scalp such as androgenetic, acute, localized, scarring, congenital or infant occipital alopecia (or pelade), alopecia areata, chemotherapy/radiotherapy-related alopecia or telogen effluvium, anagen effluvium, pilar dystrophy, trichotillomania, ringworm or greasy or dry dandruff.
The invention also relates to a process for cosmetic care of the skin and/or skin appendages and/or mucous membranes, with a view to improving their condition and/or their appearance, consisting in administering an extract according to the invention or an extract obtainable by the process according to the invention or a composition according to the invention.
In particular, the cosmetic care process firms the skin and reduces the “orange peel” effect advantageously by topical route on the skin and/or skin appendages and/or mucous membranes.
In particular, the invention relates to a process for cosmetic care of the skin and/or skin appendages, to act on the elasticity or firmness of the skin, in particular as a tensor or anti-wrinkle agent, to act on sensitive skin, or to act against pollution, consisting in applying to the skin and/or skin appendages a composition or an extract according to the present invention.
In particular, the invention relates to a process for cosmetic care of the skin and/or skin appendages, with a view to preventing damage to the barrier and dehydration thereof, consisting in applying to the skin and/or skin appendages a composition or an extract according to the present invention.
The invention relates to cosmetic skin care process, with a view to preventing aging, consisting in applying to the skin a composition or an extract according to the present invention.
The composition or extract according to the present invention can also be advantageously used in the prevention and/or treatment of vascular disorders, in particular redness and couperosis.
The composition or extract according to the present invention can also advantageously be used in the prevention and/or treatment of alterations of the adipose tissue. Alterations of the adipose tissue are in particular cellulite or the “orange peel” effect. The composition according to the invention firms up the skin.
The present invention may be illustrated in a non-limiting manner by the following examples.
A peptide extract is obtained according to the following process:
a) solution of the microalga Chlamydomonas acidophila at 10% dry matter in water;
b) hydrolysis by an alkaline protease (Alcalase from the company Lyven);
c) heat treatment at a temperature comprised between 80° C. and 100° C. to denature the enzymes;
c′) centrifugation, ultrafiltration and diafiltration on 15 kDa membranes in order to eliminate potentially-allergenic residual proteins
c″) 200 Da membrane nanofiltration to remove mineral salts or free amino acids or monosaccharides
d) recovery of the peptide extract
e) stabilization in a glycerol/1,3-propanediol mixture
The liquid peptide extract thus obtained has the following characteristics:
Dry extract (2 h, 105° C., ventilated oven): 1.2%
pH: 5.1
α-Amino nitrogen (OPA, leucine equivalent): 29%
Less than 500 Da: 40%
Greater than 3500 Da: 1%
The biological activity of the Chlamydomonas acidophila (CAP) extract obtained in Example 1 was demonstrated in vitro as described below.
These in vitro studies have shown the potential of the CAP extract on:
I. Preliminary Screening of Activity on Dermal Fibroblasts and Melanized Reconstructed Epidermis
The potential biological activities of the Chlamydomonas acidophila extract were investigated by a gene expression modulation test on dermal fibroblasts and melanized reconstructed epidermis. Thus, the expression of 96 genes of major interest in cutaneous and cosmetic physiology was studied by PCR-array on fibroblasts and melanized reconstructed epidermis.
a. Materials and Methods:
The Chlamydomonas acidophila (CAP) extract at 0.05% dry matter was added to the culture medium of normal human dermal fibroblasts (NHDFs) or melanized reconstructed human epidermis.
After 6 or 24 hours of incubation, the expression of the selected markers was assessed by quantitative RT-PCR (TaqMan microfluidic card). The change in expression of the studied markers compared with the control was expressed as a relative quantity (RQ, RQ>1: increase, RQ<1: decrease).
b. Results:
The most significant results showing the effect of the CAP extract on gene expression in reconstructed epidermis are presented in Table 1 below.
These results tend to show that the Chlamydomonas acidophila extract, by varying the gene expression of certain markers, could be of particular interest in the following activities:
Table 2 below presents the most significant results of the CAP extract on gene expression in fibroblasts.
These results show a potential activity of the CAP extract in the following areas:
II. Anti-Inflammatory Action
a. Introduction:
The inflammatory response is the normal, immediate and transient response of the body to any environmental attack.
However, in certain pathological or physiological conditions, this inflammatory reaction can be exacerbated and, if not properly controlled, can lead to tissue damage.
In the skin, the keratinocyte is one of the first cells involved in the initiation of the inflammatory reaction in response to environmental attack.
The “attacked” keratinocyte will then release:
The anti-inflammatory activity of the Chlamydomonas acidophila extract according to the invention was evaluated on a model of inflammation induced on keratinocytes by PMA (phorbol 12-myristate 13-acetate) treatment. The release of the cytokines TNFα and prostaglandin E2 (PGE2) was analyzed.
b. Materials and Methods:
Normal human epidermal keratinocytes were pretreated for 24 h with the Chlamydomonas acidophila (CAP) extract according to Example 1, at concentrations comprised between 0.0001% and 0.05% dry matter, or with the anti-inflammatory reference molecules dexamethasone at 0.1 μM or indomethacin at 0.1 μM (the latter two references serving as anti-inflammatory references for cytokines and prostaglandins, respectively).
Inflammation was then induced by addition of PMA at 10 μg/mL overnight.
A TNFα and PGE2 assay was then performed in the cell culture supernatants.
The significance of the results was checked by a one-way ANOVA followed by a Tuckey test (GraphPad Prism software version 5.02, GraphPad Software, San Diego Calif. USA).
c. Results:
PMA at 10 μg/ml significantly increased the release of TNFα in keratinocyte supernatants and thus did induce inflammation. Dexamethasone at 0.1 μM and indomethacin at 0.1 μM, as a 24 h pretreatment, did decrease TNFα release, demonstrating their anti-inflammatory effect and validating the test.
The Chlamydomonas acidophila extract, as a 24 h pretreatment at different concentrations, significantly decreased TNFα release and thus showed anti-inflammatory action against PMA.
d. Conclusion:
The anti-inflammatory effect of the Chlamydomonas acidophila extract was demonstrated through its action on the release of TNFα and prostaglandin E2 under inflammatory conditions.
a. Introduction
Nickel is the major cause of allergic contact dermatitis in the population, with a worldwide prevalence of roughly 8.6%. The objective of the study described below is to evaluate the effect of the CAP extract on the release of IL8 by nickel-stimulated keratinocytes.
b. Materials and Methods
Normal human epidermal keratinocytes were pretreated for 24 hours with CAP extract at 0.01% and 0.05% dry matter or with the anti-inflammatory reference molecule dexamethasone at 1 μM. The keratinocytes were then treated for 24 hours with nickel: NiSO4 at 10 μM. At the end of the incubation, the amount of IL8 produced by the cells was assessed by ELISA in the supernatants.
The concentration of IL8 assayed was normalized to the amount of total intracellular protein assessed by BC Assay.
Significance of results was statistically analyzed by a Student's t-test.
c. Results
The CAP extract induces a significant decrease in the release of IL8 induced by nickel stress in keratinocytes.
d. Conclusion
The Chlamydomonas acidophila (CAP) extract inhibits the release of a major cytokine, IL8, in the context of nickel-induced inflammatory stress. The extract is therefore of interest in the context of contact allergy or cutaneous hypersensitivity related to nickel.
a. Introduction
The objective of this study is to evaluate the anti-inflammatory activity of the Chlamydomonas acidophila (CAP) extract against heavy metal stress, represented by cadmium, on normal human keratinocytes.
b. Materials and Methods
Normal human epidermal keratinocytes were pretreated for 24 hours with the CAP extract at 0.001% and 0.01% dry matter or with the anti-inflammatory reference molecule indomethacin at 0.1 M. The keratinocytes were then treated for 48 hours with cadmium: CdCl2 at 100 μM. At the end of the incubation, the amount of PGE2 produced by the cells was assessed by ELISA in the supernatants.
The concentration of PGE2 assayed was normalized to the amount of total intracellular protein assessed by BC Assay.
c. Results
The CAP extract induces a decrease in the release of PGE2 induced by cadmium stress in keratinocytes.
d. Conclusion
The Chlamydomonas acidophila (CAP) extract inhibits the production of prostaglandin E2 (PGE2) induced by cadmium stress. The extract thus provides a protection of the skin toward heavy metal stress, in the context of environmental pollution, for example.
a. Introduction
The anti-inflammatory activity of the Chlamydomonas acidophila (CAP) extract was assessed on a model of inflammation induced by sodium dodecyl sulfate (SDS) treatment on reconstructed epidermis.
b. Materials and Methods
Reconstructed human epidermises (RHE) were preincubated for 24 hours in the presence of CAP at 0.01% and 0.05% dry matter. SDS at 0.025% was then applied to the surface of the epidermises which were again incubated for 24 hours, again in the presence of the CAP extract.
At the end of incubation, the cytokine tumor necrosis factor alpha (TNFα) was assayed by ELISA in the supernatants.
Gene expression of inflammatory and barrier markers was assessed by qRT-PCR.
Significance of results was statistically analyzed by a one-way ANOVA followed by a Tukey test.
c. Results
SDS treatment of reconstructed epidermis induces an increase in TNFα expression at the gene level (qRT-PCR, Table 8) and protein level (ELISA, Table 7). This proinflammatory effect is also accompanied by a decrease in keratin 1 (KRT1) expression (Table 8), testifying to an impairment of epidermal barrier function.
Under these conditions, the CAP extract significantly inhibited TNFα overproduction and increased keratin-1 expression.
d. Conclusion
These results confirm the anti-inflammatory potential of the Chlamydomonas acidophila extract and show its ability to protect the barrier from external stress.
III. Antioxidant Action
a. Introduction
The gene expression screening performed on the Chlamydomonas acidophila extract according to Example 1 having shown a potential in the stimulation of antioxidant defenses; the capacity to protect the cell from an oxidative stress was evaluated by measuring the production of reactive oxygen species (ROS) in keratinocytes submitted to oxidative stress induced by H2O2.
The evaluation of the antioxidant effect of the active agent is done through the incorporation of DCFH-DA (2′,7′-dichlorofluorescin diacetate) into cultured keratinocytes. This molecule is a non-fluorescent marker in the non-oxidized state. Under oxidizing conditions (here H2O2 stress), DCFH-DA will be degraded to DCF, a molecule that will emit fluorescence. The fluorescence measured will be proportional to the amount of reactive oxygen species produced by the cell in the presence of H2O2 and/or the extract.
b. Materials and Methods
Normal human epidermal keratinocytes were preincubated for 24 hours in the presence of the CAP extract at 0.0001% dry matter, quercetin at 10 μM or vitamin C at 500 μM (the latter two molecules serving as antioxidant reference).
The cells are then treated for 1 h in the presence of 0.5 mM DCFH-DA.
Oxidation is induced by adding 100 μM H2O2 for 20 minutes. A second treatment with the tested products is performed simultaneously with H2O2 stress (at the same concentrations as the pretreatment).
Finally, a measurement of the fluorescence density (DFU) corresponding to the amount of ROS is performed using a microplate reader.
The significance of the results was checked by a one-way ANOVA followed by a Tuckey test (GraphPad Prism software version 5.02, GraphPad Software, San Diego Calif. USA).
c. Results
An increase in ROS production was observed after H2O2 treatment, validating the model. Quercetin and vitamin C significantly decreased ROS production after H2O2 treatment. The antioxidant effect of these two references was well validated on the model.
The Chlamydomonas acidophila extract significantly decreased the production of ROS induced by H2O2 stress.
d. Conclusion:
The Chlamydomonas acidophila extract has demonstrated an antioxidant effect against H2O2-induced stress.
IV. Activity on the Barrier and Hydration
a. Introduction
The gene expression screening performed on the Chlamydomonas acidophila extract and presented above showed a potential effect on the stimulation of the expression of gene markers involved in the barrier and hydration. We sought to confirm this effect on keratinocytes.
b. Materials and Methods
Normal human epidermal keratinocytes were incubated for 48 hours in the presence of the CAP extract at 0.001% dry matter.
Gene expression of barrier function and hydration markers was assessed by qRT-PCR.
The results were statistically analyzed by a one-way ANOVA followed by a Dunnett's test.
c. Results
The Chlamydomonas acidophila extract stimulated the gene expression of the markers GBA (beta-glucocerebrosidase) and HAS3 (hyaluronan synthase-3) involved in the synthesis of epidermal lipids and hyaluronic acid, respectively. These results, in favor of an effect of reinforcement of the epidermal permeable barrier and hydration, confirm the trends observed in the context of the genomic expression screening.
Furthermore, the Chlamydomonas acidophila extract also stimulated the expression of the markers SLC6A6 and SLC5A3, encoding TAUT (taurine membrane transporter channel) and SMIT (myoinositol transporter channel), respectively.
These two genes code for osmolyte transporters and are therefore involved in maintaining skin hydration and protecting cells from external stresses.
Finally, the extract induced an increase in the gene expression of filaggrin (FLG) and PADI1 (peptidyl arginine deiminase), protein and enzyme involved in the synthesis of natural moisturizing factor (NMF) elements.
d. Conclusion
These results show that the extract of Chlamydomonas acidophila has a potential in the reinforcement of the skin barrier and the maintenance of skin hydration.
V. Evaluation of the Effects of the Chlamydomonas acidophila Extract in the Mechanisms of Allergy
The potential anti-allergic effects of the Chlamydomonas acidophila extract were investigated on:
a. Materials and Methods
Normal human epidermal keratinocytes were preincubated for 24 hours in the presence of the CAP extract at 0.01% dry matter (DM) or reference JAK inhibitor I at 10 μM. After preincubation, the cells were retreated with the CAP extract or reference and then the cells were stimulated with a Th2 cytokine cocktail (IL4+1L13+1L22+TNFα at 10 ng/ml) for 24 hours.
At the end of incubation, gene expression of markers of interest was assessed by qRT-PCR.
b. Results
In keratinocytes subjected to Th2 stress, the Chlamydomonas acidophila extract inhibited the gene expression of CCL5 (C-C motif chemokine ligand 5 or RANTES) and CCL27 (C-C motif chemokine ligand 27), encoding chemokines involved in the amplification of the cutaneous inflammatory and allergic response.
c. Conclusion
The Chlamydomonas acidophila extract modulates Th2 stress-induced inflammation in keratinocytes by inhibiting gene expression of the chemoattractant factors CCL5 and CCL27.
a. Materials and Methods
The basophil activation test (BAT) was performed using the Flow CAST® kit (BUHLMANN, item code FKCCR).
Whole blood was preincubated for 15 minutes in the presence of the CAP extract at 0.033% and 0.1% dry matter (DM) or the references (SB202190 at 30 μM; or cromoglycate at 10 mM).
The stimulant, 1 μM fMLP, was then added and the blood was incubated for an additional 15 minutes in the presence of the labeling buffer containing a mixture of monoclonal antibodies (anti-CD63-FITC and anti-CCR3-PE).
Flow cytometry analysis was then performed to count the total population of basophils (CCR3+) and activated basophils (CCR3+/CD63+).
b. Results
Stimulation with the fMLP peptide resulted in a very clear activation of basophils (40.1% activated cells, or 4527% stimulation).
In this study, 2 potential reference compounds were tested in the presence of fMLP:
Both SB202190, tested at 30 μM, and cromoglycate, tested at 10 mM, showed a significant inhibitory effect on fMLP-induced basophil activation (26% and 46% inhibition, respectively).
Under the experimental conditions of this study, the CAP extract, tested at 0.033% and 0.1%, showed a clear concentration-dependent inhibitory effect on fMLP-induced basophil activation (22% and 39% inhibition, respectively).
c. Conclusion
The Chlamydomonas acidophila extract inhibits basophil activation.
By inhibiting, on the one hand, the gene expression of chemoattractant cytokines in keratinocytes subjected to Th2 stress, and on the other hand, the activation of basophils; the Chlamydomonas acidophila extract could contribute to modulate the processes involved in the initiation of the allergic response.
The biological activity of the Chlamydomonas acidophila (CAP) extract obtained in Example 1 was demonstrated by clinical studies as described below.
All of these results demonstrate significant effects of the “CAP” active agent, such as:
These clinical studies have highlighted the potential of the CAP extract for the prevention or treatment of:
The CAP active agent (3% active matter) has demonstrated significant efficacy on the following parameters:
Intensity of Maximum Erythema 20 Minutes after Application of a 0.1% Methyl-Nicotinate Solution
The blood flow intensity measured by TiVi is significantly lower in the area treated with the active agent compared with the untreated area.
The redness measured by spectrocolorimetry is significantly lower on the area treated with the active agent compared with the untreated area.
Change in the Erythema 30 Minutes after the Maximum Intensity Erythema
The decrease in blood flow intensity measured by TiVi is significantly higher in the area treated with the active agent compared with the untreated area.
The reduction in redness measured by spectrocolorimetry is significantly greater in the area treated with the active agent compared with the untreated area.
The CAP active agent (3% active matter) has demonstrated significant efficacy on the following parameters:
Instrumental Measurements
The active agent:
Biochemical Assessments
The active agent:
Nineteen (19) subjects were analyzed for this study. The 19 subjects applied the active agent and the placebo on 2 defined areas. An untreated area was also defined.
The individuals recruited for this study were:
Application of the products by the subjects themselves, at home, twice a day (morning and evening) for 14 days from D−14 to D0t0 on each defined area of the forearm. After 14 days, subjects returned to the clinical unit. Basal D0t0 measurements are then taken. A final application of the products is performed. A chemical erythema is then induced on each area with a 0.1% methyl-nicotinate solution. Measurements on each area are then taken after 20 minutes (maximum intensity) denoted D0t20 and 50 minutes (30 minutes after the maximum erythema) denoted D0t50 following the induction of erythema.
Two parameters are analyzed for each parameter evaluated:
The method used by the TiVi 700 is based on the fact that green light is strongly absorbed by blood vessel cells, while red light is moderately absorbed. By using a polarized light source, the method does not take into account the specular reflection but only the light reflected by the skin tissue. The device produces an intensity map with each pixel representing a concentration of blood cells in the skin.
The graphs in
The graph in
Regarding the measurement of hydration, transepidermal water loss, color and questionnaire, 36 subjects were included in the analysis.
Regarding biochemical analyses, 10 subjects were included in the analysis.
The individuals recruited for this study were:
At D0:
Between D0 and D28:
At D28:
The skin barrier regulates water loss through evaporation. When this barrier is damaged, transepidermal water loss increases. Conversely, a reinforced barrier corresponds to lower transepidermal water loss.
Transepidermal water loss was measured by a Tewameter TM 300. The principle is to measure the temperature and relative humidity in a tube with one of its openings applied to the skin by 2 sensors located at 2 different heights. Fick's law is then used to determine transepidermal water loss.
The results obtained for the TEWL measurement at D0 and D28 are given in Table 14. The illustration of the changes is given in
The results obtained show that TEWL decreases significantly between D0 and D28 with the active agent and the placebo. The comparison of the change between D0 and D28 observed with the active agent versus the change observed with the placebo is statistically significant in favor of the active agent.
The measurement of skin hydration was performed by a CM 825 Corneometer. This device is based on the principle of capacitance measurement, allowing a measurement of the hydration of the superficial layers of the skin (10 to 20 μm deep).
The results obtained for hydration at D0 and D28 are given in Table 15. The illustration of the changes is given in
The results obtained show that hydration increases significantly between D0 and D28 with the active agent while it does not vary with the placebo. The comparison of the change between D0 and D28 observed with the active agent versus the change observed with the placebo is statistically significant in favor of the active agent.
The following biochemical assessments were performed:
From the swab sample:
From the D-Squames sample:
The results obtained for NMFs at D0 and D28 are given in Table 16. The illustration of the changes is given in
The results obtained show that the amount of NMFs increases significantly between D0 and D28 with the active agent, whereas the placebo significantly decreases this amount. The comparison of the change between D0 and D28 observed with the active agent versus the change observed with the placebo is statistically significant in favor of the active agent.
The results obtained for ceramides at D0 and D28 are given in Table 17. The illustration of the changes is given in
The results obtained show that the amount of ceramides decreases with the placebo, and that the active agent compensates for this decrease (the changes between D0 and D28 for the placebo and the active agent are not significant). The comparison of the change between D0 and D28 observed with the active agent compared with the change observed with the placebo is statistically significant in favor of the active agent.
The results obtained for IL1RA at D0 and D28 are given in Table 18. The illustration of the changes is given in
The results obtained show that the amount of IL1RA decreases significantly between D0 and D28 with the active agent and the placebo. The comparison of the change between D0 and D28 observed with the active agent versus the change observed with the placebo is statistically significant in favor of the active agent.
The results obtained for the Nile red/involucrin ratio at D0 and D28 are given in Table 19. The illustration of the changes is given in
The results obtained show that the Nile red/involucrin ratio does not vary significantly between D0 and D28 with the active agent and for the placebo. The comparison of the change between D0 and D28 observed with the active agent versus the change observed with the placebo is statistically significant in favor of the active agent.
| Number | Date | Country | Kind |
|---|---|---|---|
| 1874321 | Dec 2018 | FR | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/EP2019/087171 | 12/30/2019 | WO | 00 |
