Fat and/or wax activated by means of the water-insoluble fraction of <i>Carica papaya </i>sap
11478418 · 2022-10-25
Assignee
Inventors
Cpc classification
A61K2800/805
HUMAN NECESSITIES
A61K8/97
HUMAN NECESSITIES
International classification
A61K8/92
HUMAN NECESSITIES
C11B15/00
CHEMISTRY; METALLURGY
Abstract
The invention relates to a method for obtaining the water-insoluble fraction of Carica papaya sap, enriched with Carica papaya lipase, the water-insoluble fraction obtainable by this method, a method for the preparation of an activated fat and/or an activated wax by means of said water-insoluble fraction of the Carica papaya sap, the activated fat and/or the activated wax capable of being obtained by this method, a composition combining said activated fat and/or said activated wax, as well as the cosmetic use of these products.
Claims
1. A method for obtaining a water-insoluble fraction of Carica papaya sap, enriched with Carica papaya lipase, the method comprising the following steps: a) suspending dried Carica papaya sap in distilled water in a weight ratio of dried raw Carica papaya/distilled water of between 0.01 and 0.5, between 0.05 and 0.25, between 0.08 and 0.2, or 0.1, b) centrifuging the suspension obtained in step a) for a time period of between 5 minutes and 90 minutes, between 15 minutes and 60 minutes, or between 20 and 40 minutes, and at a rotation speed of between 2000 and 6000 rpm, between 3000 and 5000 rpm, or at 4000 rpm to obtain a pellet containing the water-insoluble fraction of the Carica papaya sap, enriched with Carica papaya lipase, c) recovering the pellet containing the water-insoluble fraction of the Carica papaya sap, enriched with Carica papaya lipase, and the method further comprising, after step a) and before step b), the following step: a′) stirring the suspension obtained in step a) for a time period of between 15 minutes and 240 minutes, a time period of between 30 minutes and 180 minutes, between 60 minutes and 150 minutes, or 120 minutes at room temperature.
2. The method according to claim 1, the method further comprising, after step b) and before step c), the following step: b′) drying the pellet until a particulate powder is obtained; wherein the water-insoluble fraction of Carica papaya sap, enriched with Carica papaya lipase, is recovered in step c), in the form of particulate powder.
Description
BRIEF DESCRIPTION OF THE FIGURES
(1) For the sake of completeness, the experimental data obtained by the Applicant in order to characterize the water-insoluble fraction of Carica papaya sap are presented below, within the detailed description of the invention. This experimental data refer to
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DETAILED DESCRIPTION OF THE INVENTION
Definitions
(8) Fat. Within the meaning of this invention, a fat has the definition commonly accepted in the State of the Art, namely a substance composed of molecules having hydrophobic properties. Fats are mainly composed of triglycerides which are esters derived from a molecule of glycerol and three fatty acids. The other components form what is called the unsaponifiable fraction or the “unsaponifiable”. The principal fats are: oils which are in the liquid state at room temperature because they are mainly composed of unsaturated fatty acids which have low melting points; fats that are pasty or solid at room temperature because they are mostly composed of saturated fatty acids that have higher melting points.
(9) As indicated above, for the purposes of this invention, an oil of animal, vegetable and/or marine origin, virgin or refined, advantageously virgin, is used as a fat, said oil being preferably of plant and/or marine origin, preferably of plant origin.
(10) Fats of marine origin. The marine fats may be of animal, plant, bacterial and/or may be algae (for example micro-algae, as indicated below). By way of example, mention may be made of fish oils, such as shark liver oil and cod liver oil, and algae and microalgae oils such as haemotococcus pluvialis.
(11) Oils of vegetable origin (or, more simply, “vegetable oils”). According to a particularly preferred embodiment of the invention vegetable oils are used, such as olive oil, rapeseed oil, soybean oil, corn oil, sweet almond oil, andiroba oil, Silversmiths Oil, babassu oil, laurel berry oil, borage oil, broccoli oil, buriti oil, Calophyllum inophyllum oil, Camellia oil, safflower oil, blackcurrant oil, hemp oil, coconut oil, cucumber seed oil, cranberry oil, raspberry pip oil, passion fruit oil kiwi seed oil, hazelnut oil, Brazil nut oil, shea olein oil, grape seed oil, apricot kernel oil, sesame oil, rice bran oil, nutmeg seed oil, tomato seed oil, baobab oil, evening primrose oil, Camellia oil, camelina oil, milk thistle oil, grape seed oil, carrot oil, St. John's wort oil, flaxseed oil, walnut oil, pomegranate seed oil, nigella seed oil, borage oil, squash seed oil, Perilla oil, green coffee bean oil, avocado oil, hibiscus oil, argan oil, safflower oil, rose hip oil, cloudberry oil, Chia (Salvia hispanica) oil. But also an oily macerate of flowers such as vanilla, St. John's wort, lily, carrot, Bellis, Arnica, aloe vera, calendula, prickly pear (non-exhaustive list). As indicated above, among these vegetable oils, those found during the implementation of the invention are preferred for their particularly interesting cosmetic properties, Calophyllum inophyllum oil (preferably virgin), raspberry oil (preferably virgin), Camellia oil (preferably virgin), evening primrose oil (preferably virgin), Brazil nut oil (preferably virgin), baobab oil (preferably virgin), and olive oil (preferably); with the stipulation that the first six are particularly preferred.
(12) Calophyllum inophyllum Oil (Calophyllum inophyllum L.; Tamanu). Calophyllum, a tree dubbed Tamanu by the Tahitians and Fohara by the malagasy, produces a remarkable oil for its beneficial action on the skin. It is very aromatic, and this oil is traditionally used to treat many dermatological conditions (eczema, psoriasis, zoster . . . )
(13) Numerous studies have shown that Calophyllum oil contains many active ingredients with disinfecting and protective properties, making this oil a valuable ally for skin problems.
(14) The saponifiable fraction is composed of linoleic acid (30-35%), oleic acid (30-35%), palmitic acid (15-20%), stearic acid (15-20%).
(15) The unsaponifiable fraction is composed of Inophyllin A: anti-bacterial, disinfectant action; Calaustraline and inophyllolide: powerful healing and skin repair; Polyphenols: antioxidants and healing action, they also have a very strong action on the venous circulation; vitamin E: natural antioxidant.
(16) Baobab Oil (Adansonia digitata). Baobab oil is used in the Senegalese pharmacopoeia for its anti-allergic and anti-inflammatory properties. In cosmetics, this very emollient and soothing oil is particularly effective for dry, torn and chapped skin.
(17) Renowned for healing and regeneration, it is recommended for the burn care. The saponifiable fraction is composed of linoleic acid (22-26%), oleic acid (30-40%), palmitic acid (20-25%), stearic acid (2-6%). The unsaponifiable fraction is composed of phytosterols (including beta-sitosterol) and vitamin E.
(18) Brazil Nut Oil (Bertholletia excelsa). The Brazil nut or Amazon nut comes from a South American tree called: Bertholletia excelsa. A vegetable source of phospholipids, tocopherols and phytosterols, Brazil nut oil brings softness, comfort and elasticity to the skin while acting as a natural antioxidant.
(19) The saponifiable fraction is composed of linoleic acid (40-45%), oleic acid (Omega-9) (30-35%), palmitic acid (10-15%), stearic acid (5-10%). The unsaponifiable fraction is composed of phytosterols (including beta-sitosterol): they maintain the structure and function of the cell membrane and reduce inflammation; Squalenes: the main components of the skin surface, they have emollient and antioxidant properties. Tocopherols: natural vitamin E, powerful antioxidants that prevent the rancidity of fatty acids and protect cells from free radical damage; Phospholipids: compounds similar to those that form cell membranes, emollient and protective properties; Selenium: trace element with antioxidant properties.
(20) Evening Primrose Oil (Oenothera biennis). Evening primrose oil is very rich in linoleic acid and is one of the rare oils to contain gamma-linolenic acid. These essential fatty acids, reconstructors of cell membranes, have exceptional softening, revitalizing, restructuring and anti-wrinkle properties. Also rich in vitamin E, evening primrose oil protects the skin from premature aging.
(21) The saponifiable fraction is composed of linoleic acid (70-75%), gamma-linolenic (5-10%) oleic acid (4-8%), palmitic acid (4-8%), stearic acid (1-4%).
(22) The unsaponifiable fraction (1.5 to 2%) is composed of phytosterols: healing and restorative action, they also reduce inflammation; triterpenes: anti-radical action, they protect the tissues from degeneration; Sterols including vitamin E: natural antioxidants.
(23) Camellia Oil (Camellia sinensis L.). Camellia oil, also called green tea oil is extracted from tea tree seeds. The vegetable oil of Camellia has nourishing, protective, softening and moisturizing properties for the skin.
(24) The saponifiable fraction is composed of linoleic acid (5-10%), oleic acid (75-80%), palmitic acid (5-10%), stearic acid (1-5%).
(25) The unsaponifiable fraction (1.5 to 2%) is composed of triterpenes; squalane; saponins as well as kaempferol and its glycosides: these are strongly anti-inflammatory compounds, such as quercetin. They help protect the body from damage caused by inflammatory conditions.
(26) Raspberry Oil (Rubus idaeus L.). Raspberry oil has healing properties thanks to its exceptional content of essential fatty acids (Omega-3 and Omega-6), it is also used to relieve itching and eczema. Raspberry oil is also known to absorb some of the UVA and UVB rays, providing light protection to the sun. It is rich in antioxidants and carotenoids and is also an ideal oil for sun-care and after-sun repair.
(27) The saponifiable fraction is composed of linoleic acid (50-55%) and alpha-linolenic acid (30-35%), oleic acid (Omega-9) (10-15%) palmitic acid (0-5%).
(28) The unsaponifiable fraction is composed of vitamin E (about 5600 mg/kg): natural antioxidant; gallic acid: natural antioxidant; of carotenoids (beta-carotene, lutein, and cryptoxanthin): anti-radical action, they protect the tissues from degeneration.
(29) Olive Oil (Olea europaea L.). The olive tree has been known for millennia for its exceptional longevity. It is also the first tree that is quoted in the history of the world.
(30) Obtained by cold pressing of its pulp and not of its pits, olive oil is one of the oils richest in oleic acid. Nourishing, softening and emollient, we find olive oil in the composition of traditional Aleppo and Marseille soaps.
(31) It is an excellent healing agent, it has a significant concentration of unsaponifiables (approximately 1 to 2%) that offer antioxidant, soothing and protective qualities against the harmful effects of weather and sun. Internally, olive oil has digestive and slightly laxative properties that make it recommended for digestive disorders and especially those of the stomach.
(32) The saponifiable fraction is composed of linoleic acid (15-20%), oleic acid (55-60%), palmitic acid (15-20%), stearic acid (0-5%).
(33) The unsaponifiable fraction is composed of phenolic compounds (hydroxytyrosol): majority antioxidants; phytosterols: healing and restorative action; squalenes: the main components of the skin surface, they have emollient and antioxidant properties, Vitamin E (alpha-tocopherol), Chlorophyll: natural antioxidants.
(34) When the fat used for the purposes of this invention is an oil (preferred embodiment), this oil may be virgin or refined. According to a particularly preferred embodiment of the invention, at least one virgin oil is preferably used, and preferably a virgin vegetable oil. Indeed, the technological platform developed by the Applicant has the significant advantage of being able to obtain virgin oils, preferably virgin vegetable oils, enriched with diacylglycerols and/or fatty alcohols (cosmetic active ingredients) without the need to turn to refined oils.
(35) Virgin vegetable oils. Vegetable oils called “virgin” are pure vegetable oils, extracted solely by mechanical methods from fruits, seeds, kernels, generally by first cold expression (at a maximum temperature of 60° C.) from a cultivated plant. These oils are characterized by their unsaponifiable and saponifiable fractions. The unsaponifiable fraction is the residual fraction which is insoluble in water but soluble in organic acids after saponification. The unsaponifiable fat content is generally in the range of 0.5 to 2%. It is a complex mixture comprising sterols, hydrocarbons (squalene, . . . ), triterpenes, fatty alcohols (waxes), liposoluble pigments, vitamins, etc.
(36) The unsaponifiable fraction of vegetable oils finds applications in cosmetics for its biological properties. The saponifiable fraction of oils is characterized by fatty acids, glycerides and triglycerides. Fatty acids are of two types, saturated fatty acids, unsaturated fatty acids (monounsaturated/MUFA or polyunsaturated/PUFA). Using these virgin vegetable oils as fat is particularly preferred within the meaning of this invention.
(37) Refined oils. Although, as indicated above, the use of a virgin oil, and preferably a virgin vegetable oil is preferred in the sense of this invention, the latter also works very well with refined oils. For an oil to be refined, it must undergo hot pressure. With pressing that takes place at a temperature of between 80° C. and 120° C. This produces a crude oil, which cannot be consumed as it is, it must undergo a long series of treatments in order to eliminate unwanted tastes and colors. The degumming step involves removing the substances that contribute to the instability and the production of foam and smoke during frying (free fatty acids, phospholipids), by stirring, with acidulated water, which will hydrolyze and thereby separate the substances. Then the taste is neutralized with a solution of soda. This is followed by bleaching at 90° C. with bleaching earth and filtering to rid the oil of its pigments. The method ends with deodorization using low-pressure water vapor to better preserve the qualities of the oil. In this way a clear, odorless refined oil with little flavor is obtained. These oils are only characterized by their saponifiable fraction, the unsaponifiable fraction being eliminated by the treatments.
(38) The Applicant has discovered that the components of the unsaponifiable fraction, namely sterols, hydrocarbons (squalene, etc.), triterpenes, fatty alcohols (waxes), fat-soluble pigments, and vitamins (non-exhaustive list) make it possible to increase the biological (cosmetic) activity of the activated fats and compositions containing them according to this invention. This is one of the reasons why the use of a virgin oil (preferably a virgin vegetable oil), including this valuable unsaponifiable fraction, is particularly preferred with respect to this invention, the technological platform constituting this invention being quite suitable for the enrichment/activation of a virgin oil.
(39) Activated Fat/Activated Oil. This invention aims in particular to obtain a diacylglycerol-enriched fat (preferably an oil), in particular 1,2-diacylglycerols; the diacylglycerols present in the fat/oil thus enriched acting as a cosmetic active ingredients, in the context of a cutaneous application. In other words, the diacylglycerol-enriched fats have a modified structure and increased biological (cosmetic) activity at the cutaneous level. This is why the fats/oils thus enriched are also called activated fats/activated oils.
(40) Wax. A wax is an ester of ethylene glycol and two fatty acids or a monoester of fatty acid and of a long chain alcohol. The waxes may be of animal origin, such as beeswax, of plant origin, such as jojoba, carnauba, or candelilla wax, or of mineral origin.
(41) The fatty alcohols are aliphatic alcohols with a long hydrocarbon chain having a single hydroxyl function in the terminal position. By fatty alcohol, the compounds of formula (III) is meant:
R″—OH (III)
wherein R″ represents the aliphatic chain of a fatty alcohol. This fatty alcohol is constituted by a carbon chain comprising from 10 to 34 carbon atoms.
(42) According to one particular embodiment of the invention, the fatty alcohols have a carbon chain whose carbon number is between 12 and 26. According to another preferred embodiment of the invention, the fatty alcohol has on its chain aliphatic between 16 and 22 carbon atoms.
(43) Thus, these fatty alcohols will preferably be selected from the group consisting of saturated or unsaturated average size aliphatic chain fatty alcohols.
(44) Furthermore, the aliphatic chain of fatty alcohols, that is to say the group R″, may be a linear or branched carbon chain, and/or saturated or unsaturated, the number of unsaturation being between 1 and 6. The R″ group may also be a mono- or polyhydroxylated and/or mono- or polymethoxylated and/or mono- or polyoxidized and/or mono- or poly-epoxylated chain. It can thus be present in all the existing natural forms.
(45) These fatty alcohols are found in large quantities in waxes. These waxes may be of animal origin, such as beeswax, or of plant origin, such as jojoba, carnauba, or candelilla wax, or of mineral origin.
(46) Thus, for example, the controlled hydrolysis of jojoba oil wax (abbreviated as “jojoba wax”, a wax that makes up more than 96% of jojoba oil) has the effect of releasing the main aliphatic fatty alcohols contained therein, which provides a mixture of fatty alcohols, of a saturated or unsaturated linear hydrocarbon chain.
(47) In particular, the hydrolysis of the jojoba oil wax makes it possible to obtain a mixture of fatty alcohols of a high degree of purity having, in their aliphatic chain, between 16 and 22 carbon atoms.
(48) Jojoba oil wax is the liquid wax contained in the Jojoba seed (Simmondsia chinensis), a bushy plant native to southern Arizona and California, as well as northwestern Mexico. Jojoba seeds have an oil yield of about 50% of their weight. Jojoba oil is a mixture of ceridic esters with chains of 36 to 46 carbon atoms. Each molecule consists of a fatty acid and a fatty alcohol linked by an ester bond. Jojoba oil is characterized by the presence of 10% oleic acid (C.sub.18:1), 70% gadoleic acid (C.sub.20:1), 15% erucic acid (C.sub.22:1), 5% nervonic acid (C.sub.24:1) and associated fatty alcohols, octacosenol (C.sub.18:1), eicosenol (C.sub.20:1), docosenol (C.sub.22:1) and tetracosenol (C.sub.24:1). Thus the controlled hydrolysis of jojoba wax has the effect of releasing the principal aliphatic fatty alcohols contained therein, which provides a mixture of saturated or unsaturated linear chain fatty alcohols.
(49) Thus, for example, the controlled hydrolysis of jojoba wax (a wax that makes up more than 96% of jojoba oil) has the effect of releasing the principal aliphatic fatty alcohols and fatty acids contained therein, making it possible to obtain a wax enriched in saturated and unsaturated hydrocarbon-based fatty alcohols and fatty acids. In particular, hydrolysis of jojoba oil by this invention makes it possible to obtain a fatty alcohol-enriched wax and fatty acids having between 18 and 24 carbon atoms. This hydrolysis thus makes it possible to concentrate all the active ingredients that are capable of acting on the skin.
(50) The mixture of fatty acids and fatty alcohols according to the invention will preferably be obtained from a hydrolysis of the waxes present in the jojoba oil (Simmondsia chinensis). This hydrolysis makes the release of the constituent fatty alcohols of this wax possible. This hydrolysis may be carried out enzymatically using a triacylglycerol hydrolase whose main activity is to hydrolyze the ester bond in order to release an alcohol and an acid. One of the particularly advantageous possibilities of this invention is to use the same lipase as that used for the hydrolysis of triacylglycerols.
(51) Preferably according to the invention, the active ingredients constituting the association, i.e., the diacylglycerol-enriched oil and a wax enriched in fatty acids and fatty alcohols, will be of plant, marine or animal origin.
(52) Activated Wax. This invention aims in particular to obtain a fatty alcohol-enriched wax (preferably jojoba oil wax, abbreviated “jojoba wax”), as defined above. The fatty alcohols present in the wax thus enriched act as the active cosmetic ingredients, advantageously in combination with the diacylglycerols of the activated fat/oil (see above), in the context of a cutaneous application. In other words, the fatty alcohol-enriched wax has a modified structure and an increased biological (cosmetic) activity at the cutaneous level. This is why wax thus enriched is also called activated wax.
(53) Lipase of Carica papaya. Lipases (triacylglycerol hydrolase; EC.3.1.1.3) are reversible enzymes which, in the reaction's most favorable sense, hydrolyze the glycerol esters. These enzymes can also be classified into several groups, according to their different specificities: specificity with respect to the substrate; position specificity or stereospecificity; specificity with regard to the nature of fatty acids or typo-selectivity; positional specificity or stereospecificity. In this invention, stereospecific lipase of the sn3 type is used. This lipase thus hydrolyzes the triglycerides by preferentially forming 1-2 diacylglycerol. The lipase used may be of plant origin (example: Carica papaya) or microbial (example: Penicelium cyclopium). The technological platform developed by the Applicant is based on the use of Carica papaya lipase (Villeneuve et al., JAOCS, 72, 6:753.1995).
(54) papaya (Carica papaya) is grown mainly in tropical and subtropical regions around the world. papaya is a member of the Caricaceae family, which belongs to the Brassicales family. The latex of Carica papaya is already known for its endopeptidases, a rich source of cysteine including papain, chymopapain and caricain. These proteinases can be extracted as water soluble latex proteins. The presence of lipase activity has been reported by Giordani et al. Until recently, all attempts to solubilize enzymatic activity from this latex fraction have failed. Carica papaya latex lipase (CPL) has therefore traditionally been considered a “naturally immobilized” biocatalyst. A dry powder containing lipase activity can be obtained after washing the latex particles with water and centrifuging. The stereospecificity and typo-selectivity of the Carica papaya latex raw sap were studied both during hydrolysis and during acyl transfer reactions. During the hydrolysis process, a 1,3-stereospecific activity was determined by this biocatalyst, with preferably a sn-3 stereo activity.
(55) In the context of its experimental procedures, the Applicant has demonstrated a certain typo-selectivity of this lipase for fatty acids, this invention, under the experimental conditions described, orienting it to triacylglycerols having long chain fatty acids, of a length of between 18 and 20, and having between 0 and 3 unsaturations. Indeed, tests carried out previously have shown that under the operating conditions used in the invention, the yields for obtaining a 1,2-diacylglycerol-enriched oil are greater with an oil having predominantly fatty acids with a chain length of between 18 and 20 carbons and having unsaturations (olive oil for example) than with an oil whose saturated fatty acids have a chain length of between 8 and 10 carbons respectively (caprylic/capric triglycerides—Mygliol® 812), 12 and 16 carbons (animal butter), 16 and 18 carbons (palm oil). The results are summarized in Table 1 below.
(56) TABLE-US-00001 TABLE 1 Comparison of DAG enrichment yields using the Carica papaya lipase from different fats Butter Enriched Oil Olive Oil Myglyol 812 (Milk) Palm Oil Acidity Index 17.5 10.2 3.2 13.6 (mg/g) 1,2 15 6.8 1 8.9 diacylglycerol Content 1,3 3 2 0 3.2 diacylglycerol Content
(57) As indicated above, the technological platform developed by the Applicant is based on the use of Carica papaya lipase, and more particularly on the use of the water-insoluble fraction of its sap. However, this invention also extends, more broadly, to a diacylglycerol-enriched fat, by means of any suitable lipase (preferably by means of a stereospecific lipase of the sn3 type, for example of vegetable or microbial origin), advantageously with 1,2-diacylglycerols, preferably with 1,2-diacylglycerols of the formula (I) as defined above, said fat being selected from Calophyllum inophyllum oil (preferably virgin), raspberry oil (preferably virgin), Camellia oil (preferably virgin), evening primrose oil (preferably virgin), Brazil nut oil (preferably virgin) and baobab oil (preferably virgin), said fat being Calophyllum inophyllum oil (preferably virgin). Moreover, the invention also extends to a composition comprising, consisting essentially of, or consisting of, said diacylglycerol-enriched fat (advantageously 1,2-diacylglycerols, preferably 1,2-diacylglycerols of the formula (I) as defined above) and a fatty alcohol-enriched wax (preferably of the formula (III) as defined above).
(58) Carica papaya Sap (also referred to as: “raw Carica papaya sap”). papaya sap is obtained from Carica papaya latex. The latex is collected by incising the still green fruits. The green fruit is superficially cut to collect the white latex that solidifies upon contact with the air. It is mainly composed of proteins including endopeptidase enzymes (raw papain=papain, chymopapains, papaya proteinase; papain being a protein of 212 amino acids for a molecular weight of 23000 daltons), a lipase (Carica papaya lipase, called Carica papaya lipase in English; see Villeneuve et al. JAOCS, 72, 6:753. 1995), as well as sugars and vitamins.
(59) Appendages. Appendages are visible protective products of the epidermis, characterized by intense keratinization. Hair, teeth, nails and hair are appendages.
(60) Skin. Within the meaning of this invention, the skin is understood in the broad sense as the body component constituting the coating of the body and includes notably the scalp (skin of the skull covered with hair).
(61) Characterization of Purified papaya Sap, and More Particularly of the Water-Insoluble Fraction (Non-Water-Soluble) of this papaya Sap
(62) When dried papaya sap was suspended in water, the enzymes exhibiting the lipolytic and protease activities exhibited different solubilities: the proteases being soluble unlike the enzymes responsible for the lipase activity (Giordani et al., 1991). This property made it possible to partially separate papaya sap into two fractions (see Table 2 below).
(63) TABLE-US-00002 TABLE 2 Study and characterization of the fractionation of papaya sap Proteins Dry matter Percentage/ Total Dry Total Proportion Amount/Fraction matter Amount/Fraction Raw 82% 82% 78% 64.5% Papaya Sap Insoluble 19% 17% 47% 8% Fraction Soluble 7% 59% 95% 56% Fraction
(64) The fractionation of papaya sap has been characterized by the dry matter content as well as the protein content of the fractions obtained, these values are compared with those of the sap. The sap used has 18% moisture, its suspension is 10% in water and the separation of the two fractions by centrifugation results in the recovery of 92% of the starting dry matter and 99.7% of the proteins initially present. Among these proteins, 88% are found in the water-soluble fraction and 12% in the water-insoluble particulate fraction (see Table 2 above). In a second step, the proteins obtained by the separation method have been characterized more precisely. Thus, after studying protease and lipase enzymatic activities, it was possible to determine the molecular weight of the proteins present in these two fractions. The amount of protein present in the raw sap as well as in each of the fractions was determined according to the Kjeldahl method and is reported in Table 3 below. Protease activity and lipolytic activity were then measured for papaya sap as well as in water soluble and water-insoluble fractions.
(65) TABLE-US-00003 TABLE 3 Specific and overall enzymatic activities of papaya sap as well as water soluble and water insoluble fractions Proteolytic Activity Lipolytic Activity Raw Soluble Insoluble Raw Soluble Insoluble Papaya Sap Fraction Fraction Papaya Sap Fraction Fraction Enzymatic Activity 1.5 1.35 0.1 0.67 Undetectable 0.66 (U/mg Dry Matter) Specific Enzymatic 2.35 2.4 1.3 1.05 / 7.45 Activity (U/mg Protein) Purification Factor / 1.02 / / / 7.1 Total Enzymatic Activity 150 134 11 67 / 60
(66) Suspension of papaya sap in water makes it possible to efficiently separate the protease activity from the lipase activity initially contained in the sap. Indeed, approximately 90% of the proteolytic activity and the lipolytic activity present initially are found respectively in the water soluble and water insoluble fractions.
(67) The molecular weight of proteins present in papaya sap and whose water soluble and water insoluble fractions were determined on a 12% polyacrylamide gel under denaturing and reducing conditions. The electrophoretic profile thus obtained is shown in
(68) Molecular weights of the main proteins observed were determined by correlation with those of the molecular weight marker using the representation log PM=f(Rf) (Rf=migration distance from protein/migration distance from the front of the gel).
(69) These molecular weights are presented below, in Table 4.
(70) TABLE-US-00004 TABLE 4 Molecular weights of proteins present in papaya sap, insoluble and water-soluble fractions, as well as in commercial papain. Commercial Raw Dried Insoluble Purified Papaya Sap Fraction Soluble Fraction Papain Molecular 35 23 35 35 Weight of 23 28 23 Protein 22 23 22 (kDa) 18 22 18 18 15 15
(71) papaya sap has four major proteins of molecular weight of 18, 22, 23 and 35 kDa respectively. The water insoluble fraction appears to be composed of a single protein having a molecular weight of 23 kDa. Since the water-soluble fraction of six proteins having a molecular weight of 15, 18, 22, 23, 28 and 35 kDa, the presence of a protein of 15 kDa must be due to a hydrolysis occurring during the suspension of the papaya sap. Commercial papain, which has no lipolytic activity, is made up of the same five protein forms as the papaya sap. The similarity between the electrophoretic profiles is certainly due to the fact that this commercial extract is a mixture of papaya sap endopeptidases in the form of proenzymes and/or mature enzymes.
(72) Based on these results, the Carica papaya lipase appears to have a molecular weight of 23 kDa. However, a protein of the same molecular weight is also present in the water-soluble fraction and the commercial papain wherein a single protease activity has been revealed.
(73) Characterization by Western Blot
(74) An analysis of these same samples by Western Blot was then performed in order to identify the molecular weight of the proteins corresponding to papain-type activities.
(75) The antibody used, a polyclonal antibody derived from goat serum that targets papain (Rockland, anti-Papain [Carica papaya]) mainly reveals a protein of 35 kDa. Weaker signals are observed for proteins of molecular weight between 15 and 28 kDa.
(76) Papain is described to have a molecular weight ranging from 21 to 23 kDa (Azarkan et al., 2003). The antibody used apparently bonded to the propapain, the proenzyme precursor of papain possessing an N-terminal region (134 amino acids) followed by the mature enzyme composed of 212 amino acids. Indeed, this proenzyme consists of 345 amino acids (Taylor et al., 1999, Azarkan et al., 2003).
(77) The more precise analysis of low intensity signals makes it possible to identify the presence of a less specific binding of the antibody to proteins having a molecular weight of 28 kDa for the insoluble and water-insoluble fractions as well as for a protein of 22 kDa for the water-soluble fraction.
(78) For the water-insoluble fraction, the anti-papain antibody did not bind to the protein having a molecular weight of 23 kDa. This confirms that this protein is not a protease of the papain type but the lipase of Carica papaya.
(79) Characterization by Zymogram Gel
(80) The Zymogram gel technique makes it possible to determine the presence of enzymes in a protein mixture by highlighting their own activity. A specific substrate is previously included in the polyacrylamide gel, the enzymes forming separate tracks and revealed by the same reaction are called isozymes.
(81) The implementation of two Zymogram gels (lipolytic and proteolytic) was considered in order to locate the presence of proteases and Carica papaya lipase in the various extracts.
(82) Zymogram to Reveal Proteases
(83) The Novex Casein Zymogram Zymogram gel used is composed of a separation gel composed of 12% polyacrylamide and containing 10% casein, a potential substrate for proteases. This gel is produced under non-reducing conditions in order to preserve enzymatic activities. Enzymes with proteolytic activity will locally hydrolyze the gel casein. After staining the gel with Coomassie blue, the zones where the proteins have been hydrolyzed are highlighted by their white color on a uniform blue background, as shown in
(84) The hydrolysis of casein, and therefore the presence of a more or less important protease activity, is observed for all the fractions analyzed (
(85) Zymogram to Reveal Lipases
(86) The production of Zymograms for detecting lipase enzyme activity is more complex than for proteases. The literature describes a system consisting of two gels, a first one wherein the proteins are separated and a second containing the substrate (Gilbert et al., 1991, Abousalham et al., 2000). In a first step, the proteins migrate on a polyacrylamide gel under native conditions. Then, the presence of lipases is revealed by applying an overlayer composed of a second polyacrylamide gel containing both the lipid substrate and a colored indicator, Victoria Blue, the color of which is blue in an acid medium and red in basic medium (Yadav et al., 1997). In this type of Zymogram, the presence of lipase activity is therefore displayed by the appearance of a blue color corresponding to the hydrolysis zones of the triacylglycerols of olive oil, and therefore to the production of oleic acid.
(87) Two polyacrylamide gels are implemented in parallel. A first control gel is stained with Coomassie blue (
(88) More specifically,
(89) The tracks for the Zymogram gels which are the objects of
(90) papaya sap for track 1,
(91) the water-insoluble fraction of papaya sap for track 2,
(92) the water-soluble fraction of papaya sap for track 3,
(93) commercially purchased papain (Fluka) for track 4, and
(94) to a molecular weight marker for the track (M).
(95) Under non-denaturing conditions, the migration is not precise and only two protein entities are observed. (
(96) With regard to the Zymogram itself, a blue coloration of the gel corresponding to an oleic acid production is observed for the papaya sap as well as for its water-insoluble fraction. Lipolytic activity is present only in these fractions.
(97) Commercial papain has no lipase activity. The 23 kDa protein present in this extract therefore does not correspond to a lipase as the study of the enzymatic activities of the various fractions had already suggested (see
(98) This Zymogram study makes it possible to reveal proteolytic and lipolytic activity has confirmed that the papaya endopeptidases are a group of proteins with significant proteolytic enzymatic activity in the extracts studied.
(99) This study also demonstrated that only papaya sap and its water-insoluble fraction have lipolytic activity that is bound to a protein of a molecular weight close to 23 kDa.
(100) GPC (Gas Chromatography) Method Used to Quantify 1,2-Diacylglycerol
(101) The assay is performed by gas chromatography using an internal standard, 1,3-dipalmitine. The analysis is carried out on an apolar type capillary column (HP5 (30 m×0.25 mm×0.25 mm), using Agilent 7890 gas chromatography controlled by ChemStation® software and having a flame ionization detector and an automatic injector.
(102) The vector gas being helium (1.0 ml/min), the injector and the detector being heated to 330° C., the oven being programmed in isotherm at 325° C. for 30 minutes. The volume of sample injected being 1 μl. Under these conditions, 1,3-dipalmitine has a retention time of 13 minutes, respectively. The various diacylglycerols possess a retention time determined by the use of standards, previous bibliographic studies as well as preliminary analyzes carried out by gas chromatography coupled with mass spectrometry.
(103) Determination of response coefficients, the working hypothesis being that all 1,2-diacylglycerols have the same response coefficient as 1,2-diolein, 1,2-diolein is used as the reference molecule.
(104) Standard solutions: prepare four solutions as follows by weighing exactly the following masses:
(105) TABLE-US-00005 TABLE 5 Preparation of standard solutions Product Solution 1 Solution 2 Solution 3 Solution 4 1,3-dipalmitine 20 mg 20 mg 10 mg 10 mg 2-diolein 20 mg 10 mg 20 mg 10 mg Pyridine 500 μL HMDS 400 μL TFA 50 μL
(106) Under our analysis conditions, the response coefficient of 1,2-diolein is 1.2315 (with a CV of 1.63%).
(107) The enriched oil is then analyzed as follows:
(108) TABLE-US-00006 TABLE 6 Enriched Oil Analysis Product Solution 5 Solution 6 1,2-Diacylglycerol- 100 mg 100 mg Enriched Oil 1,3-dipalmitine 15 mg 15 mg Pyridine 500 μL HMDS 400 μL TFA 50 μL
(109) The amount of 1,2-diacylglycerol contained in the solution is then determined as follows: Calculation of the mass of 1,2-diolein contained in the solution:
m(Ech)=[α(Ech)×A(Ech)×m(SI)]/A(SI)
With: m=mass (in mg) A=area under the peak α=Response coefficient Ech=sample (1,2-diacylglycerols) SI=internal standard (1,3-dipalmitine)
Or:
m(1,2-diacylglycerols)=[1,2315×ΣA(1,2-diacylglycerols)×m(1,3-dipalmitine)]/A(1,3-dipalmitine)
Calculation of content: Content (%)=[m(1,2-diacylglycerols)/m(enriched oil)]×100
Example 1—Method for Obtaining the Water-Insoluble Fraction of Carica papaya Sap, Enriched with the Lipase of Carica papaya
(110) The lipase-enriched Carica papaya extract is obtained by producing a suspension of 100 g of dried raw papaya sap in 900 g of distilled water. The dried raw papaya sap is thus a biphasic suspension comprising suspended particles and a water-soluble fraction.
(111) The mixture is agitated with an anchor-type agitator at a speed of 500 rotations per minute (rpm) for 2 hours at room temperature.
(112) The water-insoluble (non-water-soluble) fraction of papaya sap (as characterized above) is separated from the soluble fraction by centrifugation of the mixture using a centrifuge, such as a plate centrifuge with a flow rate of 200 liters per hour, for 30 minutes.
(113) The centrifugation pellet has the fraction of interest, i.e., the water-insoluble fraction of the raw papaya sap.
(114) 20 g of particulate fraction is thus obtained having a dry weight of 25%.
Example 2—Method for Preparing an Activated 1,2-Diacylglycerol-Enriched Oil
(115) The mixture is prepared in two separate steps, then bringing together the products from these two methods in appropriate proportions, by simple mixing.
(116) The 1,2-diacylglycerol-enriched oil is prepared from virgin Calophyllum inophyllum or Raspberry or Baobab or Evening Primrose or Brazil Nut or Camellia oil (because they offer excellent cutaneous cosmetic properties when they are activated, within the meaning of this invention) and an amount of water or saline solution containing a bivalent ion (such as a calcium or magnesium ion), representing from 0.5 to 50%, and preferably from 1 to 40%, of the volume of the oil. In a thermostatically controlled tank, this mixture is maintained at a temperature of between 30 and 70° C., preferably at a temperature of 50° C. This mixture is maintained under agitation using stator rotor type equipment in order to allow the formation of an emulsion between the oil and the water.
(117) This mixture then remains under agitation in the presence of a given volume of the water-insoluble fraction of the Carica papaya lipase-enriched Carica papaya sap, obtained by implementing the method of Example 1, in a volume ratio water-insoluble fraction of Carica papaya sap/fat used comprised of between about 0.01 and about 0.2, advantageously between about 0.05 and 0.015, preferably about 0.1.
(118) The agitation and the temperature are maintained for a period of between one and six hours (preferably four hours), that is to say for a time sufficient to maximize the amount of diacylglycerols formed.
(119) Monitoring of the reaction is carried out by determining the conventional fat industry indicators (measurement of the acidity index NF EN ISO 660 and peroxide index NF ISO 3976), and by the use of chromatographic methods, notably by gas chromatography for qualifying and quantifying the diacylglycerol content.
(120) The reaction medium is then purified by physicochemical purification techniques, the goal being to obtain a glossy, odorless 1,2-diacylglycerol-enriched oil that has a color closest to the oil initially used for the reaction.
(121) The reaction medium is first filtered through a series of cellulose filters of decreasing porosity (500 to 250 μm). This initially allows the emulsion to breakdown and second to clarify the medium.
(122) The residual water is then removed using an anhydrous magnesium sulfate desiccant. The oil thus obtained is then deodorized and rendered glossy with the help of an activated carbon (preferably CN1 type Cabot Norit® activated carbon).
(123) The characteristics of the oil thus obtained has a common part inherent in the method described in the invention but also specific characteristics intrinsic to the nature of the oil used. The reaction medium mainly consists of unprocessed triglycerides, monoglycerides and 1,2- and 1,3-type diacylglycerols.
(124) The oils are commonly characterized by an acidity index of between 15 and 50, a peroxide index of between 5 and 30 and a 1,2-diacylglycerol rate of between 5 and 30%.
(125) The nature of the diacylglycerols obtained is based on the triacylglycerols initially present in the oil.
(126) Tables 7 and 8 below make it possible to compare the acidity index, the peroxide index and the content of the 1,2-diacylglycerols and the 1,3-diacylglycerols of:
(127) six virgin oils, particularly preferred in the sense of this invention, with
(128) the same six virgin 1,2-diacylglycerol-enriched oils.
(129) TABLE-US-00007 TABLE 7 Acidity Index, Peroxide Index, and 1,2-Diacylglycerol and 1,3-Diacylglycerol Content of the Six Preferred Virgin Oils Initial Virgin Oil Evening Rasp- Bao- Prim- Brazil Camel- Calophyllum berry bab rose Nut lia Inophyllum Oil Oil Oil Oil Oil Oil Acidity Index 4 5 4 3 5 20 (mg/g) Peroxide Index 15 14 10 14 12 8 (meq O.sub.2/Kg) 1,2 0.5 0 0 0 0.3 2 diacylglycerol Content 1,3 diacylglycerol 0.5 0 0 0 0.4 1 Content
(130) TABLE-US-00008 TABLE 8 Acidity Index, Peroxide Index, and 1,2-Diacylglycerol and 1,3-Diacylglycerol Content of the Six Preferred Virgin Oils, after Enrichment with 1,2 DAG Oil After Enrichment 1,2 DAG 1,2 DAG 1,2 1,2 DAG 1,2 DAG 1,2 DAG Enriched- Enriched- DAG Enriched- Enriched- Enriched- Evening Brazil Enriched- Calophyllum Raspberry Oil Baobab Oil Primrose Oil Nut Oil Camellia Oil Inophyllum Oil Acidity Index (mg/g) 21.8 27.1 21.6 18.2 22.3 48.6 Peroxide Index 29.6 2.5 25.8 18.7 12.9 4.2 (meq 0.sub.2/Kg) 1,2 12 16 16.9 17.7 18 8 diacylglycerol Content 1,3 diacylglycerol 2 3.5 2.5 2 2 1.5 Content
(131) Furthermore, a liquid fatty alcohol-enriched wax is prepared by implementing mutatis mutandis the preparation method described above, namely by using a wax instead of an oil and obtaining a fatty alcohol-enriched wax. For the sake of completeness, this method is described below:
(132) Firstly jojoba wax is emulsified using a stator rotor type agitator, by mixing said wax with a quantity of water or saline solution containing a divalent ion such as a calcium or magnesium ion, representing from 0.5 to 50%, and preferably from 1 to 40% of the volume of wax, without pH modification. In a thermostatically controlled tank, this mixture is maintained at a temperature of between 30 and 70° C., preferably at a temperature of 50° C. This mixture is maintained under agitation using a stator rotor type equipment to allow the formation of a stable emulsion between the wax and the water.
(133) This mixture then remains under agitation in the presence of a given volume of the water-insoluble fraction of the Carica papaya lipase-enriched Carica papaya sap, obtained by implementing the method of Example 1, in a volume ratio water-insoluble fraction of Carica papaya sap/fat used comprised of between about 0.01 and about 0.2, advantageously between about 0.05 and 0.015, preferably about 0.1.
(134) The agitation and the temperature are maintained for a period of between one and six hours (preferably six hours), that is to say for a time sufficient to maximize the amount of fatty alcohols formed.
(135) The reaction medium is first filtered through a series of cellulose filters of decreasing porosity (500 to 250 μm). This initially allows the emulsion to breakdown and second allows the medium to clarify.
(136) The residual water is then removed using an anhydrous magnesium sulfate desiccant. The wax thus obtained is then deodorized and rendered glossy with the help of an activated carbon (preferably CN1 type Cabot Norit® activated carbon).
(137) The reaction product is carried out by measuring the acidity index NF EN ISO 660 and by using chromatographic methods, notably by gas chromatography which makes it possible to qualify and quantify the content of fatty acids and fatty alcohols.
(138) The product obtained is preferably characterized by a fatty alcohol content of between 2 and 4%, and an acidity index of 5 mg/g of wax.
(139) In a particular embodiment of the invention, the composition corresponds to a 1,2-diacylglycerol-enriched oil (activated oil) or a mixture of several 1,2-diacylglycerol-enriched oils (activated oils).
(140) In a particular embodiment of the invention, the composition corresponds to a fatty alcohol-enriched liquid wax (activated wax) or a mixture of several fatty alcohol-enriched liquid waxes (activated waxes).
(141) In a preferred embodiment, the composition according to the invention is then obtained by simply mixing the 1,2-diacylglycerol-enriched oil (activated oil) and the fatty alcohol-enriched liquid wax (activated wax) in weight proportions comprised of between 100 and 90% of enriched oil and 0 to 10% enriched liquid wax.
Example 3 Cosmetic Formulations Including Activated Oil
(142) 3.1—Eye Contour Gel
(143) TABLE-US-00009 TABLE 9 Composition of an “Eye Contour” gel according to the invention INGREDIENTS BUSINESS NAME/INCI NAME % w/w Phase A Purified Water Water/Aqua Qs 100 Activated tamanu (Proposed) Hydrolyzed Calophyllum 1.50 oil/activated jojoba Inophyllum Seed Oil and Hydrolyzed wax blend according to Jojoba Esters Example 2 Flexithix ™ polymer PVP 3.00 Phase B Si-Tec ™ DM 350 Dimethicone 3.00 silicones Si-Tec ™ RE-100 Cyclopentasiloxane (and) 10.00 silicones Dimethicone/Vinyltrimethylsiloxysilicate Crosspolymer Cyclopentasiloxane Cyclopentasiloxane 5.00 Phase C Liquid germall plus Propylene glycol (and) Diazolidinyl 0.50 preservative urea (and) Iodopropynyl butylcarbamate BPD-500 HDI/Trimethylol hexyllactone 0.50 crosspolymer & Silica Total 100.00
(144) Properties: Appearance: Smooth, semi-transparent gel pH: 5.50-6.0 Viscosity (DO) 15000-25000 (Brookfield RVT/Spindle B/5 RPM/1 minute/25° C.)
(145) This formula underwent a 3-month accelerated stability test in the laboratory. The preservation of this formula has been validated by a double efficacy test over 28 days. However, the preservatives have not been optimized to their lowest level of efficiency.
(146) 3.2—Biphasic Facial Serum
(147) TABLE-US-00010 TABLE 10 Composition of a biphasic facial serum according to the invention INGREDIENTS BUSINESS NAME/INCI NAME % w/w Phase A Purified Water Water/Aqua Qs 100 Phase B Blanose ™ 7H3SF CMC Carboxymethylcellulose 0.30 Zemea* Propanediol 1.00 Phase C Rokonsal ™/Liquapar ™ MEP Phenoxyethand (and) 0.70 preservative Methylparaben (and) Ethylparaben (and) Propylparaben Neomatrix ™ biofunctional Water/Aqua (and) Glycerin 1.00 (and) Pentapeptide (proposed name) Sodium Chloride Sodium Chloride 1.00 Unicert* Blue 05601-J (sol. Water/Aqua (and) CI 42090 0.20 0.1%) (FD&C Blue No. 1) Unicert* Yellow 08005-J (sol Water/Aqua (and) CI 19140 0.60 0.1%) (FD&C Yellow No. 5) Phase D Ceraphyl ™ ODS ester Octyldodecyl Stearate 7.00 Unicert* Green K7016-J CI 61565 (D&C Green No. 6) 0.006 Phase E Optiphen ™ preservative Phenoxyethanol (and) 0.50 Caprylyl Glycol Ceraphyl ™ 375 ester Isostearyl Neopentanoate 3.00 Activated tamanu (Proposed) Hydrolyzed 2.50 oil/activated jojoba wax Calophyllum Inophyllum Seed blend according to Example Oil and Hydrolyzed Jojoba 2 Esters Smart* 5 Isododecane (and) 8.00 Hydrogenated tetradecenyl/methylpentadecene DC* FZ-3196 Caprylyl Methicone 5.00 PF Absolute Perfection Perfum/Fragrance (and) Linalool 0.10 Total 100.00
(148) Properties: Appearance: Green and dark blue biphasic liquid—shake before use pH: 5.0-6.0 Viscosity (DO) N.A.
(149) This formula underwent a 3-month accelerated stability test in the laboratory. The preservation of this formula has been validated by a double efficacy test over 28 days. However, the preservatives have not been optimized to their lowest level of efficiency.
Example 4—Study of the Expression of Cytokeratin in Ex Vivo Human Skin, in the Presence of Different Activated Oils According to the Invention
4.1—Introduction to Cytokeratins
(150) Cytokeratins are proteins of the intermediate filaments, which together with other proteins form the cytoskeleton of the cells. These have many functions including the maintenance of epithelial structure, protection from injury, and communication with other cytoplasmic components (Fuertes L. et al., 2013).
(151) They are expressed in pairs, with different expression profiles depending on their location and are numerically ranked from 1 to 20 according to their molecular weight and isoelectric point. Cytokeratins are classified into two groups (Fuertes L. et al., 2013): Type I cytokeratins, which are acids and generally correspond to cytokeratins having a low molecular weight, Type II cytokeratins, which are basic and generally have a high molecular weight.
(152) During the process of epidermal differentiation, the keratinocytes of the basal layer lose their proliferative potential, migrate to the upper layers and the expression of keratins 5 and 14 is interrupted while that of keratins 1 and 10 increases (Paladini R D et al., 1999).
4.2—Purpose of the Study
(153) The purpose of this study is to determine the influence of various activated oils/activated wax blends according to the invention, obtained by implementing the method of Example 2, on the expression of cytokeratins in ex vivo human skin.
4.3—Oils Tested
(154) The different oils tested are:
(155) Activated tamanu oil (Calophyllum inophyllum)/activated jojoba wax blend (abbreviated in the rest of Example 4 as “activated tamanu oil”) obtained by the method of Example 2, versus native virgin tamanu oil,
(156) Activated baobab oil/activated jojoba wax blend (abbreviated in the rest of Example 4 as “activated baobab oil”) obtained by the method of Example 2, versus native virgin baobab oil,
(157) Activated raspberry oil/activated jojoba wax blend (abbreviated in the rest of Example 4 as “activated raspberry oil”) obtained by the method of Example 2, versus native virgin raspberry oil,
(158) Activated evening primrose oil/activated jojoba wax blend (abbreviated in the rest of Example 4 as “activated evening primrose oil”) obtained by the method of Example 2, versus native virgin evening primrose oil.
4.3—Protocol
(159) Human skin samples that are 6 mm in diameter are cultured in air/liquid interface. The activated oils are diluted to 0.5% in the corresponding non-activated oil. 20 μL of oil are applied topically to the skin samples (only one application), then these biopsies are incubated for 24 hours at 37° C. At the end of the culturing, the human skin samples are included in optimal cutting temperature (OCT) gel. The OCT solidifies upon contact with the cold (liquid nitrogen). These blocks containing the skin samples are stored at −20° C.
(160) Then 6 μm thick sections are taken. These sections of skin samples are then oven-dried at 37° C., and then fixed with acetone (pre-cooled to −20° C.).
(161) Immunolabeling is performed using a pancytokeratin-specific mouse monoclonal antibody (Abcam, Ref. ab27988), followed by a fluorochrome-coupled anti-mouse secondary antibody (Invitrogen, Ref A21202), followed by sections of skin samples are then examined under an Epi-fluorescence microscope (Zeiss Axiovert 200M). A quantification of the fluorescence, from the photographs obtained, was carried out with Volocity software.
4.4—Results
(162) Microscopic observations show a significantly more intense fluorescence in the epidermis of biopsies treated with activated tamanu oil, activated baobab oil, activated raspberry oil and activated evening primrose oil compared to the corresponding non-activated oil. The data obtained are presented below, in Tables 11-14.
(163) TABLE-US-00011 TABLE 11 Cytokeratin Expression (%) Activated Tamanu Oil Vs. Virgin Tamanu Oil 0.5% Activated Tamanu Oil Virgin Tamanu Oil Cytokeratin 123.3 ± 4.6 100 ± 4.7 expression (%)
(164) TABLE-US-00012 TABLE 12 Cytokeratin Expression (%) Activated Baobab Oil Vs. Virgin Baobab Oil 0.5% Activated Baobab Oil Virgin Baobab Oil Cytokeratin expression 112.7 ± 2.6 100 ± 3.1 (%)
(165) TABLE-US-00013 TABLE 13 Cytokeratin Expression (%) Activated Raspberry Oil Vs. Virgin Raspberry Oil 0.5% Activated Raspberry Oil Virgin Raspberry Oil Cytokeratin expression 124.7 ± 2.8 100 ± 2.2 (%)
(166) TABLE-US-00014 TABLE 14 Cytokeratin Expression (%) Activated Evening Primrose Oil Vs. Virgin 0.5% Activated Evening Virgin Evening Primrose Oil Primrose Oil Cytokeratin expression 124.8 ± 4.6 100 ± 4.2 (%)
(167) Evening Primrose Oil
(168) Statistical analysis was performed versus the corresponding non-activated oil (mean±sem, n=8, ***: highly significant with Student's t-test).
(169) In order to highlight the difference in expression observed between the activated oils and the corresponding virgin oils, the data obtained were plotted on the graph shown in
4.5—Conclusions
(170) The expression of cytokeratins is increased in ex vivo human skin by activated tamanu oil, activated baobab oil, activated raspberry oil and activated evening primrose oil.
(171) Thus, these activated oils stimulate epidermal differentiation and therefore have interesting cosmetic properties.
4.6—Bibliographical References
(172) Fuertes L., Santonja C., Kutzner H. and Requena L. Immunohistochemistry in Dermatopathology: a review of the most commonly used antibodies (Part I). Actas Dermo-Sifiliográficas. 104(2):99-127 (2013) Paladini R. D. and Coulombe P. A. The functional diversity of epidermal keratins revealed by the partial rescue of the keratin 14 null phenotype by keratin 16. The Journal of Cell Biology. 146 (5):1185-1201 (1999)