Water soluble and activable phenolics derivatives with dermocosmetic and therapeutic applications and process for preparing said derivatives   

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No. 12/304,212, filed Dec. 10, 2008, which is the U.S. national stage application of International Patent Application No. PCT/EP2007/055815, filed Jun. 13, 2007, the disclosures of which are hereby incorporated by reference in their entireties, including all figures, tables and amino acid or nucleic acid sequences.

FIELD OF THE INVENTION

The present invention relates to the preparation of phenolics derivatives, pharmaceutic and cosmetic compositions comprising such phenolics derivatives, and their use for the beauty of the skin and for treating diseases.

BACKGROUND OF THE INVENTION

Phenolic Compounds and their Properties

Phenolic compounds (also called phenolics), or polyphenols, constitute one of the most numerous and widely-distributed groups of substances in the plant kingdom, with more than 8,000 phenolic structures currently known. Polyphenols are products of the secondary metabolism of plants. The expression “phenolic compounds” embraces a considerable range of substances that possess an aromatic ring bearing one or more hydroxyl substituents. Most of the major classes of plant polyphenol are listed in Table 1, according to the number of carbon atoms of the basic skeleton. The structure of natural polyphenols varies from simple molecules, such as phenolic acids, to highly polymerized compounds, such as condensed tannins (HARBORNE J B (1980) Plant phenolics. In: BELL EA, CHARLWOOD BV (eds) Encyclopedia of Plant Physiology, volume 8 Secondary Plant Products, Springer-Verlag, Berlin Heidelberg N.Y. Pp: 329-395).

The three important groups for humans are phenolic acids (C6-C1, C6-C2 and C6-C3), flavonoids (C6-C3-C6) and high-molecular weight polyphenols (more than 30 carbon atoms). Indeed, the phenolics, particularly polyphenols, exhibit a wide variety of beneficial biological activities in mammals, including antiviral, antibacterial, immune-stimulating, antiallergic, antihypertensive, antiischernic, antiarrhytmic, antithrombotic, hypocholesterolemic, antilipoperoxidant, hepatoprotective, anti-inflammatory, anticarcinogenic antimutagenic, antineoplastic, anti-thrombotic and vasodilatory actions. They are powerful antioxidants in vitro.

TABLE I The major classes of phenolic compounds (or phenolics) in plants (HARBORNE JB, 1980) NUMBER OF CARBON BASIC ATOMS SKELETON CLASS EXAMPLES  6 C6 Simple phenols Catechol, hydroquinone Benzoquinones 2,6- Dimethoxybenzoquinone  7 C6-C1 Phenolic acids Gallic, salicylic  8 C6-C2 Acetophenones 3-Acetyl-6- Tyrosine derivatives methoxybenzaldehyde Phenylacetic acids Tyrosol p-Hydroxyphenylacetic  9 C6-C3 Hydroxycinnamic Caffeic, ferulic acids Myristicin, Phenylpropenes eugenol Umbelliferone, aesculetin Coumarins Bergenon Isocoumarins Eugenin Chromones 10 C6-C4 Naphthoquinones Juglone, plumbagin 13 C6-C1-C6 Xanthones Mangiferin 14 C6-C2-C6 Stilbenes Resveratrol Anthraquinones Emodin 15 C6-C3-C6 Flavonoids Quercetin, cyanidin Isoflavonoids Genistein 18 (C6-C3)2 Lignans Pinoresinol Neolignans Eusiderin 30 (C6-C3-C6)2 Biflavonoids Amentoflavone n (C6-C3)n Lignins (C6)n Catechol melanins (C6-C3-C6)n Flavolans (Condensed Tannins)

Among the phenolic acids, the most important constitutive carbon frameworks are the hydroxybenzoic (C6-C1) and hydroxycinnamic (C6-C3) structures. The hydroxybenzoic acid content of edible plants is generally very low, with the exception of certain red fruits, black radish and onions, which can have concentrations of several tens of milligrams per kilogram fresh weight. Hydroxybenzoic acids are components of complex structures such as hydrolyzable tannins (gallotannins in mangoes and ellagitannins in red fruits such as strawberries, raspberries and blackberries). The hydroxycinnamic acids are more common than are the hydroxybenzoic acids and consist chiefly of p-coumaric, caffeic, ferulic and sinapic acids. These acids are rarely found in the free form, except in processed food that has undergone freezing, sterilization or fermentation. The bound forms are glycosylated derivatives or esters of quinic acid, shikimic acid and tartaric acid. Caffeic acid and quinic acid combine to form chlorogenic acid, which is found in many types of fruit and in high concentration in coffee. Caffeic acid, both free and esterified, is generally the most abundant phenolic acid and represents between 75% and 100% of the total hydroxycinnamic acid of most fruit (MANACH C, SCALBERT A, MORAND C, REMESY C, JIMENEZ L (2004) Polyphenols: food sources and bioavailability. Am J Clin Nutr 79: 727-747).

The flavonoids consist of a large group of low-molecular weight polyphenolic substances, benzo-γ-pyrone derivatives that are diverse in chemical structure; they represent the most common and widely distributed group of plant phenolics. The flavonoids common structure is that of diphenylpropanes (C6-C3-C6); it consists of two aromatic rings (cycles A and B) linked through three carbons that usually form an oxygenated heterocycle (cycle C). FIG. 1 shows the basic structure and the system used for the carbon numbering of the flavonoid nucleus. Structural variations within the rings subdivide the flavonoids into several families: flavonols, flavones, flavanols, isoflavones, antocyanidins and others. These flavonoids often occur as glycosides, glycosylation rendering the molecule more water-soluble and less reactive toward free radicals. The sugar most commonly involved in glycoside formation is glucose, although galactose, rhamnose, xylose and arabinose also occur, as well as disaccharides such as rutinose. The flavonoid variants are all related by a common biosynthetic pathway, incorporating precursors from both the shikimate and the acetate-malonate pathways (CROZIER A, BURNS J, AZIZ A A, STEWART A J, RABIASZ H S, JENKINS G I, EDWARDS C A, LEAN M E J (2000) Antioxidant flavonols from fruits, vegetables and beverages: measurements and bioavailability. Biol Res 33: 79-88). Further modifications occur at various stages, resulting in an alteration in the extent of hydroxylation, methylation, isoprenylation, dimerization and glycosylation (producing O- or C-glycosides). Phenolic compounds act as antioxidants with mechanisms involving both free radical scavenging and metal chelation. Indeed, excess levels of metal cations of iron, zinc and copper in the human body can promote the generation of free radicals and contribute to the oxidative damage of cell membranes and cellular DNA; by forming complexes with these reactive metal ions, they can reduce their absorption and reactivity. It has to be underlined that though most flavonoids chelate Fe, there are large differences in the chelating activity. In particular, the dihydroflavonol taxifolin chelates more efficiently Fe2+ than the corresponding flavonol quercetine (VAN ACKER S A B E, VAN DEN BERG D J, TROMP M N J L, GRIFFIOEN D H G, VAN BENNEKOM, VAN DER VIJGH W J F, BAST A (1996) Structural aspects of antioxidant activity of flavonoids. Free Radic Biol Med 20: 331-342).

Flavonoids have ideal structural chemistry for free radical-scavenging activities (several studies have shown the flavonoids to act as scavengers of superoxide anions, singlet oxygen, hydroxyl radicals and lipid peroxyl radicals by rapid donation of a hydrogen atom). One important finding from the studies of the relationship between the structural characteristics of flavonoids and their antiradical activity is that a catechol moiety (3′,4′-dihydroxyphenol) on ring B is required for good scavenging activity. Recently, this statement was confirmed with nevertheless a modulation: in a study about the relationship between the structural characteristics of 29 flavonoids and their antiradical activity, it was indeed observed that the catechol structure in the B ring is not always a conditio sine qua non in achieving high free radical scavenging activity and that highly active flavonoids possess a 3′,4′-dihydroxy B ring and/or a 3-OH group (AMIC D. DAVIDOVIC-AMIC D, BESLO D, TRINAJSTIC N (2003) Structure-radical scavenging activity relationships of flavonoids. Croatica Chem Acta 76: 55-61). Flavonoids have been shown to be more effective antioxidants in vitro than vitamins E and C on a molar basis (RICE-EVANS C A, MILLER N J, PAGANGA G (1997) Antioxidant properties of phenolic compounds. Trends in Plant Science 2: 152-159). There are also reports of flavonoids inhibiting the activity of enzymes such as oxygenases.

It must be underlined that the hydrophobicity of polyphenols is intermediate between that of vitamin C (highly hydrophilic) and that of vitamin E (highly hydrophobic); polyphenols are thus expected to act at water-lipid interfaces and may be involved in oxidation regeneration pathways with vitamin C and E.

Phenolics Derivatives and their Preparation

Due to their low aqueous solubility and/or high sensitivity toward oxidation, the use of phenolics in pharmaceutical or cosmetic preparations requires adapted and specific formulations. Since these formulations must also satisfy the constraints associated with their final usage, the compromise between acceptability, concentration and stability is often difficult to reach.

More water soluble and/or oxidation resistant forms of phenolics such as the glycosides are not always available in nature and may demand, when they exist, complex procedures of extraction and purification from the plant material. Both chemical and biochemical (enzymatic) approach have been attempted to increase water solubility and/or stability. As phenolic compounds have several free hydroxyl groups, attempts for chemical modifications of phenolic compounds lead to unselective reactions, generating a panel of different molecules. Further steps of purification are then required to recover the desired product(s).

As far as the biochemical approach is concerned, three ways have been investigated to date to obtain phenolics glycosides and basically flavonoids glycosides.

The first way relies on glycosyltransferases able to transfer the sugar moiety of a sugar nucleotide to an acceptor (in the case of UDP-glucose:glucosyltransferases (UGT), glucose is transferred from uridine 5′-diphosphoglucose). These enzymes, which contribute in the synthesis of secondary metabolism in plants, have broad acceptor substrate specificities (LIM E K, HIGGINS G S, BOWLES D J (2003) Regioselectivity of glucosylation of caffeic acid by UDP-glucose:glucosyltransferase is maintained in planta. Biochem J 373: 987-92; LIM E K, ASHFORD D A, HOU B, JACKSON R G, BOWLES D J (2004) Arabidopsis glycosyltransferases as biocatalysts in fermentation for regioselective synthesis of diverse quercetin glucosides. Biotechnol. Bioeng. 87(5): 623-31). Nevertheless, this approach is impaired by the very high cost of the sugar nucleotides and the regeneration of the sugar nucleotide substrate, which is a way to decrease the substrate cost, is difficult to master at large scale.

The second way relies on saccharide—transferring enzymes able to transfer glucose from an α-glucosyl saccharide. Said enzymes are selected from the hydrolases α-glucosidase (EC 3.2.1.20) and α-amylase (EC 3.2.1.1), and from the transferase cyclodextrin-glucanotransferase (EC 2.4.1.19). Their substrates are amylose, dextrins, cyclodextrins, maltooligosaccharides and partial starch hydrolysates, all of them containing mainly or exclusively glucosyl residues linked to each other through an α 1→34 osidic bond. According to this approach, U.S. Pat. No. 5,565,435 states that α-glucosyl quercetin is obtained. It has to be underlined that the starch degrading enzymes link the glucosyl residue to the flavonoid through an α-osidic bond whereas the UDP-glucose:glucosyltransferase investigated by LIM et al. links the glucosyl residue to the flavonoid through a β-osidic bond. It has also to be underlined that in the conditions described in U.S. Pat. No. 5,565,435, the quercetin molecule could be solubilized by adjusting the pH at 8.5 and by maintaining the reaction medium at 60° C. The solubilisation of phenolics in alkaline media is due to the formation of phenolates; in these pH and temperature conditions, the stability of the substrate was achieved by operating under anaerobic conditions. It thus appears that this mode of preparation is highly difficult to control and manage and that a simple mode of preparation should be valuable.

The third way involves glucosyltransferases using sucrose (β-D-fructofuranosyl-α-D-glucopyranoside) as glucosyl donor and producing glucans with the release of fructose. Several attempts have been achieved with this class of enzymes to try to get phenolics glucosides. First, the glucosyltransferase from Streptococcus sobrinus (referenced by the authors as strain 6715, serotype g) was proven to catalyze the synthesis of 4′-O-α-D-glucopyranosyl-(+)-catechin in a strictly aqueous medium (catechin at 1 g/L in 100 mM phosphate buffer pH 6.0 containing 2% sucrose) (NAKAHARA K, KONTANI M, ONO H, KOMADA T, TANAKA T, OOSHIMA T, HAMADA S (1995) Glucosyltransferase from Streptococcus sobrinus catalyzes glucosylation of catechin. Appl. Environ. Microbiol. 61(7): 2768-70). A similar enzyme, the glucosyltransferase-D from Streptococcus mutans GS-5, was proven to be less regioselective, as it catalyzes not only the synthesis of 4′-O-α-D-glucopyranosyl-(+)-catechin but also the synthesis of 7-O-α-D-glucopyranosyl-(+)-catechin and of the diglucosylated derivative 4′,7-O-α-D-diglucopyranosyl-(+)-catechin (MEULENBELD G H, ZUILHOF H, VAN VELDHUIZEN A, VAN DEN HEUVEL R H H, HARTMANS S (1999) Enhanced (+)-catechin transglucosylating activity of Streptococcus mutans GS-5 glucosyltransferase-D due to fructose removal. Appl Environ Microbiol 65(9): 4141-7). Though several investigations regarding the acceptor specificity of Streptococcus mutans GS-5 glucosyltransferase lead the authors to infer that aromatic acceptors appear to require two adjacent aromatic hydroxyl groups (MEULENBELD G H, HARTMANS S (2000) Transglycosylation by Streptococcus mutans GS-5 glucosyltransferase-D: acceptor specificity and engineering reaction conditions. Biotechnol Bioeng 70(4): 363-9), this statement was counteracted by the identification of glucosylation at position 7 in catechin (MEULENBELD et al., 1999) and by the synthesis of non-pyrocatechol derivatives. Indeed, pinosylvin and resveratrol, respectively 3,5-dihydroxy-trans-stilbene and 3,4′,5-trihydroxy-trans-stilbene, were glucosylated by a crude glucosyltransferase preparation produced by Streptococcus mutans to form respectively 3-O-α-D-glucopyranosyl-(E)-pinosylvin and 3-O-α-D-glucopyranosyl-(E)-resveratrol (SHIM H, HONG W, AHN Y (2003) Enzymatic preparation of phenolic glucosides by Streptococcus mutans. Bull Korean Chem Soc 24(11): 1680-2). Very recently, it was claimed that the flavonols quercetin and myricetin and the flavone luteolin could be glucosylated by special glucansucrases, namely the Leuconostoc mesenteroides NRRL B-512F dextransucrase (sucrose:1,6-α-D-glucan 6-α-D-glucosyltransferase, EC 2.4.1.5) and the Leuconostoc mesenteroides NRRL B-23192 alternansucrase (sucrose:1,6(1,3)-α-D-glucan 6(3)-α-D-glucosyltransferase, EC 2.4.1.140) (BERTRAND A, MOREL S, LEFOULON F, ROLLAND Y, MONSAN P, REMAUD-SIMEON M (2006) Leuconostoc mesenteroides glucansucrase synthesis of flavonoid glucosides by acceptor reactions in aqueous-organic solvents. Carbohydr Res 341: 855-63). Conventionally, in the presence of sucrose, the former produces a glucan (dextran) in which 95% of the glucosidic bonds are α-(1→6) (skeleton of the polysaccharide) and 5% α-(1→3) (branching points), and the later a glucan (alternan) in which the glucosidic bonds are alternatively α-(1→6) and α-(1→3). The obtained flavonoid derivatives were: luteolin-3′-O-α-D-glucopyranoside, luteolin-4′-O-α-D-glucopyranoside, quercetin-3′-O-α-D-glucopyranoside, quercetin-4′-O-α-D-glucopyranoside, quercetin-3′-4′-O-α-D-diglucopyranoside, myricetin-3′-O-α-D-glucopyranoside and myricetin-4′-O-α-D-glucopyranoside. This work demonstrates that yields of glycosides derivatives synthesis not only rely on the enzyme itself (the synthesis of luteolin-O-glycosides dropped down from 44% to 8% between dextransucrase and alternansucrase), but also on slight chemical differences between two acceptors (no conversion was observed with the dextransucrase on diosmetin and diosmin).

From the above significant (though not exhaustive) state of the art regarding the experimented ways to obtain glucosylated derivatives of polyphenols in general (and flavonoids in particular) in order to overcome the main conventional drawbacks of flavonoids (poor water solubility at physiological conditions, in particular at pH ranging from 5 to 7 and 30° C. and high sensitivity to autoxidation in these biological conditions), it clearly appears that no precise guidelines can be deduced to set up the enzymatic production of a specific phenolics glycoside. On the contrary, it shows that there is no way for a man of the art to predict which flavonoid can be glucosylated with which enzyme and in which conditions to obtain high glucoside concentrations (see summary in Table 2). Indeed, though attempts have been made in order to establish a relationship between the phenolic structures and the possibility of their use as glycosyl acceptor by glycosyltransferases, it still appears that the obtention of glycosylated phenolics strongly depends on the nature of the phenolic substance and on the enzyme used for the condensation reaction. This is particularly true with glucosyltransferases synthezing conventionally α-D-glucans from sucrose (EC 2.4.1.5) for which only a very few number of polyphenolic structures have been successfully reported. Furthermore, in the case of the main glucosyltransferases studied, namely S. mutans GS-5 glucosyltransferase D and L. mesenteroides NRRL B-512F dextransucrase, it has to be mentioned that the former synthesizes a water-soluble α-glucan in a primer-stimulated and dependent manner (HAMADA N, KURAMITSU H K (1989) Isolation and characterization of the Streptococcus mutans gtfD gene, coding for primer-dependant soluble glucan synthesis. Infect Immun 56: 1999-2005) whereas the later does not (ROBYT J F, WALSETH T F (1978) The mechanism of acceptor reactions of Leuconostoc mesenteroides NRRL B 512F. Carbohydr Res 61: 433-45). These glucosyltransferases have distinct mechanism of action and consequently molecules that are acceptor for an enzyme are not necessarily acceptor for the other; in other words, as shown in the previously cited works, there is no justification to consider that the substances that act as glucosyl acceptor in the case of S. mutans GS-5 glucosyltransferase D act also as glucosyl acceptor in the case of L. mesenteroides NRRL B-512F dextransucrase and vice versa.