DE10132936B4 - Use of galloylated proanthocyanidin - Google Patents

Abstract

Use of oligomeric and polymeric proanthocyanidins for the treatment of skin and mucous membrane defects and inflammation, for regenerative skin and mucous membrane care, as well as for accelerating the regeneration of skin and mucous membranes, the proanthocyanidins being composed of the basic unit Flavan-3-ol, which is based on the C3 position is at least partially esterified with gallic acid, and the basic units are linked via (C4, C6) or (C4, C8) bonds.

Description

The present invention relates to new uses of oligomers and polymers galloylated proanthocyanidins.

The skin is the largest human organ. leading The task of human skin is in addition to maintaining a constant body temperature and protection against dehydration, especially the barrier function across from the outside world (i.e. opposite physical, chemical and microbial irritants) without the it would not be possible for the vital functions to run smoothly. This Barrier function is caused by the epidermis. By cornification and the formation of lipid layers, the epidermis matures into the horny layer approach that as the outermost layer at the interface represents the actual protective layer against the outside world. Also the Mucosa as a special epithelial form fulfills similar tasks, however due to the less pronounced and hardly stratified in comparison to the skin Epidermis in the mucous membrane in addition to the protective and drying function the selective exchange of substances between the serosal and luminal side is in the foreground.

The cellular organization within the Skin maturation represents an extremely complex one Done, with cellular components (predominantly Keratinocytes) embedded in an extracellular matrix of lipids are. The lipid precursors are also formed by the keratinocytes. In addition, change but the keratinocytes during their life cycle very strong. These skin cells become initial in an undifferentiated state from the basal cell layer formed and represent a proliferating cell mass. Since keratinocytes are continuously formed by the stratum basale, move the cells over time from the bottom towards the interface. in this connection there are characteristic changes in the cellular Appearance: The cells flatten significantly, elongate and enlarge yourself while at the same time the number of mitochondria increases. In further development cycles the Golgi development and core size are reduced, however at the same time, the tonofilament network is growing and becoming denser. Furthermore, the biochemical processes in the keratinocytes are also subject a decisive change in the course of their development. In particular The production of low molecular weight keratins increases as development progresses off while high-molecular, differentiation-specific keratins (K1 and K10) increase significantly. This differentiation towards highly keratinized Cells also work with an increased formation of lipid precursor molecules (e.g. Phospho- and sphingolipids) for the lipid bilayers along with the horny layer. These are lipids later ejected and converted enzymatically into neutral lipids. The themselves so constantly differentiating cells are above a certain keratinization density no longer viable and die apoptotically, but with the horny, dead cells (Corneocytes) next to the lipid matrix in which they are embedded, crucial for are the actual (physical) barrier function of the skin.

This results in two different ones Regulatory processes: Firstly, the proliferation of keratinocytes for permanent Renewal of the cellular Skin components and subsequently the differentiation of keratinocytes towards the horny, dead cells and associated with it to build the epidermal barrier. Damage (endogenous or exogenous Genesis) of the epidermis and the horny layer not only leads to drastic limitations the barrier properties, but also to a blunt, rough and tired Appearance of the skin.

The purpose of nourishing cosmetics consists primarily of the natural function of the skin as a barrier against external influences strengthen and the loss of the body's own Reduce substances (e.g. water, fats, electrolytes) or normal Measure this To restore loss. On the one hand, this causes a increased Resistance or resistance against external influences (e.g. against chemicals, mechanical stress, increased UV exposure or microbial Infestation), on the other hand, they also hit the skin from the outside Substances absorbed to a lesser extent. Not to be neglected is also the aspect that a skin that is washed daily by a certain Loss of lipids and moisture experiences thinning within the extracellular matrix erfäht. A changed one Regeneration of the keratinyocytes can be the result. Skin Care Products should be there accordingly help either by frequent Wash and / or compensate for loss of moisture and fat reduce skin aging, e.g. by increasing the Keratinocyte proliferation and thus the regeneration of the skin is achieved can be. Skin care products and their active ingredients serve the furthermore, the care of the skin and its supply with moisture and nutrients ensure, but above all the promotion of a faster and better regeneration of the epidermal barrier after entering damage initiate and maintain.

Proanthocyanidins are polyphenolic Biopolymers are relatively common occur in plants, i.e. these are natural substances. Is it for the basic building blocks around catechin or epicatechin, so the corresponding oligo- and Polymers also called procyanidins.

The use of proanthocyanidins from plant extracts of different compositions, but often in a mixture with other tanning agents, has already been described in the prior art. The uses described frequently relate to medical / pharmacological applications areas of application, such as virus therapy, wound healing or anti-mutagenic effects. In addition to proanthocyanidins, these extracts may contain a large number of other tanning agents and active ingredients, some of which are not yet sufficiently characterized.

Duration et al. (duration et al., Planta Medica 64 (1998), pp. 324-327) the use of extracts from Hamamelis virginiana as an anti-mutagenic agent (measurement by means of Ames test). The maximum anti-mutagenic effect of the extract is observed here in a fraction containing catechin and gallocatechin oligomers which had an average degree of polymerization of 9.2.

The EP 0 812 592 A1 describes the use of plant extracts, which include proanthocyanidin oligomers, for the treatment of cataracts, ie oxidative damage to the eye. The active ingredients used are obtained as aqueous extracts from grapes, grape skins or grains and then separated using liquid chromatography.

WO 96/30033 also describes Plant extracts enriched with proanthocyanidins which are used for inhibition of microbial adhesion on cell surfaces and thus be used as an anti-microbial agent.

Similarly, the WO 92/06695 the use of proanthocyanidin compositions as an anti-viral agent for the treatment of influenza and herpes viruses caused infections.

Finally, WO 87/00051 describes the use of witch hazel extracts to treat gingivitis (a gum disease).

The JP 100 59 846 A describes a drug for cataracts that contains proanthocyanidins. Furthermore describes the JP 200 106 4172 A an agent for the treatment of defects caused by mutations in the APC gene which contains polyphenols, in particular proanthocyanidins. Another area of application of a medicament containing different forms of proanthocyanidins is from JP 200 004 4472 A described. The drug described there is said to be used to treat diabetes to control blood sugar levels. Ultimately, that describes JP 200 1131 027 A a hair tonic and shampoo containing proanthocyanidin di-pentamers as oligomeric forms.

The JP 631 626 85 A represents an extraction process of proanthocyanidins from vegetable raw materials. In the JP 630 203 21 A an epoxy resin is described, which consists of a polyfunctional epoxy component and a proanthocyanidin.

Despite those disclosed in the prior art numerous possible applications for unfractionated or fractionated extracts, e.g. from witch hazel, chrysanthemum or can also be obtained from grapes or their skins these extracts frequently a big Number of other substances or vegetable secondary substances. It does this frequently difficult to determine a defined active ingredient and target it use. Furthermore, the presence of additives in the extract or preparation the proportion of pharmacologically active substance is reduced and thus the pharmacological efficiency is reduced.

Consequently, there is in the prior art a need to provide structurally defined proanthocyanidins in high purity, so that there is a high concentration of the desired proanthocyanidins or the content of impurities and / or by-products if possible is low.

Based on this state of the art is the object of the present invention in the utilization of substituted proanthocyanidins.

• a) aus der Grundeinheit Flavan-3-ol aufgebaut sind, welche
• b) an der C3-Position mindestens zum Teil mit Gallussäure verestert sind, und wobei
• c) die Grundeinheiten über (C4, C6)- oder (C4, C8)-Bindungen verknüpft sind, und
• d) im wesentlichen frei von anderen Zusatzstoffen ist.

Accordingly, the present invention relates to the use of oligomeric and polymeric proanthocyanidins for the treatment of skin and mucous membrane defects and inflammation, for regenerative skin and mucous membrane care, and for accelerating the regeneration of skin and mucous membranes, the proanthocyanidins from the basic unit flavan-3-ol is built up, which is at least partially esterified with gallic acid at the C3 position, and the basic units are linked via (C4, C6) or (C4, C8) bonds. The invention is therefore based on oligomeric and polymeric proanthocyanidins From the

• a) are constructed from the basic unit Flavan-3-ol, which
• b) are at least partially esterified with gallic acid at the C3 position, and wherein
• (c) the basic units are linked via (C4, C6) or (C4, C8) bonds, and
• d) is essentially free of other additives.

The use of such is also according to the invention substituted proanthocyanidins as a component of cosmetic Products to accelerate the proliferation of keratinocytes, to accelerate the proliferation of cells in cell culture and intended to accelerate the in vivo proliferation of cells.

In the structural formula shown below ( 1 ) four basic units (flavan-3-ol) are shown as examples, which are linked via a (4, 8) bond and, with the exception of the (lower) terminal basic unit, on the C3 OH group are esterified with gallic acid. The esterification of the C3 OH group of the proanthocyanidins according to the invention with gallic acid is at least partially present, ie in an advantageous embodiment at least 50% of the C3 OH groups of the basic unit present are esterified with gallic acid (galloylated), in a preferred embodiment at least 80% of the C3 OH groups of the basic units are galloylated, with a degree of galloylation of at least 90% of the C3 OH groups present being particularly preferred (the percentages are in each case based on 100% of the C3 OH present) -Groups). The possible (4, 6) linkage of the basic units also disclosed in the context of the present invention is not shown in the formula, but represents a further preferred embodiment in the context of the present invention. The numerical values which are advantageous or preferred for n are for the ( 4, 6) -linked and the (4, 8) -linked proanthocyanidins are identical, advantageous values for n being between 1 and 80. Values between 2 and 40 are preferred, values between 3 and 27 are particularly preferred. The proanthocynidines according to the invention can only occur as at least partially galloylated compounds in (4, 6) linkage than at least partially galloylated compounds in (4, 8) -Linking, as well as a mixture of these latter two options in any possible mixing ratio, the degree of galloylation (ie the percentage of C3 OH groups esterified with gallic acid being different in the (4, 6) or (4, 8) -linked molecular species The oligomeric and polymeric proanthocyanidins according to the invention are furthermore essentially free of other additives, These additives are, in particular, tanning agents which are not among the proanthocyanidins claimed according to the invention, such as hydrolyzable tannins, but also other secondary plant substances which are present as impurities in Extracts from plants or parts of v on plants can occur. The term "essentially free of" is to be understood to mean that the degree of purity of the proanthocyanidins according to the invention, ie the at least partially galloylated oligomeric and polymeric proanthocyanidins, is over 85% (ie there are 15% impurities based on the total amount of inventive Proanthocyanidins before). A degree of purity of more than 90% is preferred, a degree of purity of more than 95% is particularly preferred.

The oligomers and polymers used Proanthocyanidins have an average molecular weight in the range from 250 to 40000 Da. The average molecular weight of the proanthocyanidin refers to the mass average of all included molecular species, i.e. all at least partially galloylated at the C3 OH group, via (4, 6) or (4, 8) bonds of linked Basic units or mixtures thereof.

The invention uses an oligo and polymeric proanthocyanidin in which at least 50% of the C3 positions of the basic units with gallic acid are esterified. The percentage here again relates to the Total number of C3 OH groups present in the proanthocyanidin.

The invention also uses an oligomeric and polymeric proanthocyanidine in which, with the exception of the C3 position of one or both terminal basic units, all C3 positions are esterified with gallic acid. Terminal basic units are to be understood here as the basic units "terminating" the polymer. Thus with the in 1 The schematic molecule shown, for example, does not esterify the lower (ie final) terminal base unit with gallic acid, whereas, in contrast, the upper terminal base unit is galloylated.

It also becomes an oligomeric and polymeric Proanthocyanidin used in which all C3 positions of the basic units with gallic acid are esterified.

In another embodiment The invention relates to the use of an oligomeric and polymeric proanthocyanidin for the treatment of skin and mucous membrane defects and inflammation, for regenerative skin and mucous membrane care, as well as for acceleration regeneration of the skin and mucous membranes. The proanthocyanidin can are administered systemically (e.g. orally) or topically (locally). As medical indications regarding the use are here the post-traumatic care of abrasions, cuts and burns to call. The proanthocyanidin are suitable both for topical / local application on the affected skin areas, as in particular also for the protective or regenerative treatment of mucous membranes.

The present invention relates to in a preferred embodiment also the use of an oligomeric and polymeric proanthocyanidin as a component of cosmetic products. Proanthocyanidins are in the literature (see above) as components of so-called tannin plants known. Extracts previously used in cosmetics are mostly raw mixtures of procyanidins and other types of tannins (e.g. hydrolyzable tannins). The advantage of at least partially Galloylated procyanidins in the sense of this invention is the possibility the targeted cosmetic application of the substances disclosed here, the dermal (topical) in a concentrated and chemically defined form can be applied. The galloylation at the C3 OH group of the basic unit disclosed here improved about it beyond solubility the connections and their incorporation into topically applicable basics and stabilizes the oligomers with regard to oxidative degradation phenomena. It has been shown by in vitro and in vivo experiments that the substances are from of this invention stimulate skin regeneration and proliferation can and thus as cosmetic active ingredients, as well as proliferation promotional Agents can be used (see below, examples). Accordingly, could can be demonstrated experimentally that the proliferation rate of with the proanthocyanidins according to the invention treated cells (human keratinocytes) by 2 to 3.5 times was increased (see Example 1), and that at the cellular level the energy supply was stimulated by the mitochondria.

Accordingly, the invention relates in a further preferred embodiment also the use of an oligomeric and polymeric proanthocyanidin to stimulate the Proliferation of keratinocytes, stimulation of human primary Keratinocytes is preferred.

The present invention relates to also the use of the oligomeric and polymeric proanthocyanidins according to the invention to accelerate the proliferation of cells in cell culture, as well the use of oligomeric and polymeric proanthocyanidins for acceleration the in vivo proliferation of cells. As from the exemplary embodiments 1 and 2 (see below), the proanthocyanidin increases the proliferation of human keratinocytes significantly.

Primary keratinocyte cultures will be more frequently used in the context of skin grafts. With large-scale injuries or burns where there is not enough intact skin material for transplantation to the injured areas disposal stands are common skin pieces taken, then created from these in vitro and ex vivo keratinocyte cultures, which are then on inert carrier materials cultivated to massive confluence and finally on the injured skin areas of the patient are transplanted (see in this context also: Horch R, Bannasch H, Stark GB, "Transplantation of cultured autologous keratinocytes in fibrin sealant biomatrix to resurface chronic wounds ", transplant. Proc., 33 (2001), 642; Lee KH, "Tissue-engineered human living skin substitutes: development and clinical application. "Yonsei Med. J. 41 (6) (2000), 774, review article; Tanczos E, Horch RE, Bannasch H, Andree C, Walgenbach KJ, Voigt M, Stark GB, "Keratinocyte transplantation and tissue engineering. New approaches in treatment of chronic wounds. "Zentralbl. Chir. 1999; 124 (1999), Suppl 1: 81-6, review article).

As part of the present invention by adding the oligomeric and polymeric procyanidin to the culture media the proliferation of skin cells can be accelerated this means that the transplant can be done earlier. The latter is associated with considerable advantages for the patient, since its wounds earlier can be supplied which reduces the risk of infection (e.g. sepsis) and the overall prognosis improved.

In a further embodiment, the oligomeric and polymeric proanthocyanidins can be used as a medicament or for the manufacture of a medicament for the treatment of skin and mucosal defects, and for stimulating the proliferation of cells. The possible dosage forms here include oral dosage forms such as capsules and tablets, as well as parenteral dosage forms, see above such as in particular topical or local forms of application in the form of lozenges, douches, suppositories, stili, ointments and creams.

• i) Extraktion von Pflanzenmaterial enthaltend Proanthocyanidine mit einem Gemisch umfassend Aceton,
• ii) weitere Behandlung des Extraktes aus Schritt i) mit wenigstens einem organischen Lösungsmittel und Verwerfen der organischen Phase,
• iii) Auftragen der verbliebenen wässrigen Phase aus Schritt ii) auf wenigstens eine Säule,
• iv) Waschen der beladenen Säule aus Schritt iii) mit einem geeigneten Lösungsmittel,
• v) Elution der Säule aus Schritt iv) mit einem Aceton/Wasser Gemisch oder Methanol oder zunächst mit Methanol und anschließend mit einem Aceton/Wasser Gemisch oder zuerst mit einem Aceton/Wasser Gemisch und anschließend mit Methanol.

The oligomeric and polymeric proanthocyanidins used can be prepared and isolated in the following way:

• i) extraction of plant material containing proanthocyanidins with a mixture comprising acetone,
• ii) further treatment of the extract from step i) with at least one organic solvent and discarding the organic phase,
• iii) applying the remaining aqueous phase from step ii) to at least one column,
• iv) washing the loaded column from step iii) with a suitable solvent,
• v) Elution of the column from step iv) with an acetone / water mixture or methanol or first with methanol and then with an acetone / water mixture or first with an acetone / water mixture and then with methanol.

The extraction of plant material which contains the galloylated oligomeric and polymeric proanthocyanidins takes place in step i). In a preferred embodiment, crushed bark of Hamamelis virginiana is used and extracted at room temperature using a suitable light protection known to those skilled in the art using a mixture comprising acetone, an acetone / water mixture (preferred ratio acetone / water = 7: 3 (v / v)) is preferred. A preferred embodiment of the method for extracting the galloylated proanthocyanidins is in 8th shown. A preferred exhaustive extraction method for obtaining the galloylated pro anthocyanidin of the present invention, which leads to good yields, is percolation. However, other extraction methods known to the person skilled in the art can also be used within the scope of the present invention.

The use of methanol as an extractant should within the scope of the present invention in the first extraction steps avoided or the methanol exposure of the solution should be avoided be kept short, as this leads to the methanolysis of the isolate Gallussäureester of proanthocyanidine (Bruneton, J, "Pharmocognosy, Phytochemistry, Medicinal Plants. ", (1995) Lavoisier, London, New York, 313-334.

After removing the acetone at maximum 40 ° C in step ii) the aqueous Phase extracted with at least one organic solvent, in a preferred embodiment petroleum ether is used, then the mixture is extracted with ethyl acetate. The organic phases are discarded. The proanthocyanidin is located now in the watery Phase, the yield of the aqueous Phase after drying usually up to 8%, based on the Total dried plant material used.

Subsequently, the raw mixture is on a column applied (step iii), with Sephadex LH20 from Pharmacia or an equivalent material is used. Alternatively, a corresponding batch process can also be used. It will always first Washed with 96% ethanol until the flow was colorless (Step iv), the flow then being discarded. following will exclusively eluted with an acetone-water mixture (step v), if appropriate exclusively with methanol. Alternatively, an elution sequence takes place, first elution with methanol and then with an acetone / water mixture takes place or vice versa. Typical yields of a methanol eluate (excluding methanol) are> 30% based on the amount of raw product used, that of an acetone / water eluate (excl. acetone / water) is> 10%. The general rule is that by methanol elution Proanthocyanidin fractions obtained with a smaller molecular weight be and by acetone-water elution higher molecular weight proanthocyanidins be eluted. By combining the two elution methods (first methanol, then acetone or vice versa) different galloylated oligomeric or polymeric proanthocyanidins can be obtained in the average molecular size (DP) differ.

To narrowly limited fractions Obtaining molecular weight ranges can optionally further column chromatography Fractionation on Sephadex LH20 columns or equivalent materials, preferably using an ethanol-water-acetone mixture, respectively.

The present invention is also intended are explained using the following examples and illustrations, in particular their advantageous applicability both in the field skin regeneration and cell culture, as well as in the field of Cosmetics.

explanation of the pictures

1 shows structural features of the galloylated oligomeric and polymeric proanthocyanidins, whereby there is a (4, 8) linkage of the basic units.

2 shows the influence of oligomeric, galloylated proanthocyanidins of different molecular weights in doses of 1 and 10 μg / ml on the cell proliferation of human keratinocytes after a nine-day incubation period. The results were determined by BrdU incorporation into the cellular DNA and then the ELISA evaluation. The stated values represent the mean values +/- the standard deviation from n = 10 with * p <0.1 and ** p <0.05 compared to the untreated control.

3 shows the influence of galloylated proanthoycyanidines on the cell proliferation of NHK over a 10-day feeding period. The results were determined by BrdU incorporation into the cellular DNA and subsequent ELISA evaluation. The stated values represent the mean values +/- the standard deviation from n = 10 with * p> 0.1 and ** p> 0.05 compared to the untreated control.

4 describes the influence of a galloylated proanthocyanidin on the mitochondrial activity of human keratinocytes in doses of 1 and 10 μg / ml after a nine-day incubation period. Evaluation using MTT test; the values given represent the mean values from n = 10 with * p <0.1 compared to the untreated control.

5 shows the influence of various high molecular weight galloylated proanthocyanidins (substances B and C) on the cell differentiation of human keratinocytes in doses of 0.1 to 50 μg / ml after nine days of incubation.

4A : Absorbance measurements, determined by means of keratin ELISA;

4B : Quantification of the amount of differentiation-specific keratins 5A ,

6 shows delta TEWL after 3 days of cumulative irritation of skin areas; GPC = galloylated proanthocyanidins.

7 shows the clinical score after 3 days of cumulative irritation GPC: galloylated proanthocyanidins.

8th shows a colorimetry after 3 days of cumulative irritation of skin areas.

9 shows schematically a preferred purification process to obtain the galloylated proanthocyanidins.

example 1

galloylated Proanthocyanidins stimulate the proliferation of human keratinocytes in vitro

Galloylated were used as test substances Proanthocyanidins with average molecular weights of 1000 Da (substance B) and 5000 Da (substance C) used.

It became an investigation of the influence of the two proanthocyanidin fractions on human keratinocytes (natural human keratinocytes, NHK). For this purpose primary cultures were created from donor keratinocytes and these cultures in vitro with the above substances B and C to be investigated, dissolved in physiological Medium, offset. Treated in the same way served as negative controls Cultures to which only pure medium has been added.

The system of the "submerged" keratinocyte culture was chosen for all in vitro investigations. Keratinocytes were either obtained from commercial suppliers (Cell Systems, St. Katharinen, Germany) or obtained from human skin from surgical resections. In the latter case, the skin pieces were taken directly after removal a solution which decontaminates 0.2% fungicone, 5.9 μg / ml gentamycin and 7.5% sodium hydrogenphosphate in DMEM medium (Gibco, Karlsruhe, Germany), the tissue was mechanically disrupted and the dermis and epidermis were separated by dispensing treatment Epidermis was trypsinized (15 min, 34 ° C trypsin 0.05%, NaEDTA 0.53mM). The reaction was stopped by adding TNS (Cell Systems, St. Katharinen, Germany). The generation cells were centrifuged off (463 × g, 5 min), resuspended in PBS, centrifuged again and sown in KGM2 medium The culture was carried out at 37 ° under a 5% CO ₂ atmosphere.

At about 60 to 70% confluence rates secondary cultures were established after trypsinization. All cultures were essentially free of contaminations, especially those The absence of fibroblasts and melanocytes was routinely checked. The Secondary cultivation period was 9 to 14 days.

The proanthocyanidins were added to the keratinocytes after dissolving in KGM-2 / DMSO. Highly concentrated calcium medium with 1.5 mM Ca ²⁺ served as positive controls for the detection of differentiation processes. The untreated cultures served as controls.

Keratinocyte physiology was characterized by determining the growth rates of the cells after trypsinization. Mitochondrial activities were determined using the MTT method according to Mosmann (Mosmann, M, "Rapid colorimetric assay for cellular growth and survival: applications to proliferation and cytotoxicity assays.", J. Imm. Methods 65 (1983), 55-163) , Lactate dehydrogenase titers were determined using an LDH assay kit (Sigma, Steinheim, Germany) (Amador E, Dorfman LE, Wacker WEC, "Serum lactic dehydrogenase. An analytical assessment of current assays.", Clin. Chem. 9 (1963), 391-395). Cell proliferation was determined using a BrdU kit (Boehringer, Mannheim, Germany) according to Porstmann T., Ternyck T., Avrameas S, "Quantitation of 5-bromo-2-deoxyuridine into DNA an enzyme immunoassay for the assesment of the lymphoid cell proliferative response. ", J. Immunol. Methods 82: 169-179 (1985). The differentiation-specific Keratins K1 and K10 were determined as follows: the cells were washed and the keratins were solubilized using urea 8 M, β-mercaptoethanol 0.2 M and Tris 1.5 mM. 50 μL of the solubilizate were transferred to 96-well microtiter plates and incubated at 4 ° C. for 18 h. Thereafter, it was treated with block medium (milk powder 7% in PBS with 0.05% Tween 20) for 1 hour, followed by the addition of 100 μL of a monoclonal mouse anti-human keratin antibody (Dako, Hamburg, 1: 400 in block medium) , This is followed by washing 3 times with 150 μL PBS containing 0.05% detergent. POD-bound goat anti-mouse IgG antibody (Sigma, Steinheim, Germany) was then added. After washing three times, 100 μL of substrate solution (phenylenediamine, hydrogen peroxide) were added, the reaction time was 10 min at room temperature, and the reaction was then ended by adding 100 μl of sulfuric acid. The evaluation was carried out by determining the UV absorption at 492 nm, the calibration was carried out with standard keratins.

All cultures that had been treated with the proanthocyanidin fractions showed clear and rapid formation of confluent monolayers after 9 days of incubation. The untreated control groups showed only about 40% confluency within this nine-day test period. As expected, this result was also reflected in the cell proliferation rate, which was quantified by BrdU incorporation (bromo-deoxy-uridine) (see 1 ).

From the in 1 The data shown clearly shows that the proanthocyanidins used, here substances B and C, increase the cell proliferation rate by two to three and a half times compared to the untreated control and are therefore advantageously used in the context of a cell culture to increase the proliferation rate and thus achieve confluency more quickly can. As a result, the proliferation rates when adding the galloylated proanthocyanidins were increased threefold compared to the untreated control. However, this also means that these substances, if they are added to the nutrient media or directly to the cultures, can generally be used as growth enhancers for cells, cell cultures and tissues. As a result, the cell cultures or tissues grow faster, making their production more efficient and cheaper. Another advantage of using the proanthocyanidins is the faster and more efficient possibility of providing transplanting cells or tissues for, for example, skin transplant patients.

Example 2

High molecular, galloylated Proanthocyanidins need for one Proliferation stimulation human skin cells are continuously supplied

If the cell proliferation of human keratinocytes is examined over time in the presence or in the absence of the at least partially galloylated proanthocyanidins, it is found that significant differences between treated and untreated groups appear after only six days of incubation and that the treated cells continue to show significantly stronger growth ( 2 ). However, this also means that the proanthoycyanidins do not stimulate cell proliferation as a single stimulus after a single dose, but must be constantly supplied to the cells.

Example 3 Galloylated proanthocyanidins stimulate the energy supply the cell

The proliferation rates achieved by means of the high-molecular, galloylated procyanidins go hand in hand with a significantly increased mitochondrial activity, i.e. with increased energy production, at the subcellular level. This was demonstrated by determining the mitochondrial activity of treated and untreated cells after nine days of incubation using the MTT test ( 3 ).

Example 4 galloylated proanthocyanidins have no influence on human differentiation processes skin cells

To investigate the extent to which the high molecular weight galloylated procyanidins also influence differentiation processes, were comparative studies on differentiation-specific Keratins K1 and K10 performed using the ELISA technique (Muzarelli et al. (1999) EXS 87, 251-264).

Again, NHK were treated either with various procyanidins or pure medium, the cells were disrupted after nine days of incubation, the keratins were solubilized and quantified with specific antibodies ( 4 ). This clearly shows that influencing keratin formation and thus also differentiation is not triggered by the proanthocyanidins. The effect of the galloylated proanthyocyanidins can thus be attributed to an increase in proliferation and energy metabolism, while undesired differentiation processes are not induced.

As a result, it should be noted that the differences that go beyond the amount of differentiation specific keratins (ELISA) were determined, are not significant between the individual groups and therefore there is no influence on the cellular differentiation processes by the proanthocyanidins.

Example 5 Galloylated proanthocyanidins show no detectable in vitro toxicities in the dose range from 1 to 100ug / ml

An important requirement for nursing Cosmetics are always compatibility. For examination the extent to which cellular toxicities can be observed, keratinocytes with high molecular weight, galloylated Proanthocyanidins incubated in a dose range of 1 to 100 μg / ml and necrotic processes over the measurement of extracellular Lactate dehydrogenase determined. The concentration interval used goes far beyond the doses in the upper dose range a topical application actually come into contact with the cells. Necrotic processes could occur in no dose interval was found. All groups turn out to be as not significantly different compared to the untreated ones Control groups. No microscopic cell deformations were possible or other evidence of cell damage to be watched.

Keratinocytes cultured in vitro are generally used as an internationally accepted test system for evaluation more cytotoxic and skin damaging Effects used (Sandt S, Roguet R, Cohen C, Esdaile D, Ponec M, Corsins E, Barker C, Fusenig, N, Liebsch M, Benford D, Fartasch M, "The use of keratinocytes and human skin models for predicting skin irritation. ", European Center for the validation of alternative methods ECVAM, ATLA 27 (1999), 723-743).

Example 6 Galloylated proanthocyanidins cause increased regeneration in vivo damaged epidermis

By far the most common cause of damage to the Skin barrier is the repeated contact with in relatively little Dimensions irritating acting substances, such as detergents in water.

Already exists in the literature a variety of in vivo models for proof of effectiveness of skin protection products. So-called cumulative models perform the actual exposure situation best with detergents.

The skin situation is assessed predominantly through a clinical evaluation of the irritation (clinical scoring), a quantification of the erythema (colorimetry) and by Measurements of the barrier disturbances over the transepidermal water loss (TEWL). In a validated cumulative-irritative Model (Schnetz E, Diepgen TL, Elsner P, Frosch PJ, Klotz AJ, Kresken J, Kuss 0, Merk H, Schwanitz HJ, Wigger-Alberti W, Fartsch M, Contact Dermatitis 42: 336-343 (2000); Fartasch M, Schnetz E, Ennen J, Lanzendörfer G, Diepgen TL, journal f. Skin diseases, H + G, 74 (1999), 1-6; Fartasch M, Schnetz E, Diepgen TL, J. Invest. Dermatol., Symposium Proceedings, Vol. 3, No. 2 (1998), 121-127) were the investigations on the volar forearm 3 days. This model offers the possibility the effectiveness of skin care products and their active ingredients in a short time recognizable reproducibly.

This cumulative-irritative test was used to test the effectiveness of galloylated proanthocyanidins to test as active ingredients in cosmetic preparations. The testing occurs at a concentration of the galloylated proanthycyanidines of 1% in a cosmetic basis (Eucerin cum aqua, according to the requirements of the European Pharmacopoeia) to the injured Skin (0.5% sodium lauryl sulfate, SDS, SLS). Comparative values were a negative control by treating the skin with aqua purificata, and a positive control due to skin irritation with 0.5% SLS. As a control for the applicability of the test procedure were the measurement values of the untreated Skin captured.

test procedure

Day 1: Double application of 50μl of a 0.5% aqueous sodium lauryl sulfate solution (sodium lauryl sulfate, SLS) to the volar right and left forearm in 11 mm Finnchambers ^® on filter paper discs with 30min occlusion and 3h ± 30min pause between applications. The test product was applied to the test sites 10 min beforehand (0.05 g / ² cm ² ). This scheme was repeated on the 2nd and 3rd days. The test areas were permuted clockwise between the test subjects.

Skin condition assessment (TEWL, Colorimetry and clinical score) was performed before the first application of the test substances on the 1st day and about 3 to 4 hours after the last Irritation performed on day 3.

policies

Clinical scoring 0.5 = very weak erythema - somewhat shiny surface - no scaling - no edema - no fissures; 1 = Erythema weak, diffuse or follicular - slightly rough surface - minimal scaling - slight edema - fine fissures; 2 = medium erythema - medium roughness, scaling and edema, with wide fissures; 3 = clear erythema, rough scaling, pronounced edema, pronounced fissures with hemorrhage and exudation.

The TEWL was measured by one by Tewameter of type TM 210 (Courage and Khazaka, Cologne) under standardized conditions in an air-conditioned room. Duplicate. Before there was a 20 minute Test subjects rest.

The colorimetry was done with a Colorimeter from Minolta, according to the European guidelines (see Fullerton A, Fischer T, Lauti A, Wilhelm KP, Takiwaki H, Serup J, Contact Dermatitis 35 (1996), 1-10).

7 volunteers, skin-healthy individuals (2 male, 5 female, ages 22 to 38 years, average 29.8 years) Available. There was no known type IV allergy in any of the subjects. There was no atopic skin diathesis and no previous eczema diseases.

Scoring System

The results of the TEWL measurements and the colorimetry are shown in 5 shown as boxplots. The boxplots show the median, the 25% percentile, the 75% percentile and values that lie outside the percentile. The lower limit of the box is the 25% percentile, the upper limit of the 75% percentile, the line inside the box is the median. Lines are drawn from the boundaries of the box to the highest and lowest observed values that do not yet represent "outliers" or extreme values. Values that lie between 1.5 and 3 times the box length above or below the limit of the box are called "outliers" (o) and are not taken into account. If values are more than 3 times outside the box, they are considered extreme values and are also disregarded.

The results of the clinical score are shown in a bar chart (see 6 ). The bars show the mean. The error bars indicate the standard error of the mean

Results

TEWL:
The results from the TEWL measurements were in good agreement with the results of the clinical score and colorimetry. In all cases, irritation with SLS - with or without care products - led to an increase in the TEWL values compared to the untreated control and water. The cosmetic preparation examined, containing 1% of galloylated proanthocyanidins according to the invention, however, led to delta TEWL increases of different strengths.

The delta TEWL values for the pure The basis containing the proanthocyanidin preparation were significant lower than the values after pure irritation with SLS. Using on a pure basis (without proanthocyanidins) could not be significant Humiliation compared to pure irritation.

The percentage reduction in the delta TEWL (mean) when applying skin protection (delta TEWL with pure irritation = 100%) was as follows:

The results of the TEWL measurements are in 5 played.

Clinical score

Irritation of the skin with 0.5% SLS causes an increase in the values of the clinical scoring. This cannot be completely prevented by the investigated compositions with proanthocyanidins be changed. The different products, however, show different effects against irritation with SLS. When applying a pure basis with the galloylated proanthocyanidins according to the invention, the induced erythema was markedly less pronounced than when applying a pure basis. The difference between the products was significant. The graphical representation of the results is in 6 played.

colorimetry

Also the quantification of the erythema shows the same results as the clinical evaluation of the skin. An irritation with SLS leads to an increase in the values caused by the cosmetic test product, containing the galloylated according to the invention Proanthocyanidins dissolved in basis, not completely can be prevented.

When using the test product (with galloylated proanthoyanidines), however, it was found that the majority of the values were significantly lower than with pure irritation or the use of a pure basis. The graphical representation of the results is in 7 played.

It became a pure foundation (Eucerin cum aqua) and basis with the addition of 1% of galloylated according to the invention Proanthocyanidins as an active ingredient in a three-day cumulative test a regenerative effect on skin-healthy test subjects (number of people n = 7) tested after irritation with 0.5% SLS.

Thus it is evident that the addition of 1% of the galloylated proanthocyanidins advantageous as a basis is, and not just regarding the reduction of potential irritation, but also regarding the faster and more efficient regeneration induced by this the epidermal barrier (determined by the TEWL) after entering damage brought about faster improvement of the (skin) condition.

Example 7

manufacturing the proanthocyanidins and subsequent analysis of the obtained Verbidnungen

insulation

The extraction of plant material, which contains the galloylated oligomeric and polymeric proanthocyanidins at room temperature under light protection using an acetone / water mixture (in the ratio of 7: 3). An exhausting one Extraction method, which leads to good yields, provides percolation represents, but other technical methods are also conceivable here.

The use of methanol as an extractant will first avoided, as this leads to the methanolysis of the gallic acid esters can come (Bruneton, 1995).

After removing the acetone at one Maximum temperature of 40 ° C becomes the watery Phase extracted with at least one organic solvent here petroleum ether is used, then the mixture is extracted with ethyl acetate. The organic phase is discarded. The proanthocyanidins are located now in the watery Phase (the yield of the aqueous Phase after drying is about 8%, based on the dried Starting plant material.

Subsequently, the raw mixture is on applied a column filled with Sephadex LH20 (Pharmacia). Then will the pillar washed with 96% ethanol until the flow rate appears clear (at least Wash with three column volumes). The flow rate (wash solution) will subsequently discarded. Subsequently, either with methanol or with a Acetone-water mixture (ratio 7: 3) eluted. The typical product yield of a methanol eluate are around 30%, based on the amount of raw product used, and that of an acetone / water eluate is about 10%. By methanol elution Procyanidin fractions with a smaller molecular weight are obtained by acetone-water elution high molecular procyanidins are eluted. Through combinations of the two elution methods (first methanol, then acetone 70%) can different products are obtained, the galloylated oligo- or polymeric proanthocyanidins, but in the middle Molecular size (DP) differ.

To use more narrowly defined fractions To obtain narrower molecular weight ranges, there is now another by column Fractionation on Sephadex LH20 using an ethanol-water-acetone (7: 7: 6) mixture.

Analysis of the received used proanthocyanidins

The acidic hydrolysis of the product with ethanolic hydrochloric acid results in the occurrence of the anthocyanins cyanidine and delphinidine in equal parts. Thus the proanthocyanidins used consist of units which are both di- and trihydroxylated in ring B. The identification of gallic acid by Hy Drolysis of the oligomeric and polymeric proanthyocyanidins with 2N ethanolic hydrochloric acid for 60 min at reflux and subsequent thin-layer chromatographic identification of free gallic acid was carried out in comparison with an authentic reference substance. Clear amounts of free gallic acid were detected in all samples examined. The chain end units and chain extension units were identified after acid-catalyzed, thiolytic cleavage with benzyl mercaptan as the nucleophile (2 g sample in 15 ml ethanol 96%, 1350 μl benzyl mercaptan, 660 μl glacial acetic acid, nitrogen fumigation, 95 ° C, 5 days in a sealed ampoule) and subsequent HPLC - Separation and quantification of the fission products. After a two-day reaction period, only 3-O-galloylepigallocatechin-4-benzylthioether and 3-O-galloyl-epicatechin-4-benzylthioether were detectable. The amounts of epigallocatechin-4-benzylthioether and epicatechin-4-benzylthioether were <1%. The following percentage distribution of the thioethers formed in complete thiolysis of two selected fractions resulted after a five-day reaction period (calculation from area integral HPLC; data in%):

Analytical data of the W _m2 sub-fraction

Individual components of the polymer (thiolysis, HPLC):
3-O-galloyl-epigallocatechin, 3-O-galloyl-epicatechin as main components of the chain links, small amounts of free catechin and epicatechin as terminal end groups
Interflavan linkage (partial thyolysis, HPLC): 4⇒6 and 4⇒8 linkage
Average degree of polymerization (GPC): 12 ^
Product content (UV)> 90%, high purity

That determined by anthocyanin cleavage Occurrence of di- and trihydroxylation in the B rings in the ratio of about 1: 1 could thus be confirmed become. The composition of the individual fractions fluctuated of about 10% very similar.

Epicatechin-4-benzylthioether and Epigallocatechin-4-benzylthioether originated only in minor Dimensions. Due to the long reaction time (5 days, 95 ° C) it can be assumed that these from the corresponding compounds galloylated at position 3, arise. Having these two connections in the first two Days of the reaction were undetectable and free gallic acid in the thiolysis batch detected, can almost certainly be assumed be that the polymeric proanthocyanidins on the chain extension units at position 3 completely are galloylated.

The chain ends sat down biggest part composed of catechin (95%), only about 5% of gallocatechin in front.

• a) Durch Gelpermeationschromatographie der peracetylierten Verbindungen an Styrol-Divinylbenzol-Copolymeren
• b) Durch vollständige Thiolyse (3 mg Probe in 300 μl Ethanol, 30 μl Benzylmercaptan, 15 μ Eisessig, Stickstoffbegasung, 95°C) und Molekulargewichtsbestimmung über das Verhältnis der Endgruppen zu den Kettenglieder

The average molecular weights were determined using two different methods:

• a) By gel permeation chromatography of the peracetylated compounds on styrene-divinylbenzene copolymers
• b) By complete thiolysis (3 mg sample in 300 μl ethanol, 30 μl benzyl mercaptan, 15 μ glacial acetic acid, nitrogen fumigation, 95 ° C.) and molecular weight determination via the ratio of the end groups to the chain links

Results:
Fraction C2 (elution on LH20 column with 70% acetone, obtaining sub-fraction 2): average degree of polymerization 20.0
Fraction M2 (elution on LH20 with methanol, sub-fraction 2): average degree of polymerization 12.9

The above results were additionally confirmed by MALDI-TOF mass spectroscopy. The determinations The interflavan linkage was achieved by partial thiolytic degradation of the oligomers and polymers under gentle conditions (6 g sample in 45 ethanol 96%, 4050 μl benzyl mercaptan, 1980 μl glacial acetic acid, nitrogen fumigation, 95 ° C, 48 hours in a closed ampoule) and HPLC -Analysis of the resulting gap mixture. Identification of the cleavage products by comparison and co-chromatography with authentic reference substances.

Result for one faction M2:

The following fission products were found:
catechin
gallocatechin
Epicatechin 4β-benzyl thioether
3-O-galloyl-epicatechin 4β-benzyl thioether
3-O-galloyl epigallocatechin-4SS-benzyl thioether
Procyanidine B1
Procyanidine B3

Due to the appearance of dimeric procyanidins B2 (4.8-linked Catechins) and B3 (4,6-linked catechins) there is an Interflavan link via positions 4⇒8 as well 4⇒6. This Verknüfungstypen were also about the 1H-NMR spectra of the polymeric proanthocyanidins were detected (Doublets at 6.49, 6.51, 6.85 ppm, corresponding to the H-6 and H-8 the "upper" molecular unit, and signals at 5.35 and 5.42 ppm, corresponding to resonance H-2 the "lower" unit).

Example 8

Stability of the galloylated proanthocyanidins

An advantage of the galloylated oligo- and polymeric proanthocyanidins of the present invention in their pronounced Stability, compared to the unesterified (non-galloylated) compounds clearly elevated is. Serious instabilities of the unesterified compounds were from Rohr (Rohr G, 1999, dissertation ETH Zurich, Diss. ETH No. 13020). Castillo et al. show that the free 3-OH groups of the flavan-3-ol polymer skeleton important functions within the scope of the radical scavenger property of the unesterified procyanidins (Castillo, J. et al., J. Agric. Food Chem. 48 (2000), 1738-45). This means that oxidation, radical or radiation stress can be mitigated by these procyanidins, but this on Because of the specific substance properties for oxidation or for radical destruction of procyanidine got to.

These rapid substance degradations are in the galloylated oligomeric and polymeric procyanidins not observed. As relevant to stability analytical parameters can especially the color intensity, the average molecular weight and the UV adsorption of the compound be used. Typical signs of degradation go with it oxidation to the corresponding o-quinones (i.e. to oxidized flavanols) associated. This can be detected on the basis of the corresponding UV adsorption become. As a subsequent reaction, high-molecular, amorphous pigments arise from red to brown-black color, the so-called Phlobaphene. Corresponding reactions are due to changes in the VIS adsorption spectra clearly recognizable (significant adsorption shift and increase in adsorption to 500 to 610 nm).

To demonstrate the high stability of at least partially galloylated proanthocyanidins, a freshly obtained one Sample opposite a sample stored for 2 years at room temperature (20-25 ° C) was examined. It was found by UV spectroscopy no relevant differences.

To investigate the short-term stability of the sample under stress conditions, UV spectra of a 1% solution at pH 7, as well as in the acidic range (HCl 0.05 N), in an alkaline environment (NaOH 0.05 N) and under oxidative conditions (3% H ₂ O ₂ ) recorded over 16 hours. There were no changes in the neutral and acidic environment or under oxidative influences. Significant decomposition was detected in the alkaline range.

This shows a pronounced stability of the galloylated oligomeric and polymeric proanthocyanidins, both in acid and in the neutral pH range.

Claims (5)

Use of oligomeric and polymeric proanthocyanidins for the treatment of skin and mucous membrane defects and inflammation, for regenerative skin and mucous membrane care, as well as for accelerating the regeneration of skin and mucous membranes, the proanthocyanidins being based on the basic unit Flavan-3-ol, which is based on the C3 position is at least partially esterified with gallic acid, and being the basic units are linked via (C4, C6) or (C4, C8) bonds. Use of proanthocyanidins according to claim 1 as a component of cosmetic products. Use of proanthocyanidins according to claim 1 to accelerate the proliferation of keratinocytes. Use of proanthocyanidins according to claim 1 to accelerate the proliferation of cells in cell culture. Use of proanthocyanidins according to claim 1 to accelerate the in vivo proliferation of cells.