DE10161253A1 - Use of dihydropinosylvin and its derivatives as inhibitors - Google Patents

Abstract

Disclosed are new uses of compounds of formula (I) or mixtures of substances comprising one or more compounds of formula (I), wherein R<sb>1</sb> and R<sb>2</sb>, independent of one another, are selected from the group consisting of hydrogen, methyl, straight-chained or branched, saturated or unsaturated alkyl with 2-10 C atoms and alkyl interrupted by one or more oxygen and/or sulfur atoms with 2-10 C atoms, and R<sb>3</sb> and R<sb>4</sb>, independent of one another, are selected from the group consisting of hydrogen, hydroxy and methoxy. Said compounds are suitable as agents for: (a) inhibiting enzymes selected from the group consisting of tyrosinase, tyrosinase isoenzymes and the mixtures thereof and/or (b) treating microorganisms selected from the group consisting of (i) dandruff-causing microorganisms; (ii) body odor-causing microorganisms, (iii) acne-causing microorganisms, (iv) mycosis-causing microorganisms and their mixtures (v). Further disclosed are methods for preserving perishable articles and for cosmetic lightening of the human skin using the above-mentioned compounds.

Description

• - Wasserstoff,
• - Methyl,
• - geradkettiges oder verzweigtes, gesättigtes oder ungesättigtes Alkyl mit 2-10 C-Atomen und
• - durch ein oder mehrere Sauerstoff- und/oder Schwefelatome unterbrochenes Alkyl mit 2-10 C-Atomen

besteht, und
R₃ und R₄ sind unabhängig voneinander ausgewählt aus der Gruppe, die aus

• - Wasserstoff,
• - Hydroxy und
• - Methoxy

besteht. The present invention relates primarily to new uses of dihydropinosylvin and certain derivatives of dihydropinosylvin, namely of compounds of the formula 1 below


where:
R ₁ and R ₂ are independently selected from the group consisting of

• - hydrogen,
• - methyl,
• - Straight-chain or branched, saturated or unsaturated alkyl with 2-10 C atoms and
• - Alkyl with 2-10 C atoms interrupted by one or more oxygen and / or sulfur atoms

exists, and
R ₃ and R ₄ are independently selected from the group consisting of

• - hydrogen,
• - hydroxy and
• - methoxy

consists.

There is a constant need in the cosmetic industry Inhibitors for certain enzymes and microorganisms.

An example of an enzyme whose inhibition among the most varied For example, the cosmetic condition is desirable Tyrosinase (phenolase). It is one of the Key enzymes in the melanin biogenesis pathway. Melanins, which are made from the amino acid Tyrosine are formed by means of the tyrosinase via the L-dopaquinone the pigments responsible for tanning the skin. It is known that depigmentation or inhibition of tyrosinase Can lighten the skin, for example around freckles or Combat age spots. The cosmetic industry is therefore constantly on the Search for substances that inhibit the enzyme tyrosinase and thus a key step in melanin synthesis, namely conversion from tyrosine to L-dopaquinone and the subsequent oxidation too Stop dopaquinone.

Human skin is populated by a variety of different microorganisms. Most of these microorganisms are not pathogenic and are irrelevant to the physiological condition of the skin and its smell. Others, on the other hand, can significantly influence the healthy condition of the skin. Some microorganisms that strongly influence the human skin flora are shown in Table 1. Table 1

Due to the bacterial degradation contained in the body's own sweat Fabrics such as B. unsaturated fatty acids arise from more or less weak smelling precursors Decomposition products that can have a major impact on physical well-being. to Prevention of the development of those responsible for body odor Substances are used in cosmetics products that either Prevent the formation of body sweat (so-called antiperspirants) or Substances that contribute to the growth of odors inhibit responsible bacteria of human skin (deodorants). Bacterial species such as Staphylococcus epidermidis, Corynebacterium xerosis and Brevibacterium epidermidis are essential for the formation of axillary and foot odor, or body odor in general. In the cosmetic industry there is therefore a constant need for new ones Agents to treat this and other body odors (including Armpit and foot odor) causing microorganisms.

An acne-causing microorganism is Propionibacterium acnes, at which is an anaerobically growing germ. The cosmetic industry is constantly looking for agents to treat this germ and other microorganisms that cause acne.

All areas of human skin can be affected by mycoses (especially Dermatomycoses and nail mycoses). Particularly common areas of the skin are affected, which can be affected by wearing Clothing, footwear or jewelry can accumulate moisture and heat. Fungal diseases of the are found to be particularly unpleasant Finger and toenail areas. Responsible for the formation of Mycoses are often different trichophyton and epidermophyton Species. The cosmetic industry is constantly looking for new means Treatment of these and other mycoses Microorganisms.

In the area of hair care, there is also an intense search for substances for the treatment of those responsible for the formation of dandruff Germ causing Malassezia furfur and other dandruff Microorganisms searched.

In the context of the present text, "treatment" includes each Form of influence on the microorganisms concerned understood, in which the multiplication of these microorganisms is inhibited and / or the microorganisms are killed.

There is also in the cosmetic and pharmaceutical industry otherwise a constant need for preservatives perishable cosmetic or pharmaceutical items. Preferably a such preservative against several of those in Table 1 listed microorganisms to be effective for the parallel use of further To make active substances unnecessary.

• - toxikologisch unbedenklich,
• - gut hautverträglich,
• - stabil (insbesondere in den üblichen kosmetischen und/oder pharmazeutischen Formulierungen),
• - vorzugsweise geruchlos und
• - preiswert herstellbar (d. h. unter Einsatz von Standardverfahren und/oder ausgehend von Standardprekursoren)

sein müssen. When looking for appropriate antimicrobial and / or preservatives it should be noted that the substances used in cosmetic and / or pharmaceutical products

• - toxicologically harmless,
• - well tolerated by the skin,
• - stable (especially in the usual cosmetic and / or pharmaceutical formulations),
• - preferably odorless and
• - inexpensive to manufacture (ie using standard processes and / or starting from standard precursors)

have to be.

The search for suitable (active) substances that contain one or more of the have sufficient properties mentioned is the Expert complicates that no clear dependency between the chemical structure of a substance on the one hand and its biological Activity against certain microorganisms (germs) and their On the other hand, there is stability. Furthermore, there is no predictable one Relationship between the antimicrobial effect, the toxicological Harmlessness, skin tolerance and / or stability.

A special requirement for the use of an active substance in the Practice is their stability towards chemical substances, which as Accompanying substances are commonly used in cosmetics the oxygen contained in the ambient air and towards light.

It was therefore the object of the present invention, an active ingredient indicate which (a) a good antimicrobial (i.e. inhibitory) effect compared to at least one, but preferably several of the above microorganisms discussed, (b) a tyrosinase inhibiting Has effect, and thus as a cosmetic skin lightener or cosmetic Depigmentation agent can be used and (c) against the influences shows good stability of air and light.

• a) Inhibierung von Enzymen, die aus der Gruppe ausgewählt sind, die aus Tyrosinase, Tyrosinase- Isoenzymen und deren Mischungen besteht, und/oder
• b) Behandlung von Mikroorganismen, die aus der Gruppe ausgewählt sind, die aus (i) Schuppen verursachenden Mikroorganismen, (ii) Körpergeruch verursachenden Mikroorganismen, (iii) Akne verursachenden Mikroorganismen, (iv) Mykosen verursachenden Mikroorganismen und deren Mischungen besteht,

eingesetzt werden und weisen dabei in kosmetischen und pharmazeutischen Produkten eine außerordentlich hohe Stabilität gegenüber Luftoxidation sowie gegenüber lichtinduzierten Isomerisierungs- und/oder Abbaureaktionen auf. Surprisingly, it has now been shown in our own investigations that the compounds of the formula 1 mentioned at the outset (hereinafter also referred to as bisbenzyl derivatives or dihydropinosylvin derivatives) and substance mixtures which comprise one or more compounds of the formula 1, the groups R ₁ to R ₄ each have the meaning given above and are suitable for solving the problems set. Accordingly, they can be used as a means of

• a) inhibition of enzymes selected from the group consisting of tyrosinase, tyrosinase isoenzymes and mixtures thereof, and / or
• b) treatment of microorganisms selected from the group consisting of (i) dandruff microorganisms, (ii) body odor microorganisms, (iii) acne causing microorganisms, (iv) mycosis causing microorganisms and mixtures thereof,

are used and in cosmetic and pharmaceutical products have an extraordinarily high stability against air oxidation as well as against light-induced isomerization and / or degradation reactions.

From DE Fagboun et al .; Phytochemistry 26 (12), 3187, 1987 already knew that dihydropinosylvin has an antimicrobial effect against, for example, Aspergillus niger and Escherichia coli. However, the publication does not give any indication of the antimicrobial activity of dihydropinosylvin or another compound of the formula 1 (with the meanings given of the groups R ₁ to R ₄ ) with respect to the other microorganisms mentioned above. There is also no indication of a tyrosinase-inhibiting activity of compounds of the formula 1. Overall, there is no indication of the usability of dihydropinosylvin or another compound of the formula 1 as a cosmetic agent.

The antimicrobial activity of the stilbene derivatives which are structurally related to the compounds of formula 1, such as pinosylvin (formula 2) and pinosylvinmonomethyl ether (formula 3), has long been known (Nature, Vol 155; April 14; p. 454; 1945). In the publication mentioned it is described that the substances Pinosylvin and Pinosylvinmonomethylether, which occur naturally in many Pinus species, have a strong antimicrobial activity against various Bacillus and Penicillium species.

About the antimicrobial effectiveness of stilbenes of the type Pinosylvins and their general importance as phytoalexins is also described in the following publications: K. Ratanabanangkoon et al., J. Sci. Soc. Thailand, 2: 202-205 (1976); H. Erdtmann, TAPPI 32 (7), 305-310 (1949); C. C. Celimene, Phytochem. 56, 161-165 (2001); T. P. Schultz, Phytochem. 31 (11), 3801-3806 (1992).

T. Suga, Phytochem 33 (6), 1395-1401 (1993) describes Pinosylvin- Monomethyl ether and dihydrogen-pinosylvin monomethyl ether as nematicidal substances. Use of the substances mentioned as however, no antimicrobial agent or tyrosinase inhibitor is mentioned.

EP 1 029 530 A1 describes the use of 3,5-dihydroxystilbene and 3-hydroxystilbene described as deodorants in cosmetic products. The EP 1 068 864 A1 describes the use of certain style levels as Inhibitors of glycosidation of certain proteins (e.g. skin proteins). Both EP documents give no indication of the use of the corresponding bisbenzyl derivatives.

Own studies on the antimicrobial effectiveness of Solvent extracts from Pinus species and stilbene derivatives isolated from them led to results that correspond to the results known from the literature largely correspond. The antimicrobial effectiveness of stilbene Plant extracts as well as from Pinosylvin and Pinosylvinmonomethylether was confirmed.

However, the fact that the investigated stilbenes for photoisomerization and degradation reactions tend to make their use in cosmetic and pharmaceutical In practice, products are only possible to a limited extent. The own Studies have shown that naturally occurring trans- Stilbenes of the Pinosylvin type are extremely light sensitive and within isomerize to the corresponding cis compounds in a short time Dimerize cyclobutane derivatives and to unspecified Remove follow-up products. Not only the low stability of the stilbene but also the fact that their toxicological potential and the dermatological risks of photoisomerization and degradation Subsequent products are difficult to estimate are of great disadvantage for the potential use of stilbenes such as Pinosylvin and Pinosylvinmonomethyl ether in topical products.

Surprisingly, on the other hand, it was found that the compounds of the formula 1 with the abovementioned meanings of the groups R ₁ to R ₄ have excellent stability to light and oxygen and their antimicrobial activity is not or at least not significantly behind stilbenes such as pinosylvin and pinosylvinmonomethyl ether. The use of the bisbenzyl derivatives according to the invention in pharmaceutical and cosmetic compositions is therefore possible without any problems.

Bisbenzyl derivatives such as B. dihydropinosylvin (formula 4) or dihydropinosylvin monomethyl ether (formula 5) can be selected by selective catalytic hydrogenation z. B. with Pd / H ₂ . The educts here are crude extracts containing stilbene from plants containing stilbene (e.g. Pinus, Alnus, Polygonum, Dalbergia, Scutellaria or Lindera species), chromatographic fractions of plant extracts with an increased content of stilbenes and stilbene obtained from plant extracts in highly pure form into consideration. Alternatively, bisbenzyl derivatives of the formula 1 can also be isolated directly from plants containing bisbenzyl derivatives, such as, for example, Pinus or Dioscorea species. In addition, bisbenzyl derivatives of the formula 1 can of course also be produced in a targeted manner using synthesis processes known from the literature.

Efficacy studies with catalytic hydrogenation plant extracts containing bisbenzyl (as mixtures of substances) and from them isolated pure bisbenzyl derivatives such as dihydropinosylvin (formula 4) and dihydropinosylvinmonomethyl ether (formula 5) are examples of that substances of Formula 1 (as well as their mixtures) are strong antimicrobial effect against odor-producing microorganisms human skin such as Corynebacterium xerosis, Staphylococcus own epidermidis and Brevibacterium epidermidis. Because of their high Contrary to, they can have stability towards light and oxygen the corresponding stilbene derivatives are excellent as an alternative or as a supplement to known antimicrobial agents (such as Farnesol or triclosan) in cosmetic products and the like as deodorants Find use.

The compounds of formula 1 according to the invention are also also against Propionibacterium acnes, Malassezia furfur and mycoses causing germs such. B. Trichophyton rubrum well effective, so that they are also used to treat (combat) acne, as Antidandruff or in the treatment of mycoses (especially Dermatomycoses) can be used.

Because of their broad spectrum of activity and in particular because of their activity also against Gram-negative bacteria such as Escherichia coli and Pseudomonas aeruginosa, against yeasts like Candida albicans and against fungi like Aspergillus niger (Table 2; effectiveness already known from the literature, see above), the compounds according to the invention Formula 1 also excellent means for preserving, for example, cosmetic and / or pharmaceutical formulations. The use of co-preservatives is therefore usually not necessary when using compounds of the formula 1 (where R ₁ to R _{4 have} the meaning given above).

In addition to the use as an antimicrobial active ingredient, dihydropinosylvin derivatives of the formula 1 (where R ₁ to R _{4 have} the meanings given above) are also suitable as (cosmetic) agents, in particular for skin lightening. They are suitable for inhibiting the enzyme tyrosinase and thus preventing a key step in melanin synthesis, namely the conversion of tyrosine to L-dopaquinone and the subsequent oxidation to dopaquinone. In the presence of the compounds of formula 1 according to the invention (where R ₁ to R _{4 have} the meanings given above), less dopaquinone is formed, which leads to lower melanin production, which in turn results in skin lightening. Therefore, the said dihydropinosylvin derivatives can be used as cosmetic agents or effective components of cosmetic agents, in particular for the treatment of age spots and freckles.

Corresponding own series of tests showed that both plant extracts which comprise one or more compounds of the formula 1 (where R ₁ to R _{4 have} the meanings given above) and pure bisbenzyl derivatives of the formula 1 (where R ₁ to R _{4 have} the meanings given) possess) have tyrosinase inhibiting properties. Dihydropinosylvin is about 10 times more effective than commercially available skin lightening agents such as B. hydroquinone or kojic acid. Compared to the stilbene Pinosylvin, which has also been shown to lighten the skin, dihydropinosylvin is more effective and also advantageous in use due to its significantly higher stability.

• - Methyl,
• - geradkettiges oder verzweigtes, gesättigtes oder ungesättigtes Alkyl mit 2-10 C-Atomen und
• - durch ein oder mehrere Sauerstoff- und/oder Schwefelatome unterbrochenes Alkyl mit 2-10 C-Atomen

besteht. For the treatment of the microorganisms mentioned and / or for the treatment (inhibition) of tyrosinase, tyrosinase isoenzymes and their mixtures, preference is given to compounds of the formula 1 in which R _{1 is} hydrogen and R _{2 is} selected from the group consisting of

• - methyl,
• - Straight-chain or branched, saturated or unsaturated alkyl with 2-10 C atoms and
• - Alkyl with 2-10 C atoms interrupted by one or more oxygen and / or sulfur atoms

consists.

• - Wasserstoff oder
• - Methyl

ist. Compounds of formula 1 in which R _{1 is} hydrogen and R ₂ are further preferred

• - hydrogen or
• - methyl

is.

• a) eines Enzyms, das aus der Gruppe ausgewählt ist, die aus Tyrosinase, Tyrosinase-Isoenzymen und deren Mischungen besteht und/oder
• b) eines Mikroorganismus, der aus der Gruppe ausgewählt ist, die aus (i) Schuppen verursachenden Mikroorganismen, (ii) Körpergeruch verursachenden Mikroorganismen, (iii) Akne verursachenden Mikroorganismen, (iv) Mykosen verursachenden Mikroorganismen und deren Mischungen besteht,

werden das Enzym und/oder der Mikroorganismus mit einer inhibierend wirkenden Menge einer Verbindung der Formel 1 oder einer Substanzmischung, die eine oder mehrere Verbindungen der Formel 1 umfasst, kontaktiert. Die Gruppen R₁ bis R₄ besitzen hierbei die vorstehend angegebenen Bedeutungen, wobei die Angaben zu den bevorzugten Gruppen R₁ bis R₄ ebenfalls zutreffen. Die Inhibierung erfolgt hierbei regelmäßig aus kosmetischen Gründen, kann aber in Ausnahmefällen auch einen therapeutischen Charakter besitzen. In a method for inhibition according to the invention

• a) an enzyme selected from the group consisting of tyrosinase, tyrosinase isoenzymes and mixtures thereof and / or
• b) a microorganism selected from the group consisting of (i) dandruff microorganisms, (ii) body odor causing microorganisms, (iii) acne causing microorganisms, (iv) mycosis causing microorganisms and mixtures thereof,

the enzyme and / or the microorganism are contacted with an inhibiting amount of a compound of the formula 1 or a mixture of substances which comprises one or more compounds of the formula 1. The groups R ₁ to R ₄ have the meanings given above, the information relating to the preferred groups R ₁ to R ₄ also being applicable. The inhibition occurs regularly for cosmetic reasons, but in exceptional cases can also have a therapeutic character.

An important area of application of bisbenzyl derivatives of the formula 1 (where R ₁ to R _{4 have} the meanings given) is the inhibition of the bacteria responsible for the formation of body odor (including armpit and foot odor) (in particular Staphylococcus, Corynebacterium and Brevibacterium -Species). In addition, bisbenzyl derivatives of the formula 1 can also be used to inhibit mycosis-causing skin and nail fungi (dermatomycoses, nail mycoses; trichophyton and epidermophyton species), to inhibit microorganisms responsible for dandruff formation (Malassezia furfur, Syn .; Pityrosporum ovale or P. orb ) and used to treat acne (inhibiting the growth of Propionibacterium acnes). Efficacy also against fungi such as Aspergillus niger and yeasts like Candida albicans, and in concentrations of at least 2200 ppm also against gram-negative bacteria such as. B. Escherichia coli or Pseudomonas aeruginosa also allows the use of the bisbenzyl derivatives of the formula 1 described as preservatives (it being possible to dispense with co-preservatives with antimicrobial activity against the microorganisms mentioned).

The compounds of formula 1 (or corresponding mixtures of such Connections), in particular as far as they counteract body odor causing germs and / or for skin lightening are used in the Usually topically in the form of solutions, creams, lotions, gels, sprays or the like. Applied. For other purposes, oral is in some cases (Tablets, capsules, powder, drops), intravenous, intraocular, intraperitoneal or intramuscular application or an application in the form of a impregnated association useful.

The concentration of active ingredients (bisbenzyl derivatives) of formula 1 in the Formulations to be applied (topically) are preferably in the range of 0.0008% -20% by weight and preferably in the range of 0.05% -0.5% by weight. The antimicrobial complex of active ingredients can be (a) prophylactic or (b) are used if necessary.

The concentration of the z. B. daily amount of active ingredient to be applied varies and depends on the physiological condition of the subject as well as individual-specific parameters such as age or body weight. Bisbenzyl derivatives of formula 1 can be used alone, as mixtures or also in combination with other antimicrobial substances are used.

However, the compounds of formula 1 according to the invention are of course not only for application on human or animal Body provided, but are also used to treat, for example Microorganisms on or in inanimate matter and for conservation perishable item suitable.

A method according to the invention for the preservation of a perishable article against attack by microorganisms selected from the group consisting of (i) dandruff microorganisms, (ii) body odor causing microorganisms, (iii) microorganisms causing acne, (iv) mycosis causing microorganisms and mixtures thereof, comprises providing the perishable article with an amount of a compound of the formula 1 which has an inhibiting or killing effect on the said microorganisms, the statements made above regarding the residues R ₁ to R ₄ (also with regard to the preferred selection) being applicable.

The compounds of formula 1 (wherein R ₁ to R _{4 have} the meanings given above and what has been said above with regard to the preferred meanings of R ₁ to R ₄ ) can also be used as a constituent of cosmetic agents and fragrance compositions (fragrance compositions), for example give a perfumed finished product an antimicrobial effect. A particularly preferred fragrance composition comprises (a) a sensorially effective amount of a fragrance, (b) an antimicrobial amount of one or more compounds of formula 1 (where R ₁ to R ₄ can have the meanings given above) and optionally (c) one or several carriers and / or additives. Since the proportion of perfume in a finished cosmetic product is frequently in the range of approximately 1% by weight, a perfume which contains a compound of the formula 1 is preferably approximately 5 to 50% by weight of one or more compounds of the Formula 1 exist. It had been found to be particularly advantageous that the substances of Formula 1 have only a weak intrinsic odor or are completely odorless; because this property predestines it for use in a fragrance composition.

Preferred configurations and further aspects of the present Invention result from the appended claims and the following examples.

Examples 1 to 4 Production of bisbenzyl-containing extracts and Bisbenzyl derivatives of formula 1 example 1 Extraction of Pinus sylvestris heartwood

2 kg of Pinus sylvestris heartwood chips are mixed with 14 kg of ethanol for 1 hour extracted under reflux. After filtration and concentration to dryness 100 g of dry extract are obtained (content according to HPLC: 1.8% pinosylvin, 6.8% pinosylvinmonomethyl ether). This is in 250 ml of methanol recorded and extracted five times with 250 g of hexane each. Narrow down to Drying gives 25 g Pinus sylvestris extract (HPLC content: 7.2% Pinosylvin, 29.5% Pinosylvinmonomethylether, next to it a number of Diterpene acids).

Example 2 Isolation of Pinosylvin and Pinosylvinmonomethylether from Pinus sylvestris extract

20 g of Pinus sylvestris extract according to Example 1 are on silica gel 60 chromatographed (eluent: hexane / diethyl ether gradient 70:30 → 30:30), whereby the two enriched fractions I and II are obtained. Out Fraction I is by RP18 medium pressure chromatography (column: YMC ODS- AQ, eluent: methanol / water 80: 20 + 0.5 ml acetic acid / l, λ 270 nm) Pinosylvinmonomethyl ether isolated (purity: 97%). According to RP18 Medium pressure chromatography (column: YMC ODS-AQ, eluent: Methanol / water 60: 40 + 0.5 ml acetic acid / l, λ 270 nm) from fraction II Obtain Pinosylvin (purity 98%). Spectral data for Pinosylvin and Pinosylvinmonomethyl ether: cf. Table 2.

Example 3 Catalytic hydrogenation of Pinus sylvestris heartwood extract according to example 1

1 g of Pinus sylvestris extract according to Example 1 is in 50 ml of ethanol recorded and in the presence of palladium (5% on activated carbon) with Hydrogen hydrogenates. Yield: 935 mg.

Example 4 Catalytic hydrogenation of the stilbene pinosylvin isolated according to Example 2 and pinosylvinmonomethyl ether

1 g of stilbene is taken up in 50 ml of ethanol and hydrogenated with hydrogen in the presence of palladium (5% on activated carbon). Yields: Dihydropinosylvin 940 mg (purity: 97%); Dihydropinosylvinmonomethyl ether: 925 mg (purity 96%). Spectral data for dihydropinosylvin and dihydropinosylvinmonomethyl ether: cf. Table 2. Table 2 ¹³ C NMR data for pinosylvin (2), pinosylvinmonomethyl ether (3), dihydro-pinosylvin (4) and dihydropinosylvinmonomethyl ether (5)

Example 5 A: Studies on the antimicrobial effect of bisbenzyl-containing Pinus sylvestris extract, dihydropinosylvin (4) and Dihydropinosylvinmonomethyl ether (5)

The realization that bisbenzyl derivatives of formula 1 (where R is each of the have the meanings given above) excellent for Fight germs that are responsible for body odors are based on series of studies, in which the particular relevant germs Staphylococcus epidermidis, Corynebacterium xerosis and Brevibacterium epidermidis were treated. However, below not just the results for Staphylococcus inhibition epidermidis, Corynebacterium xerosis and Brevibacterium epidermidis, but also the results of further tests. It turned out that the activity of the bisbenzyl derivatives mentioned also against others Test germs such as Malassezia furfur, Trichophyton rubrum, Propionibacterium acnes, Pseudomonas aeruginosa, Escherichia coli, Candida albicans and Aspergillus niger is high, resulting in additional uses result.

General test conditions

Evidence of the antimicrobial activity of those according to Examples 1-4 synthesized substances was carried out using the agar dilution method in Based on DIN 58 940 / ICS and DIN 58 944 / ICS. There were Petri dishes of 5.5 cm diameter with 8.7 ml freshly made and at 50 ° C. liquid Mueller Hinton agar (Merck, Art. 1.05437 or Wilkins- Chalgren Agar Boillon, Oxoid, Art. Cm 643, supplemented with 10 g agar Agar / liter) to which the various concentrations of the diluted samples in 3.3 vol .-% = 0.3 ml were added. For the test germ Malassezia furfur, Mueller-Hinton agar was used, the 3% Tween80 (Merck, Art. 8.22 187).

2.6 ml of the 6.6% samples were mixed with ethanol (96%; Merck, Art. 1.00971) diluted. By continuous 2: 1 dilution with ethanol (96%) were the further test concentrations of the respective Dilution series created in the form of geometric series.

By further dilution with the test agar (0.3 ml sample or corresponding dilutions + 8.7 ml agar) were 30 times each lower final concentrations reached (corresponds to an initial concentration of 2200 ppm each). The concentrations given below refer to the pure substance and are converted into ppm. Per Test concentration and nutrient medium were poured into 2 agar plates.

The following tests were carried out with 2 agar plates each:
K1: 9.0 ml Mueller-Hinton agar (Uninoculated) K2: 8.7 ml Mueller-Hinton agar + 0.3 ml ethanol (96%) (Uninoculated) K3: 8.7 ml Mueller-Hinton agar + 0.3 ml ethanol (96%) (Inoculated) K4: 9.0 ml Mueller-Hinton agar (Inoculated) K5: 9.0 ml Wilkins-Chalgren agar (Uninoculated) K6: 8.7 ml Wilkins-Chalgren agar + 0.3 ml ethanol (96%) (Uninoculated) K7: 8.7 ml Wilkins-Chalgren agar + 0.3 ml ethanol (96%) (Inoculated) K8: 9.0 ml Wilkins-Chalgren agar (Inoculated) K9: 9.0 ml Wilkins-Chalgren agar (Uninoculated) K10: 8.7 ml M.-H. Agar + 3% Tween80 + 0.3 ml ethanol (96%) (Uninoculated) K11: 8.7 ml M.-H. Agar + 3% Tween80 + 0.3 ml ethanol (96%) (Inoculated) K12: 9.0 ml M.-H. Agar + 3% Tween80 (Inoculated)

After solidification and drying (approx. 1 h at 37 ° C.), the test plates were inoculated punctually with 1 μl of the test germ suspensions listed in the examples below. To check purity and identity, the aerobically growing bacteria (Brevibacterium epidermidis, Corynebacterium xerosis, Staphylococcus aureus; Pseudomonas aeruginosa, Staphylococcus epidermidis) were used on Columbia blood agar (BioMérieux, Art. 43049). The mold Aspergillus niger, the yeast Candida albicans and the two skin fungi Trichophyton rubrum and Epidermophyton floccosum were cultivated on Sabouraud agar (BioMérieux, Art. 43555). Malassezia furfur was grown on Sabourad HLT agar with deflowering agents (addition of Tween80: 1%; lecithin: 0.3%; histidine: 0.1%; Merck, Art. 1.18368). Propionibacterium acnes was cultivated on Schaedler agar (BioMérieux, Art. 43273). Further information on the treated test germs can be found in Table 3. Table 3 Test germs (strain names) and bacterial counts

The test germ suspensions of the aerobically growing bacterial germs were prepared by incubating Mueller-Hinton broth (Merck, Art. 1.10293) at 36 ° C., which had been inoculated with a few individual colonies of the respective test germs. After a clear turbidity was reached, so much sterile nutrient broth was added to the suspensions that their turbidity corresponded to the McFarland standard 0.5 (approx. 1.5 × 10 ⁸ CFU / ml).

To prepare the remaining test germ suspensions, the Test strains cultivated on the above-mentioned, solid culture media, using sterile Harvested swabs and taken up in so much Mueller-Hinton broth or diluted that the turbidity of the suspensions the McFarland Standard corresponded to 0.5.

All test germ suspensions, with the exception of Propionibacterium acnes, were again diluted 1:10 with sterile broth and their bacterial count in Surface method determined using a spiralometer (results: see table 1).

The inoculated plates were incubated under the conditions given in Table 4 and then evaluated. The lowest active substance concentration at which macroscopically no growth is present was regarded as the MIC (minimum inhibitory concentration). Minimal, barely visible growth or a few small single colonies were rated as inhibitions. Table 4 Inoculation and Incubation

B: MIC values of Pinus sylvestris extract hydrogenated, dihydropinosylvin and Dihydropinosylvinmonomethylether

The MIC values of Pinus sylvestris extract, dihydropinosylvin and dihydropinosylvin monomethyl ether were determined in accordance with the general test conditions described under A and are summarized in Table 5 below.

A significant inhibition of gram-positive, body odor growth causing bacteria such as Staphylococcus epidermidis, Corynebacterium xerosis, Brevibacterium epidermidis already in concentrations smaller 0.015% could be observed for the substances listed in Table 5 become. The greatest effectiveness was shown here Dihydropinosylvinmonomethyl ether (Staphylococcus epidermidis: 69 ppm = 69 µg / ml; Corynebacterium xerosis: 69 ppm = 69 µg / ml; Brevibacterium epidermidis: 69 ppm = 69 µg / ml). The effectiveness over that responsible for acne Propionibacterium acnes, on the other hand, is special with Pinus sylvestris extract pronounced (35 ppm = 35 µg / ml), although also with dihydropinosylvin (138 ppm = 138 µg / ml) and a clear one for dihydropinosylvinmonomethyl ether Effectiveness can be observed. Also against yeast and Mold (Candida albicans, Aspergillus niger, Trichophyton rubrum, Epidermophyton floccossum) considerable effects have been observed become. Because of the observed antimicrobial effect too bisbenzyl derivatives of Malassezia furfur. formula 1 can also be used as a dandruff. Towards Pseudomonas aeruginosa and Escherichia coli proved in the chosen Concentration range only the dihydropinosylvin at a dosage of 2200 ppm 2200 µg / ml as effective. Since the substances mentioned or also Mixtures of substances over a very broad spectrum of effectiveness can also be used as a preservative.

Example 6 Skin lightening effect of bisbenzyl derivatives of formula 1 Preliminary note

Tyrosinase is one of the key enzymes in the melanin biogenesis pathway. When looking for skin-lightening effectiveness, the aim is Find substances that inhibit the enzyme tyrosinase and thus one Key step in melanin synthesis, namely the conversion of Tyrosine to L-dopa and the subsequent oxidation to dopaquinone prevention. In the presence of substances with depigmenting Properties less dopaquinone arises, so less as a result Melanin produces what ultimately lightens the skin.

Investigation of the tyrosinase inhibitory effect of stilbene or bisbenzyl-containing plant extracts as well as pure stilbenes and bisbenzyl derivatives:
The skin-lightening effectiveness of selected substances is described in more detail below. The results obtained result from test series 1 and 2 with differently active tyrosinase (see Table 5: Values for the substance known as tyrosinase inhibitor kojic acid, which was used as a positive control: 100 μmol and 390 μmol, respectively). In order to be able to compare the IC50 values of all the extracts and pure substances examined, in addition to the absolute values (values in µg / ml or for pure substances also in µmoles), the efficacies relative to the effectiveness of the kojic acid are also given (see Table 5 and 6).

Experimental Procedure Test 1

• 1. Preparation of the test solutions:
  • a) kojic acid (positive control) or one of the test substances to be examined,
  • b) tyrosinase (SIGMA T7755, isolated from fungi; 10 units, preincubated for 10 minutes together with positive control or test substance on ice) and
  • c) 5 mM tyrosine (SIGMA T8909) are placed together in 50 mM phosphate buffer pH 7.5
• 2. The test solutions are incubated at 37 ° C for 1 h
• 3. The conversion of tyrosine to dopaquinone is spectrophotometric determined: λ = 450 nm

dosage Positive control and test substances

• a) Positive control: Kojic acid 10 mM, 5 mM, 0.5 mM, 0.05 mM and 0.005 mM in phosphate buffer
• b) Pinosylvin or dihydropinosylvin each 10% in ethanol: 0.015%, 0.05%, 0.15%, 0.5% and 1.5%;

Result

The IC50 value (value of the concentration at which a 50% inhibition was reached) for Kojic acid in test 1 was 100 µmol. The value for Pinosylvin was 94 µmol (= 21.2 µg / ml). The IC50 value for dihydropinosylvin could not be determined exactly in test series 1 because it was <70 µmol (i.e. < 14.9 µg / ml) was below the lowest concentration still tested (see Tables 5 and 6). Because of this, the IC50 was designed for Dihydropinosylvin exactly determined in test 2.

Test 2

• 1. Preparation of the test solutions:
  • a) kojic acid (positive control) or one of the test substances to be examined,
  • b) tyrosinase (SIGMA T7755, isolated from fungi; 10 units, preincubated for 10 minutes together with positive control or test substance on ice) and
  • c) 5 mM tyrosine (SIGMA T8909)
  are presented together in 50 mM phosphate buffer at pH 7.5
• 2. The test solutions are incubated at 37 ° C for 1 h
• 3. The conversion of tyrosine to dopaquinone is spectrophotometric determines λ = 450 nm

dosage Positive control and test substances

• a) Positive control: Kojic acid 10 mM, 5 mM, 1 mM, 0.1 mM and 0.01 mM in phosphate buffer
• b) Dihydropinosylvin 10% in ethanol: 0.0015%, 0.005%, 0.015%, 0.15%;
• c) Pinus sylvestris extract or Pinus sylvestris extract hydrogenated 10% in Ethanol: 0.015%, 0.05%, 0.15%, 0.5%, 1.5%

Result test 2

The IC50 value for kojic acid in test 2 was 390 µmol. The IC50 value for dihydropinosylvin was 38.0 µmol (= 8.10 µg / ml). The IC50 values for Pinus sylvestris extract and Pinus sylvestris hydrogenated extract were> 1500 µg / ml and 140 µg / ml, respectively (see Tables 5 and 6). Table 5

Table 6

The studies show that stilbenes such as Pinosylvin, bisbenzylhaltige Plant extracts and bisbenzyl derivatives of the dihydropinosylvine type strongly inhibit tyrosinase and therefore used as a skin lightening agent can find. The greatest effectiveness was shown here Dihydropinosylvin with IC50 values of 38 µmol corresponding to an application concentration of approx. 8 µg / ml. As the relative values show, dihydropinosylvin is thus about 10 times more effective than the known tyrosinase Inhibitor kojic acid. The examined plant extracts containing bisbenzyl still show relatively good efficacy, but are less effective as kojic acid.

Claims (9)

1. Use of a compound of the formula 1 or a mixture of substances which comprises one or more compounds of the formula 1,


in which
R ₁ and R _{2 are} independently selected from the group consisting of
Hydrogen,
Methyl,
straight-chain or branched, saturated or unsaturated alkyl with 2-10 C atoms and
alkyl with 2-10 C atoms interrupted by one or more oxygen and / or sulfur atoms
exists and
R ₃ and R _{4 are} independently selected from the group consisting of
Hydrogen,
Hydroxy and
methoxy
consists,
as a means of a) inhibition of enzymes selected from the group consisting of tyrosinase, tyrosinase isoenzymes and mixtures thereof and / or b) treatment of microorganisms selected from the group consisting of (i) dandruff microorganisms, (ii) body odor microorganisms, (iii) acne microorganisms, (iv) mycosis causing microorganisms and (v) mixtures thereof. 2. Use according to claim 1, wherein
R _{1 is} hydrogen and
R _{2 is} selected from the group consisting of
Hydrogen,
Methyl,
straight-chain or branched, saturated or unsaturated alkyl with 2-10 C atoms and
alkyl with 2-10 C atoms interrupted by one or more oxygen and / or sulfur atoms
consists. 3. Use according to claim 1 or 2, wherein
R _{1 is} hydrogen and
R _{2 is} hydrogen or
methyl
is. 4. Method of inhibition a) an enzyme selected from the group consisting of tyrosinase, tyrosinase isoenzymes and mixtures thereof and / or b) a microorganism selected from the group consisting of (i) dandruff-causing microorganisms, (ii) body odor-causing microorganisms, (iii) acne-causing microorganisms, (iv) mycosis-causing microorganisms and (v) mixtures thereof, wherein
the enzyme and / or the microorganism is contacted with an inhibiting amount of a compound of the formula 1 or a mixture of substances which comprises one or more compounds of the formula 1,


in which
R ₁ and R _{2 are} independently selected from the group consisting of
Hydrogen,
Methyl,
straight-chain or branched, saturated or unsaturated alkyl with 2-10 C atoms and
alkyl with 2-10 C atoms interrupted by one or more oxygen and / or sulfur atoms
exists and
R ₃ and R _{4 are} independently selected from the group consisting of
Hydrogen,
Hydroxy and
methoxy
consists. 5. The method of claim 4, wherein the inhibitory amount of Compound or mixture of substances topically on a human or animal body is applied. 6. A method of preserving a perishable article against attack by microorganisms selected from the group consisting of (i) dandruff microorganisms, (ii) body odor causing microorganisms, (iii) acne causing microorganisms, (iv) mycosis causing microorganisms and (v) their mixtures,
wherein the perishable article contains an amount of a compound of formula 1 which has an inhibiting or killing effect on the microorganisms


is equipped, whereby
R ₁ and R _{2 are} independently selected from the group consisting of
Hydrogen,
Methyl,
straight-chain or branched, saturated or unsaturated alkyl with 2-10 C atoms and
alkyl with 2-10 C atoms interrupted by one or more oxygen and / or sulfur atoms
exists and
R ₃ and R _{4 are} independently selected from the group consisting of
Hydrogen,
Hydroxy and
methoxy
consists. 7. A process for the cosmetic lightening of human skin, comprising the topical application of a tyrosinase-inhibiting amount of one or more compounds of the formula 1,


in which
R ₁ and R _{2 are} independently selected from the group consisting of
Hydrogen,
Methyl,
straight-chain or branched, saturated or unsaturated alkyl with 2-10 C atoms and
alkyl with 2-10 C atoms interrupted by one or more oxygen and / or sulfur atoms
exists and
R ₃ and R _{4 are} independently selected from the group consisting of
Hydrogen,
Hydroxy and
Methoxy. 8. Perfume composition, comprising a) a sensorially effective amount of a fragrance, b) an antimicrobial amount of one or more compounds of the formula 1


in which
R ₁ and R _{2 are} independently selected from the group consisting of
Hydrogen,
Methyl,
straight-chain or branched, saturated or unsaturated alkyl with 2-10 C atoms and
alkyl with 2-10 C atoms interrupted by one or more oxygen and / or sulfur atoms
exists and
R ₃ and R _{4 are} independently selected from the group consisting of
Hydrogen,
Hydroxy and
methoxy
consists. 9. Use of a compound of the formula 1 or a mixture of substances which comprises one or more compounds of the formula 1,


in which
R ₁ and R _{2 are} independently selected from the group consisting of
Hydrogen,
Methyl,
straight-chain or branched, saturated or unsaturated alkyl with 2-10 C atoms and
alkyl with 2-10 C atoms interrupted by one or more oxygen and / or sulfur atoms
exists and
R ₃ and R _{4 are} independently selected from the group consisting of
Hydrogen,
Hydroxy and
methoxy
consists,
as a cosmetic.