Gentiopicroside free gentiana extract

Abstract

The present invention relates to gentiopicroside free Gentiana acaulis and/or Gentiana septemfida extracts. Furthermore, it relates to cosmetic compositions comprising such gentiopicroside free Gentiana acaulis or Gentiana septemfida extract, as well as mixtures thereof.

Claims

1. A method for combatting skin aging by reducing wrinkles, inducing collagen synthesis, and/or reducing intracellular reactive oxygen species production in skin of a patient in need thereof, wherein the method comprises topically applying to the skin of the patient an effective amount of a cosmetic composition comprised of a substantially gentiopicroside-free extract of Gentiana acaulis and/or Gentiana septemfida, and observing the skin aging combatting effects thereof, wherein the substantially gentiopicroside-free extract comprises gentiopicral hydrolyzed from gentiopicroside.

2. The method according to claim 1, wherein the cosmetic composition comprises between 0.001 and 20 wt. % in dry matter, based on the total weight of the cosmetic composition, of the substantially gentiopicroside-free extract of Gentiana acaulis and/or Gentiana septemfida.

3. The method according to claim 1, wherein the cosmetic composition further comprises glycerin.

4. The method according to claim 1, wherein the gentiopicral is present in the cosmetic composition in an amount of less than 1%.

5. The method according to claim 1, wherein the cosmetic composition comprises less than 0.1% concentration of gentiopicroside.

6. The method according to claim 1, wherein the cosmetic composition comprises no detectable gentiopicroside.

7. A method for combatting skin aging by reducing wrinkles, inducing collagen synthesis, and/or reducing intracellular reactive oxygen species production in skin of a patient in need thereof, wherein the method comprises topically applying to the skin of the patient an effective amount of a cosmetic composition comprised of a substantially gentiopicroside-free extract of Gentiana acaulis and/or Gentiana septemfida in a mass ratio of Gentiana acaulis to Gentiana septemfida between 5/95 and 50/50, and observing the skin aging combatting effects thereof.

8. The method according to claim 7, wherein the mass ratio of Gentiana acaulis to Gentiana septemfida is between 10/90 and 30/70.

9. The method according to claim 7, wherein the mass ratio of Gentiana acaulis to Gentiana septemfida is between 15/85 and 25/75.

Description

EXAMPLES

Example 1: Skin Care Composition

(1) TABLE-US-00001 2.1 2.2 O/W-Emulsions Wt. % Wt. % Cetearyl Alcohol + Sodium Cetylstearylsulfat 3.00 2.00 Glycerylstearat SE 2.00 4.00 Octyldodecanol 2.00 2.00 C12-15 Alkylbenzoate 1.00 1.00 C13-16 Isoparaffin 3.00 3.00 Caprylic acid-/Capric acid triglyceride 2.00 2.00 Glycerine 5.00 6.00 Dimethicone 0.50 0.50 Sodium ascorbylphosphate 0.10 Ethylhexylsalicylate 0.50 0.50 Glycyrrhetic acid 0.10 Gentiana acaulis extract 1.00 1.50 Grape seed oil 0.50 0.50 Dihydroxyacetone 2.00 Paraffinum Liquidum + Ginkgo Biloba Extract 0.25 0.25 Citric acid 0.09 0.09 Sodium citrate 0.17 0.17 Xanthan gum 0.10 Ammonium Acryloyldimethyltaurate/VP Copolymer 0.40 Carbomer 0.30 Parabene 0.30 0.30 Phenoxyethanol 0.50 0.50 Alcohol Denat. 3.50 3.50 Perfume q.s q.s Water ad 100 ad 100 2.3 2.4 Sprayable emulsions Wt. % Wt. % Isoceteth-20 5.00 3.00 Glycerylisostearate 3.00 2.00 Mineral Oil 5.00 4.00 Glycerine 4.00 5.00 Tocopherylacetate 0.50 0.40 Gentiana acaulis extract 0.20 0.50 Natriumcitrate 0.40 0.40 Phenoxyethanol 0.40 0.40 Citric acid 0.20 0.20 DMDM Hydantoin 0.20 0.20 Perfume q.s. q.s. Water ad 100 ad 100 2.5 2.6 2.7 O/W-Emulsions wt. % wt. % wt. % Stearic acid 2.00 3.00 3.00 Glycerylstearat 2.00 2.00 1.00 Sorbitanstearat 1.00 Dicaprylyl Ether 3.00 3.00 Caprylic acid-/Capric acid triglyceride 3.00 3.00 Cetearyl Alcohol 2.00 2.00 Cetyl Alcohol 1.00 Stearyl Alcohol 3.00 Hydrogenated Coco-Glycerides 4.00 Mineral Oil 3.00 PEG-100 stearat 1.00 0.50 Trisodium EDTA 1.00 Glycerine 4.00 6.00 10.00 Dimethicone 1.00 Glycyrrhetic acid 0.20 Gentiana acaulis/Gentiana septemfida 0.20 0.50 0.10 extract in a ratio 15/85 Erythrulose 4.00 Phenoxyethanol 0.40 0.40 0.40 Parabene 0.20 0.20 0.20 Citric acid 0.09 Carbomer 0.20 0.20 0.20 Perfume q.s. q.s. q.s. Water ad 100 ad 100 ad 100 2.8 2.9 O/W-Emulsions Wt. % wt. % Glycerylstearate 3.00 4.00 C12-15 Alkylbenzoate 4.00 4.00 Caprylic acid-/Capric acid triglyceride 3.00 2.50 Isopropylstearat 2.00 2.50 Cetyl Alcohol 2.00 2.00 Stearyl Alcohol 2.00 2.00 Glycerin 3.00 5.00 Dimethicone 0.50 2.00 Glycyrrhetic acid 0.10 Gentiana acaulis/Gentiana septemfida extract 1.00 0.50 in a ratio 15/85 Phenoxyethanol 0.40 0.40 Parabene 0.20 0.20 Carbomer 0.10 0.10 Perfume q.s. q.s. Water ad 100 ad 100 2.10 Tanning Spray Wt. % Butyl methoxydibenzoylmethane 5.00 Octocrylene 10.00 Homosalate 5.00 Glycerine 0.50 Gentiana acaulis/Gentiana septemfida extract in a ratio 15/85 0.50 Perfume q.s. Ethanol ad 100 2.11 Emulsions-Fluid Wt. % Stearic acid 2.00 Dicaprylylether 3.00 Octyldodecanol 2.00 C12-15 Alkylbenzoate 4.00 Cetylalcohol 2.00 Cetearyl Ethylhexanoate + Isopropyl myristate 2.00 Glycerine 5.00 Ethylhexylmethoxycinnamate 2.00 TiO2 1.00 Cetylpalmitate 1.00 Glyceryl Stearate 1.00 Phenoxyethanol 0.40 Butyl Methoxydibenzoxylmethane 2.00 Perfume q.s. EDTA 0.20 Carbomer 0.20 Magnesium Aluminium Silicate 0.20 Paraben 0.20 Vitamin E Acetat 0.10 Gentiana acaulis/Gentiana septemfida extract in a ratio 15/85 0.10 Paraben 0.05 BHT 0.05 DHA 1.00 2.12 O/W-Emulsion: night cream Wt. % Glycerylstearatcitrate 2.00 Stearylalcohol 2.00 Cetylalcohol 2.00 Hydrogenated Coco Glyceride 1.00 Caprylic acid-/Capric acid triglyceride 3.00 Ethylhexylkcoco fatty acid esters 2.00 Dicaprylylether 2.00 C12-15 Alkylbenzoate 3.00 Tocopherylacetate 1.00 Ubichinon (Coenzyme Q10) 0.10 Sodium ascorbylphosphate 0.10 Gentiana acaulis/Gentiana septemfida extract in a ratio 15/85 1.00 Parabene 0.40 Methylpropandiol 1.00 Carrageenan 0.10 Carbomer 0.20 Tapioca starch 2.00 EDTA 0.20 Glycerine 5.00 Waster and/or oil soluble dyes 0.05 Filling agents/Additives 0.50 Perfume q.s. Water ad 100 The pH of the formulation is adjusted to about pH 6.5 2.13 2.14 O/W-Emulsion: day cream Wt. % Wt. % PEG-40-Stearat 1.00 1.00 Glycerylstearate 3.00 3.00 Cetearylalcohol 2.00 2.00 Dimethicone 1.00 1.00 Hydrogenated Coco Glycerides 2.00 2.00 Caprylic acid-/Capric acid triglyceride 2.00 2.00 Octyldodecanol e 2.00 2.00 Dicaprylylcarbonate 2.00 2.00 C12-15 Alkylbenzoate 3.00 3.00 Ethylhexyl methoxycinnamate 4.00 4.00 Butyl methoxydibenzoylmethane 2.00 2.00 Tocopherylacetate 1.00 1.00 Panthenol 0.50 0.50 Sodium ascorbylphosphate 0.10 0.10 Ammonium Acryloyldimethyltaurate/VP Copolymer 0.40 Glycyrrhetic acid 0.10 Gentiana acaulis/Gentiana septemfida extract in a 1.00 0.50 ratio 15/85 Nylon-12 3.00 Distarch phosphate 2.00 Parabene 0.40 0.40 Methylpropandiol 1.00 1.00 Carbomer 0.20 0.20 Xanthan gum 0.10 0.10 EDTA 0.20 0.20 Glycerine 8.00 8.00 Tapioca starch 0.05 0.05 Filling agents/Additives 0.30 0.30 Perfume q.s. q.s. Water ad 100 ad 100 2.15 2.16 O/W-Emulsion: facial cream Wt. % Wt. % Polyglyceryl-3-Methylglucosedistearate 2.00 2.00 Sorbitanstearate 3.00 3.00 Cetylalcohol 2.00 2.00 Myristylmyristate 1.00 1.00 Dicaprylylether 3.00 3.00 Octyldodecanol 2.00 2.00 C12-15 Alkylbenzoate 3.00 3.00 Cetearyl Ethylhexanoat + Isopropylmyristate 2.00 2.00 Ethylhexyl methoxycinnamate 2.00 2.00 Ethylhexyltriazone 1.00 1.00 Butyl Methoxydibenzoxylmethane 2.00 2.00 Magnesium Aluminium Silicate 0.20 0.20 Glycerine 5.00 5.00 Phenoxyethanole 0.40 0.40 Parabene 0.30 0.30 Vitamin E Acetate 0.10 0.10 Glycyrrhetic acid 0.10 Gentiana acaulis/Gentiana septemfida extract in a 1.50 1.00 ratio 15/85 BHT 0.05 0.05 EDTA 0.20 0.20 Carbomer 2.00 0.20 Perfume q.s. q.s. Water ad 100 ad 100

Example 2: Preparation of a Gentiopicroside/Gentiopicral Free Gentiana Extract

(2) aPreparation of the Enzyme Bound on Solid Phase (Step Known from the Literature)

(3) The enzyme -glucosidase (EC 3.2.1.21) (100 mg) was incubated with 15.4 mg of glucose dissolved in 82 ml of buffer solution (pH=7.75) at room temperature for 1 hour.

(4) The resin Purolite ECR 8214F is mixed separately with 30 ml of buffer solution (pH=7.75) for 5 minutes and then filtered (washing step).

(5) The filtered resin was then added to the buffer solution containing the enzyme at 25 C. and mixed gently for 24 hours. After 24 hours, the solution was filtered and the resulting resin was washed with 60 ml of buffer solution (pH=7.75) and twice 60 ml of deionized water. The resin was then mixed in 80 ml of buffer solution (pH=7.75) and 1 ml of BSA (Bis(trimethylsilyl)acetamide) was added. The suspension was mixed gently for 24 hours. After 24 hours, the resin was filtered and washed twice with 100 ml deionized water.

(6) The buffer solution is a mixture of 14.4 ml of solution KH.sub.2PO.sub.4 (68 g/500 ml) and 86.6 ml of solution K.sub.2HPO.sub.4 (87.1 g/500 ml).

(7) bPreparation of the Gentiopicroside Free Gentiana Extract: Transformation of the Gentiopicroside into the Gentiopicral in Gentiana Extract.

(8) b-1) An extract of 50 g of the aerial parts (flower, leaf, stem) of rare alpine plants from the Gentian species (Gentiana acaulis and/or Gentiana septemfida) was prepared as followed: chopped dried plants were extracted twice with 10 times (w/w) of 60% ethanol (with active carbon) for 2 hours at 50 C. The extract was then filtered and concentrated in a Rotavapor device at 50 C. and 50 mbar atmosphere to obtain a concentrated aqueous solution. Pure ethanol (5 times the quantity in weight of the aqueous solution) was added into this concentrate in order to precipitate sugars and proteins, then, after filtration, ethanol was removed once again in a Rotavapor device resulting in about 54 g of an aqueous extract comprising about 36% (area %) of gentiopicroside (determined by UPLC: C18 reversed phase column, eluen:water/acetonitrile, detection: 254 nm) which was used in in the subsequent step b-2):

(9) b-2) The aqueous solution obtained as described in b-1) was placed in a flask at 28 C. (bath) and the enzyme (5 g) prepared in step a) (white powder) was added to extract solution. The reaction was done during a few days and was followed by UPLC. The percentage of transformation of the gentiopicroside was closely monitored. If necessary, a supplementary amount of bound enzyme was adde. When the gentiopicroside was completely transformed into gentiopicral, the reaction was terminated by filtration and subsequent washing of the filtered resin with 50 ml of deionized water. The washing solution was collected together with the main solution.

(10) The solid phase bound with the enzyme was washed and dried (lyophilized) in order to be re-used.

(11) cRemoval of the Gentiopicral.

(12) The obtained solution in step b-2) (100 ml) was extracted twice with an equal amount of ethyl acetate (100 ml). The organic and aqueous phase were then separated. The aqueous solution was analyzed by UPLC: C18 reversed phase column, eluent: water/acetonitrile, detection: 254 nm.) to determine if gentiopicral was completely removed. If necessary a third extraction with 100 ml ethyl acetate was performed. The obtained aqueous phase was distilled under vacuum in order to remove remaining traces of ethyl acetate. The aqueous solution finally obtained can be used for formulation directly or for dry extract manufacturing.

(13) The studies were performed with the extracts from individual Gentian or as mixtures of both plants in a ratio of Gentiana acaulis/Gentiana septemfida, 15/85 mass/mass or 25/75 mass/mass.

Example 3: Effect of a Gentiana Extract on Hyaluronan Production

(14) Gentiana extract (Gentiana acaulis/Gentiana septemfida, 25/75 m/m) was used. Normal human fibroblasts (NHF) P2+1 were seeded at 5000 cell/well in 96 well cell culture plates (Nunclon) in MEM 10% FCS (Pan Biotech). After three days of growth, Gentiana extract solution was added in medium without FCS and incubated the cells for an additional three days. Secreted hyaluronan in the growth medium was measured using a Hyaluronan Assay Kit (K-1200, Echelon, Salt Lake City, Utah).

(15) Compared to untreated cells we showed a 264.9% increase in hyaluronan production with 0.001% Gentiana extract and TGFbeta1 (10 ng/ml), served as positive control, stimulated by 45.61.1%.

Example 4: Effect of a Gentiana Extract on CPD Repair (Cyclobutane Pyrimidine Dimer)

(16) An average of 400000 cells were seeded into 6 well plates. After over-night incubation in growth medium cells were washed twice with PBS, overlaid with 1 ml PBS, and irradiated with 20 mJ/cm2 UVB. Afterwards, the cells were incubated with growth medium and Gentiana extract. At defined time-points cells were trypsinized, and DNA was isolated using a DNA-Isolation Kit from Qiagen. 200 ng of DNA was spotted onto ELISA-plates and CPDs were detected using a biotinylated anti-CPD-antibody from Kamiya at 1:2000 dilution. The extract consists of Gentiana acaulis & Gentiana septemfida, 25/75 m/m.

(17) These results in table below show a 40% increase of CPD repair after 4 hours with 0.1% Gentian extract.

(18) TABLE-US-00002 Relative Hours after UVB Relative CPD staining of CPD staining of treated (20 mJ/cm2) control (Untreated) with 0.1% Gentian extract 0 100% 100% 2 95.4% 101.1% 4 75.0% 33.9% 24 29.2% 6.7%

Example 5: Effect of a Gentiana Extract on Pro-Inflammatory Cytokines & MMPs

(19) Material & Method

(20) Measurement of Cytokines and MMPs

(21) Normal human keratinocytes (NHK) P4+1 were grown to sub-confluency in CnT-07 growth medium (CELLnTEC, Berne, Switzerland). After irradiation with 100 mJ/cm.sup.2 UVB with a solar light simulator (SOL500RF, Dr. Hoenle, Germany), cells were incubated again for 24 hrs in CnT-07 containing 0.1% Gentiana extract (Gentiana acaulis & Gentiana septemfida, 25/75 m/m) or vehicle. Cell culture supernatant was collected over time and frozen at 20 C until cytokines and MMPs were measured in a Luminex100 device. Luminex100 measurements were performed using a multiplex custom made kit (Panomics, Italy) according to manufacturer's instructions.

(22) An inhibition of pro-inflammatory cytokines IL-1, IL-8, TNF, MMP-1 release was found as highlighted in the table below

(23) Table with results for example 8: %-Inhibition of cytokine release of UVB-irradiated cells treated with 0.1% Gentiana extract compared to UV irradiatedbut untreated cells.

(24) TABLE-US-00003 IL-1beta TNFalpha IL-8 MMP-1 2 h 78% 0% 80% 83% 4 h 87% 31.2% 74.3% 82.3% 8 h 80.7% 95.7% 80% 94% 24 h 37% 77.9% 31% 56.9%

Example 6: Reduction of Intracellular Reactive Oxygen Species Production by a Gentiana Extract

(25) Detection of Reactive Oxygen Species (ROS)

(26) Intracellular ROS generation was determined using ROS specific probe H.sub.2DCF-DA (Molecular Probes). Oxidation of H.sub.2DCF-DA to the fluorescent product DCF serves an indicator of the overall degree of intracellular oxidative stress.

(27) Primary human fibroblasts were seeded at 5000 cells/well in 96 well cell culture plates in DMEM, 10% FCS, 1% Pen/Strep. After 24 h Gentiana extract (Gentiana acaulis & Gentiana septemfida, 25/75 m/m) was added solution and incubated the cells for additional 72 h. After compound treatment we incubated the cells with H.sub.2DCF-DA (25 M) and Hoechst 33258 (1 ug/ml) in HBSS/2.5% HEPES for 45 min at 37 C. in the dark. We washed the cells twice with HBSS/HEPES to remove any H.sub.2DCF-DA which has not been internalised and subjected them to 30% H.sub.2O.sub.2. ROS production was measured in a fluorescent plate reader (SpectraMax GeminiXS) after 30 min (at 520 nM), and normalized to the DNA content.

(28) In this study we showed compared to untreated cells a 30% reduction of intracellular ROS production in cells treated with 0.05% Gentiana extract.

(29) All this data demonstrate a positive effect on photo-aging and anti-aging in vitro.