SEED EXTRACT OF ANNONA CHERIMOLA

Abstract

The present invention generally relates to the field of cosmetics. More particularly, the invention relates to a cosmetic skincare composition comprising a seed extract of the plant Annona cherimola. The invention is based on the finding that such seed extract has several favourable effects on skin. Amongst others, a seed extract of Annona cherimola exerts calming, soothing, moisturizing and anti-itching effect. In another aspect, the invention therefore provides a cosmetic method of improving skin appearance or reducing skin dryness in a subject, comprising administering a skin care composition of the present invention. In yet another aspect, the invention provides a cosmetic method of soothing or calming irritated skin in a subject, comprising administering a skin care composition of the present invention. The invention also relates to the use of a seed extract of Annona cherimola for skin care, and in particular for soothing or calming irritated skin, or improving skin appearance or reducing skin dryness and itching.

Claims

1. Cosmetic skin care composition comprising a seed extract of the plant Annona cherimola.

2. Cosmetic composition of claim 1, wherein the extract is an aqueous extract.

3. Cosmetic composition of claim 2, wherein the extract is present in the composition in an amount of 0.05 to 25.0% (w/w), and preferably 0.5 to 5.0% (w/w).

4. Cosmetic composition according to claim 1, wherein said composition is formulated for topical application.

5. Cosmetic composition of claim 4, wherein the said composition is formulated for topical application to the skin of the face or body.

6. Cosmetic composition according to claim 1, wherein said composition is formulated as an ointment, cream, lotion, paste, gel, hydrogel, foam or powder.

7. Cosmetic composition according to claim 1, wherein said composition comprises an emollient selected from the group consisting of olive oil, palm oil, soybean oil, sesame seed oil, rapeseed oil, evening primrose oil, sunflower seed oil, avocado oil, olive oil, coconut oil, castor oil, safflower seed oil, myristyl lactate, isopropyl myristate, polyethylene glycol, isopropyl palmitate, isopropyl stearate, isobutyl palmitate, isocetyl stearate, or cetyl alcohol.

8. Cosmetic composition according to claim 1, wherein said composition has a pH in the range from about 2.5 to about 6.5, preferably from about 4.0 to about 6.0,

9. Cosmetic composition according to claim 1, wherein said composition comprises a humectant selected from the group consisting of glycerine, polyethylene glycol ethers of glycerine, amino acids, such as proline and arginine, sugar and sugar alcohols, such as glucose, mannose, trehalose, and polyglycerol sorbitol, 1,3-butylene glycol, propylene glycol, diglycerol, glycerol monopropoxylate, glycogen, sodium hyaluronate, sodium poly-aspartate, sodium polyglutamate, sorbeth 20, sorbeth 6, and hydrogenated starch hydrolysates.

10. Cosmetic composition according to claim 1, wherein said composition comprises an exfoliating compound selected from the group consisting of urea, alpha-hydroxy acids and beta-hydroxy acids, and their esters, anhydrides, and salts.

11. A cosmetic method of improving skin appearance or reducing skin dryness in a subject, comprising administering a composition of claim 1 to the skin of said subject.

12. A cosmetic method of soothing or calming irritated skin in a subject, comprising administering a composition of claim 1 to the skin of said subject.

13. A cosmetic method of claim 11, wherein said administration comprises topical administration to the skin of the face or body of said subject.

14. Use of a composition of claim 1, for cosmetic skin care.

15. Use of a composition of claim 1, for soothing irritated skin, calming irritated skin, improving skin appearance and/or reducing skin dryness.

Description

BRIEF DESCRIPTION OF THE FIGURES

[0033] FIG. 1 shows that the extract F230 acts as a cannabinoid receptor-2 agonist. 293T-CB2-CRE-luc cells were treated with the extract F230 or the positive control WIN-55,212-2 for 15 min. Subsequently, cells were incubated with 10 μM Forskolin. After six hours incubation, the cells were lysed and luciferase activity was measured. The specific transactivation observed for F230 is expressed as [%] of Forskolin stimulation.

[0034] FIG. 2 shows that the extract F230 has a STAT3-inhibiting activity. HeLa-STAT3-luc cells were treated with F230 and stimulated with IFN-γ. After 6 h the cells were lysed and the luciferase activity was measured. The results are expressed as percentage of the inhibition of STAT3 activity induced by IFN-γ (100% activation). The experiments for each F230 concentration were made in triplicate.

[0035] FIG. 3 shows that the extract F230 effectively reduces the capsaicine-induced release of the cytokine IL-1β in a co-culture of keratinocytes and human sensory neurons.

[0036] FIG. 4 shows that the extract F230 effectively reduces the capsaicine-induced release of the cytokine IL-8 in a co-culture of keratinocytes and human sensory neurons.

[0037] FIG. 5 shows that the extract F230 reduces capsaicine-induced CGRP expression. Increasing amounts of F230 result in higher levels of CGRP expression inhibition.

[0038] FIG. 6 shows that the extract F230 reduces skin itching significantly compared to placebo already 1 minute after application.

[0039] FIG. 7 shows that the extract F230 reduces skin redness significantly compared to placebo 17 days after application.

[0040] FIG. 8 shows that the extract F230 increases the perception threshold of the skin compared to placebo.

EXAMPLES

[0041] The following examples describe certain preferred embodiments of the present invention. It is however to be noted that the invention is not limited to such embodiments.

Example 1: Preparation of Annona cherimola Seed Extract

[0042] Extracts of Annona cherimola were prepared as follows. Fruits from Annona cherimola were opened and the fruit pulp was removed. The seeds were harvested and subjected to drying overnight at a temperature of 40° C. After drying, seeds were disintegrated by milling. The disintegrated seeds were then extracted for 4 h at 50° C. using a sodium-citrate buffer comprising 0.1 M citric acid monohydrate (C.sub.6H.sub.8O.sub.7.H.sub.2O) and 0.1 M trisodium citrate, dihydrate C.sub.6H.sub.5O.sub.7Na.sub.3.2H.sub.2O), pH 6.0.

[0043] After extraction, the liquid phase was separated from solid plant material using a decanter CA 22 device (Westfalia Seperator AG). The liquid fraction was kept for 16 hours at room temperature for stabilization and subsequently subjected to a final filtration step using a 0.2 μm celluloseacetate membrane. The extract so obtained is referred to as “F230” in the following. Mass spectrometry analysis of the extract F230 revealed that it contains cherimolacyclopeptides A, B, C, D, E and F (see references 1-10) as well as alkaloids isoboldine, reticuline, corydine, michelalbine, and riboflavin.

Example 2: Cannabinoid Receptor-2 Agonistic Activity

[0044] The cannabinoid receptor 2 (CB2) is part of a complex regulation system that is involved in inflammation. Amongst others, the activity of this receptor is involved in thermoregulation, cell growth, host defense, apoptosis, pruritus, pain, and wound healing (Soethoudt et al. (2017), Nat. Commun. 8, 13958; Ibsen (2017), Cannabis and Cannabinoid Research 2:1, 48-60). The activation of CB2 leads to suppression of inflammatory cytokines and pain inducing neuropeptides like substance CGRP (Calcitonin Gene-Related Peptide). Therefore, compounds that enhance the activity of CB2 are potentially useful for mitigating inflammatory states of the skin that are associated, for example, with atopic dermatitis, psoriasis and pruritus.

[0045] For assessing the potential effect of an F230 extract prepared as described in Example 1, 293T-CB2 cells which had been stably transfected with cDNA encoding the human cannabinoid receptor 2 (CB2) were incubated in 24-well plates at a density of 1×10.sup.5 cells/ml and transiently transfected with 0.1 μg/ml of the plasmid CRE-luc containing six consensus cAMP responsive elements (CRE) linked to firefly luciferase. Transient transfection was performed with Rotifect (Carl Roth GmbH, Karlsruhe, Germany) according to the manufacturer's instructions. Cells were harvested 24 h after transfection.

[0046] To measure the CB2 agonistic activity of F230, 293T-CB2-CRE-luc cells were treated either with increasing concentrations of the F230 extract or the positive control WIN-55,212-2 (Sigma Aldrich, Taufkirchen, Germany) for 15 min and then with 10 μM Forskolin (Sigma Aldrich, Taufkirchen, Germany). After six hours of stimulation, the cells were lysed in 25 mM Trisphosphate pH 7.8, 8 mM MgCl.sub.2, 1 mM DTT, 1% Triton X-100, and 7% glycerol. The luciferase activity was then measured using an Autolumat LB 9501 (Berthold Technologies, Bad Wildbad, Germany) following the instructions of the luciferase assay kit (Promega, Madison, USA). The background obtained with the lysis buffer was subtracted in each experiment, and the specific transactivation was expressed as [%] of Forskolin stimulation (the latter of which was set to 100%).

[0047] Results: The results are depicted in FIG. 1. The assay results reflect a CB2 agonist activity as inhibition of Forskolin-induced CRE-Luc activity. It can be seen that the positive control WIN-55,212-2 acts as a CB2 agonist and partially antagonizes the effect of Forskolin. The same effect is also observed for F230, whereas the level of Forskolin antagonization increases with increasing amounts of F230. Consequently, the assay demonstrates that F230 exerts a CB2 agonistic activity.

Example 3: STAT3 Inhibition Activity

[0048] The signal transducer and activator of transcription 3 (STAT3) is a human transcription factor encoded by the STAT3 gene. STAT3. During skin inflammation, IFN-γ stimulates the expression of STAT3 which in turn induces the expression of additional proinflammtory cytokines, thereby contributing to inflammation (Archer et al. (2017), The Journal of Immunology, 198 (1 Supplement) 197.4). The inhibition of IFN-γ-stimulated STAT3 expression is thought to reduce an inflammatory immune response of the skin.

[0049] For assessing the potential effect of an F230 extract prepared as described in Example 1 on STAT3 activity, HeLa-STAT3-luc cells that had been stably transfected with the plasmid 4×M67 pTATA TK-Luc were used. The cells were seeded at a density of 2×10.sup.4 cells/ml in 96-well plates the day before the assay. Then the cells were treated with F230 for 15 min and subsequently stimulated with 25 U/ml IFN-γ.

[0050] After 6 h the cells were washed twice with phosphate buffered saline (PBS) and lysed by incubation in 50 μl lysis buffer containing 25 mM Tris-phosphate (pH 7.8), 8 mM MgCl.sub.2, 1 mM DTT, 1% Triton X-100 (Sigma Aldrich, Taufkirchen, Germany), and 7% glycerol (Sigma Aldrich, Taufkirchen, Germany) for 15 min at RT in a horizontal shaker.

[0051] Luciferase activity was measured using a plate reader Berthold/LB 941 (Berthold Technologies) following the instructions of the luciferase assay kit (Promega, Madison, Wis., USA). The relative luminescence unit (RLU) was calculated and the results expressed as percentage of inhibition of STAT3 activity induced by IFN-γ (100% activation). The experiments for each concentration of the test items were done in triplicate wells.

[0052] Results: The results are depicted in FIG. 2. As can be seen from the figure, F230 counteracts the IFN-γ-induced STAT3 activity. Increasing amounts of F230 result in higher levels of STAT3 inhibition.

Example 4: Inhibition of IL-1β and IL-8 Expression

[0053] As pro-inflammatory cytokines, IL-1β and IL-8 are involved in numerous skin diseases that are associated with inflammation, such as psoriasis, atopic dermatitis, neutrophilic dermatoses, and eczema (Jensen (2010); Current opinion in investigational drugs (London, England: 2000); 11(11), 1211-20); Bou-Dargham et al. (2017), Med. Res. Rev., 37: 180-216; Amarbayasgalan et al. (2013), Int Arch Allergy Immunol, 160:63-74). Accordingly, inhibition of the production of these cytokines is helpful for mitigating skin inflammation.

[0054] To examine whether an F230 extract prepared as described in Example 1 is able to inhibit IL-1β and IL-8 expression, a co-culture system of keratinocytes and human sensory neurons was used. Human sensory neurons were derived from human induced pluripotent stem cells (hiPS cells). hiPS cells were obtained by transfecting human fibroblasts which were reprogrammed into neuronal cells. hiPS cells were plated in 6 wells plates coated by a thin layer of Matrigel® (Corning GmbH, Kaiserslautern, Germany) at a density of 250,000 cells in a medium composed by DMEM-F12 (Pan-Biotech, Aidenbach, Germany) supplemented with 10% Knockout Serum Replacement (Life technologies, Carlsbad, USA), 0.1 μM retinoic acid (Sigma Aldrich, Taufkirchen, Germany), 1% P/S (Pan-Biotech) and a cocktail of inhibitors. The culture was maintained for 6 days at 37° C. and 5% of CO.sub.2.

[0055] After 6 days of differentiation, hiPS cells were plated at a density of 20,000 cells in the differentiation medium using 96-well plates that had been coated with a thin layer of Matrigel®. After 9 days, the culture medium was replaced by a maturation medium, i.e. a DMEM-F12 supplemented with 1% N2 (Life technologies, Carlsbad, USA), 10 ng/mL Neurotrophin-3 (NT-3), 10 ng/mL glial cell-derived neurotrophic factor (GDNF, Pan-Biotech), 10 ng/mL of brain-derived neurotrophic factor (BDNF, PanBiotech) and 10 ng/mL Nerve Growth Factor (NGF, Sigma). Commercial human keratinocytes were thawed and propagated in 75 cm.sup.2 culture flasks in keratinocyte growth medium. At the end of propagation, the cells were dissociated and frozen in liquid nitrogen. At day 14 of the neuronal culture, keratinocytes were thawed again and plated in 96-well plates on top of the neurons at the density of 30,000 cells per well in a medium composed by ⅔ of maturation medium and ⅓ of keratinocyte growth medium. The medium was changed every 2 or 3 days. On day 18, the medium was removed and fresh medium was added, either (i) control medium, or (ii) medium containing capsaicine, or (iii) medium containing capsaicine and capsazepine, or (iv) medium containing capsaicine and F230 in 3 different concentrations. After 1 hour of capsaicin stimulation, supernatants were removed and assayed for cytokine release. Samples were evaluated by cytometry flux (BD Bioscience) and compared to non-treated controls.

[0056] Results: The results of measuring IL-1β secretion are depicted in FIG. 3. Incubation with capsaicine strongly induced IL-1β expression. To the contrary, a treatment of the co-cultured cells with capsazepine before or during cell activation with capsaicin completely abrogates IL-1β release. Similarly, increasing amounts of F230 resulted in increasing levels of inhibition of IL-1β expression. The results of measuring IL-8 secretion are depicted in FIG. 4. Incubation with capsaicine strongly induced IL-8 expression, while treatment of the co-culture with capsazepine before or during cell activation with capsaicin significantly reduced IL-8 release. Similarly, increasing amounts of F230 resulted in increasing levels of inhibition of IL-8 expression.

Example 5: Inhibition of CGRP

[0057] The calcitonin gene-related peptide (CGRP) is a member of the calcitonin family of peptides that acts as a neuropeptide. The release of CGRP is associated with neurogenic inflammation by blood vessel dilatation resulting in erythema and pain. CGRP production is induced by capsaicine. The above-described co-culturing approach with human sensory neurons and keratinocytes was used to measure the amount of CGRP released into the supernatant after 30 minutes of stimulation with 10 pM capsaicine. For CGRP detection, an ELISA (Antibodies-online) was used. The results obtained were compared to non-treated cells.

[0058] Results: The results are depicted in FIG. 5. As can be seen from the figure, F230 counteracts the capsaicine-induced CGRP expression. Increasing amounts of F230 result in higher levels of CGRP expression inhibition.

Example 6: Reduction of Skin Itching

[0059] For in-vivo studies a composition containing F230 and a placebo composition were prepared.

[0060] The composition with 3% F230 was prepared as follows:

[0061] A. Deionized water 237.875 g

[0062] B. Xanthan gum 2.50 g

[0063] C. Euxyl PE9010 2.00 g

[0064] D. F230 7.5

[0065] E. Citric acid (10%) 0.125 g

[0066] Components A and B are mixed and dispersed to homogeneity. Subsequently, components C-E are added in the given order. Finally, the pH is adjusted to 5.71.

[0067] The placebo composition was prepared as follows:

[0068] A. Deionized water 238.00 g

[0069] B. Xanthan gum 2.50 g

[0070] C. Euxyl PE9010 2.00 g

[0071] D. Deionized water 7.50 g

[0072] Components A and B are mixed and dispersed to homogeneity. Subsequently, components C-D are added in the given order. The pH of the composition was adjusted to 5.70.

[0073] To examine the effect of F230 on skin itching, the composition containing 3% F230 was applied to itching skin. A double blind study with 42 healthy volunteers was conducted (9 males and 33 females). 22 volunteers were assigned to F230 treatment, and 20 volunteers were assigned to placebo treatment. Some of the volunteers had a history of dry and itchy skin, including 11 subjects with atopic dermatitis, 8 subjects with type IV allergy, 1 subject with psoriasis, and 1 subject with diabetes type II. 12 volunteers had sensitive skin. 9 volunteers had normal skin. Itching was scored at the time points t=0, 1 min, 5 min and 24 hours. The baseline at t=0 was set to 0%. The frequency of itching was assessed by the test subjects on a 5-point scale before and after use of the test compositions.

[0074] Results: The results are depicted in FIG. 6. It can be seen that itching was significantly reduced by F230 compared to placebo already 1 minute after application.

Example 7: Reduction of Skin Redness

[0075] To examine the effect of F230 on skin redness, a formulation containing 3% F230 was applied to skin with abnormal redness. The volunteers used in Example 6 were used for the study. The formulation was applied twice daily. Redness was scored at t=0 and after 17 days of application. The baseline at t=0 was set to 0%. Before and after the test period, a trained grader evaluated any erythema that occurred at the skin of a subject due to scratching.

[0076] Results: The results are depicted in FIG. 7. It can be seen that the redness due to scratching was reduced by F230 compared to placebo after two weeks of application (Day 17) compared to baseline (Day 1).

Example 8: Reduction of Skin Sensitivity

[0077] To examine a neurosoothing effect of F230 on skin sensitivity, a formulation containing 3% F230 was applied to skin. The volunteers used in Example 6 were used for the study. The formulation was applied one time. The skin sensitivity was determined by measuring the current perception threshold (CPT) with a Neurometer® CPT/C device (Neurotron Inc., Baltimore, USA) at 250 HZ and 5 Hz at t=0 (i.e. immediately before application) and 40 min after application of test formulations or placebo. The baseline at t=0 was set to 0%.

[0078] Results: The results are depicted in FIG. 8. It can be seen that both at 250 Hz and at 5 Hz, the perception threshold is much higher after treatment of the skin with F230 compared to placebo.

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