TERPENE-CONTAINING COMPOSITION AND ITS COSMETIC USE

Abstract

The present invention relates to a composition comprising beta-caryophyllene, alpha-humulene, and linoleic acid. Moreover, the present invention refers to a composition comprising a blend of one or more oils from the genus Copaifera and one or more oils from the genus Passiflora. The present invention further relates to a cosmetic product comprising such composition. Furthermore, the present invention relates to a method for preparing such composition and to the use of the composition or the cosmetic product of the present invention for reducing inflammatory responses.

Claims

1. A composition comprising of: (A) 45 to 80% by weight of beta-caryophyllene as component A; (B) 8 to 15% by weight of alpha-humulene as component B; (C) 5.5 to 8% by weight of linoleic acid as component C; and optionally (D) 0 to 41.5% by weight of one or more other components which are different from (A), (B) and (C) and which are miscible with (A), (B) and (C) as component D.

2. A composition comprising a blend of: (a) one or more oils from the genus Copaifera as component (a); and (b) one or more oils from the genus Passiflora as component (b).

3. The composition of claim 2, wherein said composition consists of a blend of: (a) one or more oils from the genus Copaifera as component (a); and (b) one or more oils from the genus Passiflora as component (b).

4. The composition of claim 1, wherein said composition comprises or consists of a blend of (a) one or more oils from the genus Copaifera as component (a); and (b) one or more oils from the genus Passiflora as component (b).

5. The composition of claim 2, wherein the weight ratio of components (a):(b) is in the range of from 80:20 to 98:2.

6. The composition of claim 2, wherein the component (a) is oil from Copaifera officinalis.

7. The composition of claim 2, wherein the component (b) is oil from Passiflora edulis.

8. The composition of claim 2, wherein the component (a) is oil from Copaifera officinalis and component (b) is oil from Passiflora edulis.

9. The composition of claim 2, wherein at least one of the components (a) and (b) is a seed oil, a resin oil or a combination thereof, or wherein both components (a) and (b) are each a seed oil, a resin oil or a combination thereof.

10. A cosmetic product comprising or consisting of: (I) 0.1 to 100% by weight of a composition of any of claims 1 to 9; and optionally (II) 0 to 99.9% by weight of one or more cosmetically acceptable carriers, filers, or combinations of carriers and fillers; and optionally (III) 0 to 50% by weight of one or more further cosmetically acceptable additives, in particular selected from the group consisting of fragrances, dyes, pigments, emulsifiers, lubricants, chelating agents, acidity regulators, antimicrobial agents, preservatives, antioxidants, and combinations of two or more thereof.

11. The cosmetic product of claim 10, which is a cosmetic product for external topical use on the skin.

12. The cosmetic product of claim 10, wherein said cosmetic product is selected from the group consisting of emulsions, gels, ointments, tonics, liquid soaps, bar soaps, bath oils, shower oils, massage oils, makeups, scalp treatments, aftershaves, shaving products, deodorants, shower gel, shampoos, and combinations of two or more thereof.

13. A method for preparing a composition of, comprising the steps of: (i) providing plant parts of the genus Copaifera and plant parts of the genus Passiflora; (ii) distilling the plant parts to obtain one or more oils for each genus; (iii) mixing the one or more oils from the genus Copaifera the one or more oils from the genus Passiflora and optionally one or more further components which are miscible with said oils.

14. The method of claim 13, wherein distilling comprises steam distilling conducted at a temperature range of between 100 and 300° C.

15. The method of claim 13, wherein said method further contains a step of mixing one or more oils with one or more organic solvents in which the one or more oils are solved and separating the one or more organic solvents from the one or more oils.

16. Use of the composition of claim 2 for reducing inflammatory responses.

17. The use of claim 16, wherein inflammatory responses are inflammatory responses of the skin.

18. The use of claim 16, wherein the level of inflammatory cytokines, in particular decreasing levels of interleukin 1 beta (IL-1β), interleukin 6 (IL-6), or both is reduced.

19. The use of claim 16, wherein the level of interleukin 1 beta (IL-1β), interleukin 6 (IL-6), or both is reduced.

20. The use of claim 16, wherein the level of beta-endorphin is increased.

21. The composition of any of claim 2 for use in a method for reducing inflammatory responses.

22. The composition or cosmetic product for use of claim 21, wherein inflammatory responses are inflammatory responses of the skin.

23. The composition or cosmetic product for use of claim 21, wherein the level of inflammatory cytokines, in particular decreasing levels of interleukin 1 beta (IL-1β), interleukin 6 (IL-6), or both is reduced.

24. The composition or cosmetic product for use of claim 21, wherein the level of interleukin 1 beta (IL-1β), interleukin 6 (IL-6), or both is reduced.

25. The composition or cosmetic product for use of claim 21, wherein the level of beta-endorphin is increased.

Description

EXAMPLES

Example 1—Preparation and Technical Effects of a Composition of the Present Invention Material and Methods

Part of the Plant Use for the Generation of Oil

[0119] Copaiba officinalis oil was obtained from the resin of Copaiba officinalis. In the present example, the resin was an exudate obtained by means of trunk tree excision and the collection of the amorphous mass formed as resin. This can also be considered as resin oil

[0120] The part of the plant used for obtaining Passiflora edulis oil is based on the seeds of the Passion fruit (Passiflora edulis). This can also be considered as seed oil.

Obtaining Oils from the Plant Part

[0121] The procedure for obtaining standardized vegetable oils experimentally used herein comprises several steps including as extraction (by physical process), purification and standardization in order to obtain reproducible and quality oils batch by batch.

[0122] The extraction of Copaifera officinalis resin oil was obtained based by means of steam distillation. The extraction was performed by means of direct and constant heating (increasing temperature from 140 to 250° C.) of the Copaifera officinalis resin to its boiling point, forming steam under a controlled pressure of approximately 700 to 760 mmHg (933.26 to 1013.25 hPa)). The volatile compounds were diffused to the steam and they were dragged in an upward vertical direction in the distillation column. This cycle was repeated several times and in top of the column, the more volatile compounds were condensed. On the base of column, the compounds which bear higher boiling points (i.e., less volatile compounds) were collected. Finally, these compounds were condensed and collected in a vessel, obtaining a composition of volatile and fixed compounds in a liquid form. This provided the Copaifera officinalis resin oil. The process was finished after homogenization by means of adding tocopherol to the oil in order to keep the oxidative stability of the product and finally, the oil was bottled under nitrogen.

[0123] Regarding passion fruit oil, the extraction process was performed by means of cold press of the seeds. Alternatively, it can be performed by batch or continuous (expeller) extraction. The seeds were collected, selected and dried (in the sun or in vacuum oven). The drying was perpetuated until an appropriate residual mass (approximately 10%) was maintained in order to keep the quality of seeds and the oil to be pressed accordingly. A hydraulic press was used at room temperature to obtain the Passiflora edulis seed oil by applying mechanical pressure on the seeds on an endless shaft that grind the seeds. The (fixed) oil (product of the extraction) was released. Subsequently, the oil was subjected to the process of purification (“oil bleaching”). This was performed by means of adsorbent compounds, herein used: diatomaceous earth. The process comprised decreasing phospholipids and colorant materials (as example chlorophyll) that degrade the oil due to the oxidation process with a consequent darkening and loss of quality. After diatomaceous earth adding (use of heat and vacuum in order to a better incorporation) to the oil, the generated product was filtered by mechanical press with the using of filtering elements (generally paper) with pore size (grammage) of about 100 to 150 μm. The purification process was performed by a deodorization of the oil at a temperature in the range of 140 to 150° C.) at a pressure of 700 to 760 mmHg (933.26 to 1013.25 hPa). Volatile compounds (aldehydes, ketones and free fatty acids) were vaporized and thereby removed from the oil. Finally, due to the loss of antioxidant elements of the oil during the process, tocopherol was added to keep the oxidative stability and finally the oil was bottled under nitrogen.

[0124] The purified Copaifera officinalis resin oil (a) obtained from the above process and the Passiflora edulis seed oil (b) were mixed in a weight ratio of components (a):(b) of 90:10. This provided the composition of the present invention which is designated as “Blend of Oils” in the below experiments.

[0125] It was found that this composition of the present invention which is designated as “Blend of Oils” contained mostly beta-caryophyllene (50 to 70% by weight) and further 8 to 15% by weight of alpha-humulene, 5.5 to 8% by weight of linoleic acid and less than 36.5% of further ingredients such as mainly other sesquiterpenes, other terpenes, fatty acids and tocopherol.

Experimental Procedure Assays Based on Human Cell Culture

[0126] In an in vitro (also: ex vivo) model system to simulate in effects in vivo, i.e., a human skin culture, the effects of the composition of the present invention on non-irritated and irritated skin were determined. For this purpose, the established pro-inflammatory markers IL-1β and IL-6 as well as the marker for a pleasant sensation beta-endorphin were detected

Experimental Design

[0127] The skin culture was treated with the composition of the present invention (Blend of Oils) and stimulated with lipoteichoic acid (LTA) for inflammatory microenvironment induction. The protein synthesis of interleukins IL-1β and IL-6 and synthesis of beta-endorphin were measured by standard enzyme-linked immunosorbent assay (ELISA).

Material

[0128] Human skin culture was obtained from skin fragments derived from an abdominoplasty from a healthy woman (58 years old). After surgery the skin fragments were collected in plastic bags containing an isotonic 0.9% saline solution and kept on freeze for up to 24 hours. The use of human skin fragments from elective surgeries for the study was performed in agreement with the Ethics Committee in Research.

[0129] Skin Fragments Protocol

[0130] Skin fragments were fractioned into pieces of about 1.5 cm.sup.2 and incubated in culture medium. Then, the fragments were treated once a day during three consecutive days with the composition of the present invention (Blend of Oils) at 25±2 μL/cm.sup.2 and rub for 30 seconds. Immediately after the third day treatment, the skin culture was concomitantly stimulated with 100 μM lipoteichoic acid (LTA) for the inflammatory microenvironment induction. The treated skin culture was kept in an incubator at 37° C. in the presence of 5% (v/v) of CO.sub.2 for 72 hours. After this incubation, the fragments were collected for determining the levels of the markers. Tissue homogenate was prepared

[0131] Quantification of IL-1β, IL-6 and β-endorphin in the tissue homogenate were performed by means of a standard enzyme-linked immunosorbent assay (ELISA), using a kit from R&D systems (Phoenix Pharmaceuticals, Inc., USA). The absorbance measurement was evaluated at 450 nm in Monochromator Multiskan GO (Thermo Scientific). Values were normalized by the total proteins of the sample, measured by a routine Bradford assay.

[0132] Statistical analysis was performed by means of an ANOVA test, with measurement of variance of results and comparison of results between groups. Subsequently, a post-test according to Bonferroni was performed to confirm results presented by ANOVA test. Level of significance used: 5% (Graph Pad Prism v.6).

Results

[0133] The effect of the composition of the present invention (Blend of Oils) on the reduction of production of pro-inflammatory markers (exemplified as IL-1β and IL-6) as well as the recovery and increase of production of beta-endorphin in human skin culture (in vitro) was investigated. In particular such effects were investigated when the human skin culture is stimulated with a skin irritating stimulus (exemplified as lipoteichoic acid (LTA)). The results are depicted in Tables 1 to 3 below.

TABLE-US-00001 TABLE 1 Effect of the composition of the present invention (blend of oils) on the reduction of production of IL-1β in human skin culture stimulated with lipoteichoic acid (LTA). Herein, the IL-1β (pg/mg of protein) is depicted. Blend of Reduction of Stimulated Blend of Oils + IL-1β by cells Control with LTA Oils LTA stimulated with LTA 9.199 32.525 6.052 14.971 54.0 8.759 38.033 6.595 15.011 60.5 7.501 35.934 9.024 17.494 51.3 Average 8.486 35.497 7.223 15.825 55.4 (pg/mg) Deviation 0.881 2.78 1.582 1.445

[0134] It was found that the composition of the present invention (blend of oils) slightly decreased IL-1β in human skin culture which was not stimulated with an irritating stimulus. In case the skin culture is stimulated with the irritating stimulus lipoteichoic acid (LTA), IL-1β production increased by 4.18-fold (p<0.001). The treatment with the composition of the present invention was surprisingly capable to avoid the exacerbated production of TIL-1β (p<0.001), decreasing its production in 55.42% when compared to the group stimulated with LTA. These values represent a protection rate when compared to the control group of 72.830.

TABLE-US-00002 TABLE 2 Effect of the composition of the present invention (blend of oils) on the reduction of production of IL-6 in human skin culture stimulated with lipoteichoic acid (LTA). Herein, the IL-6 (pg/mg of protein) is depicted. Blend of Reduction of Blend of Oils + IL-6 by cells Control LTA Oils LTA stimulated with LTA 169.568 246.178 156.861 205.365 16.6 184.419 309.501 163.872 200.212 35.3 173.12 274.234 185.845 214.941 21.6 Average 175.702 276.638 168.86 206.84 25.2 (pg/mg) Deviation 7.755 31.73 15.122 7.474 —

[0135] It was found that the composition of the present invention (Blend of Oils) slightly decreased IL-6 in human skin culture which was not stimulated with an irritating stimulus. In case the skin culture is stimulated with the irritating stimulus lipoteichoic acid (LTA), IL-6 production increased by 1.57-fold (p<0.001). The treatment with the composition of the present invention was surprisingly capable to avoid the exacerbated production of IL-6 (p<0.001) decreasing its production in 25.23% when compared to the group stimulated with LTA. These values represent a protection rate when compared to the control group of 69.15%.

TABLE-US-00003 TABLE 3 Effect of the composition of the present invention (Blend of Oils) on the recovery and increase of production of beta-endorphin in human skin culture stimulated with lipoteichoic acid (LTA). Herein, the beta-endorphin (pg/mg of protein) is depicted. B-endorphin (pg/mg of protein) Blend Blend of Increase of of Oils + beta-endorphin by cells Control LTA Oils LTA stimulated with LTA 15.273 8.683 28.068 16.671 92.0 14.493 7.341 22.955 19.456 165.0 14.435 10.912 25.876 15.833 45.1 Average 14.734 8.979 25.633 17.32 92.9 (pg/mg) Deviation 0.468 1.804 2.565 1.896

[0136] It was found that the composition of the present invention (blend of oils) increased beta-endorphin to 173.98% in human skin culture which was not stimulated with an irritating stimulus. In case the skin culture is stimulated with LTA, beta-endorphin production decreased by 39.06% (p<0.001). The treatment with the composition of the present invention was surprisingly capable to Increase its production by 92.90% when compared to the group stimulated with LTA. These values represent a protection of rate about 100% of the control group and an even higher stimulation, which is desirable due to a particularly good skin sensation.

[0137] The composition of the present invention (blend of oils) was surprisingly found to be able to act on the skin inflammatory response control, with a significant 55.4% and 25.2% reduction in the inflammatory interleukins IL-1β and IL-6 respectively, besides a 92.9% significant increase of beta-endorphin stimulus.

[0138] In summary, it was found that the composition of the present invention is surprisingly beneficial for decreasing inflammation (exemplified by the pro-inflammatory markets IL-1β and IL-6) and surprisingly beneficial for stimulating desirable beta-endorphin release. Thus, these results indicate an anti-inflammatory activity that favors both skin healing and re-epithelization. The skin sensation (also: skin feeling, skin feel) on the composition is particularly beneficial as well. Thus, the composition of the present invention and a product comprising such can promote an anti-inflammatory activity that may support the healing and re-epithelization of the skin and may promote cutaneous wellbeing due to recovery and/or maintain neuroimmunology homeostasis.

Example 2—Complementary Tests on Effects of a Composition of the Present Invention in Comparison to Other Cosmetic Agents

[0139] The composition of the present invention (“Blend of Oils”) is prepared as described in Example 1 above.

[0140] In an in vitro (also: ex vivo) model system to simulate in effects in vivo, i.e., a human skin culture, the effects of the composition at different concentrations of the present invention on non-irritated and irritated skin are determined when in comparison with other compounds, as CBD (cannabidiol, at different concentrations) and alpha-bisabolol (at different concentrations). For this purpose, the established pro-inflammatory markers IL-1β and IL-6 as well as the marker for a pleasant sensation beta-endorphin are evaluated together with the present invention, and the positive control, the solvent control, and the untreated control.

Experimental Design

[0141] The skin culture is treated with the composition of the present invention (Blend of Oils in different concentrations) and also with other compounds known as anti-inflammatory activity agent such as alpha-bisabolol (at different concentrations) and CBD (cannabidiol, at different concentrations). When in contact with lipoteichoic acid (LTA), an inflammatory microenvironment induction occurs. The protein synthesis of interleukins IL-1β and IL-6 and synthesis of beta-endorphin are measured by standard enzyme-linked immunosorbent assay (ELISA) and values are compared.

Material:

[0142] Human skin culture is obtained from skin fragments derived from an abdominoplasty from a healthy person. After surgery the skin fragments are collected in plastic bags containing an isotonic 0.9% saline solution and kept on freeze for up to 24 hours. The use of human skin fragments from elective surgeries for the study is performed in agreement with the Ethics Committee in Research.

Skin Fragments Protocol

[0143] Skin fragments are fractioned into pieces of about 1.5 cm.sup.2 and incubated in culture medium. Then, the fragments are treated once a day during three consecutive days with the composition of the present invention (Blend of Oils), as well other fragments in contact with alpha-bisabolol (at 5%) and CBD (at 3 and 5%) at 25±2 μL/cm.sup.2 and rub for 30 seconds. Immediately after the third day treatment, each skin culture concomitantly is stimulated with 100 μM lipoteichoic acid (LTA) for the inflammatory microenvironment induction. Each treated skin culture is kept in an incubator at 37° C. in the presence of 5% (v/v) of CO.sub.2 for 72 hours. After this incubation, the fragments are collected for determining the levels of the markers. Tissue homogenate is prepared with each composition.

[0144] Quantification of IL-1β, IL-6 and β-endorphin in each tissue homogenate are performed by means of a standard enzyme-linked immunosorbent assay (ELISA).

[0145] The first experiments indicate that the composition of the present invention (Blend of Oils) leads to technical effects which are widely comparable with alpha-bisabolol and CBD (cannabidiol). This particularly refers to an anti-inflammatory action as can be indicated by the decrease of the anti-inflammatory markers IL-1β and IL-6.

[0146] Furthermore, there are indications that a constant production of beta-endorphin is stimulated by the composition of the present invention (Blend of Oils) even at lower concentrations. This supports the finding that the composition of the present invention is beneficial for wellness formulations. It can be beneficial for skin recovery as well the use for sensitive skin.

[0147] Based on the results, it is possible to observe also the beneficial effect to have a composition including specific fatty acids from Passiflora edulis, to keep the constant absorption of the components, favoring the skin healing.

[0148] In summary, also these findings indicate that the composition of the present invention is beneficial for decreasing inflammation (exemplified by the pro-inflammatory markets IL-1β and IL-6) and for stimulating desirable beta-endorphin release. The composition of the present invention is particularly beneficial for a cosmetic product having anti-inflammatory activity and promoting cutaneous wellbeing. Compounds such as alpha-bisabolol and CBD (cannabidiol) can be avoided and replaced by the composition of the present invention. This allows the reduction of undesired side effects while maintaining the desired beneficial effects