COMPOSITION CONTAINING NERO DI TROIA POMACE EXTRACT

Abstract

It is herein described a gel composition for topical use comprising active ingredients for the prevention of oxidative stress damage due to sun exposure and for the prevention of skin tumours. The invention relates in particular to a composition containing a glyceric extract of Vitis vinifera obtained from the pomace of the Nero di Troia vine and xanthan gum, optionally with the addition of sunscreens, wherein, optionally, the glyceric extract is obtained by ultrasound extraction.

Claims

1. A gel composition for topical use comprising xantham gum, a glyceric extract of the pomace Vitis vinifera from Nero di Troia vine and pharmaceutically acceptable excipients.

2. The composition according to claim 1, wherein the glyceric extract is present in amounts from 10% to 60% by weight.

3. The composition according to claim 1, wherein the xantham gum is present in an amount ranging from 0.5% to 4% by.

4. The composition according to claim 1, comprising UVA, UVB sunscreens.

5. The composition according to claim 4, wherein the sunscreens are present in sufficient quantity to achieve a SPF50 protection.

6. Method of preventing skin diseases in a subject in need thereof with the composition according to claim 1, said method comprising applying to said subject a pharmaceutically effective amount of said composition and preventing said skin diseases.

7. The method according to claim 6, wherein the skin disease is a form of skin cancer.

8. A method for the prevention damage from oxidative stress due to sun exposure of an individual in need thereof with the composition according to claim 3, said method comprising: applying a pharmaceutical effective amount of the composition according to claim 3 to said individual.

9. A glyceric extract of pomace of Vitis vinifera from Nero di Troia vine obtainable by ultrasonic extraction at room temperature for 4-10 minutes from a mixture of 50-80% by weight of glycerin and 20-50% of water.

10. A process for preparing a glyceric extract of the pomace of Vitis vinifera from Nero di Troia vine by ultrasonic extraction at room temperature for 4-10 minutes from a mixture of 50-80% by weight of glycerin and 20-50% by weight of water.

11. (canceled)

12. A process for the preparation of a composition according to claim 1, comprising the mixture of xanthan gum, a glyceric extract of Vitis vinifera pomace from Nero di Troia vine and pharmaceutically acceptable excipients.

13. The composition according to claim 1, wherein the glyceric extract is present in amounts from 20% to 50% by weight.

14. The composition according to claim 1, wherein the xantham gum is present in an amount ranging 1% to 2% by weight.

15. The method according to claim 7, wherein the skin disease is melanoma.

Description

DESCRIPTION OF THE DRAWINGS

[0029] FIG. 1FIG. 1 reports the 3D pie charts representing the content of flavonoids and terpenoids of glyceric extracts obtained as described in Example 1 from pomaces of Nero di Troia (NdT) (1a) and Primitivo (1b).

[0030] FIG. 2FIG. 2 reports the 3D pie charts representing the content of flavonoids and terpenoids of glyceric extracts obtained as described in Example 1 from pomaces of Aglianico (2a) and Falanghina (2b).

[0031] FIG. 3FIG. 3 reports the ESI (Electrospray ionization) mass spectrum (positive ion scan) of the NdT pomace extract highlighting highest m/z signals (with a cutoff at 10% abundance).

[0032] FIG. 4FIG. 4 reports the ESI mass spectrum (positive ion scan) of the Primitivo pomace extract highlighting highest m/z signals (with a cutoff at 10% abundance).

[0033] FIG. 5FIG. 5 reports the ESI mass spectrum (positive ion scan) of the Aglianico pomace extract highlighting highest m/z signals (with a cutoff at 10% abundance).

[0034] FIG. 6FIG. 6 reports the ESI mass spectrum (positive ion scan) of the Falanghina pomace extract highlighting highest m/z signals (with a cutoff at 10% abundance).

EXAMPLES

Example 1Preparation of the Glyceric Extract of the Pomace of Vitis vinifera from the Nero Di Troia Vine

[0035] The glyceric extract of pomace of Vitis vinifera of the Nero di Troia vine was obtained by preparing a mixture containing 60% by weight of glycerin, 25% by weight of pomace and 15% water.

[0036] To this mixture, in order to obtain the extract, ultrasounds were subsequently applied at room temperature for 5 minutes. Subsequently the extract was filtered, obtaining a clear or slightly opalescent liquid with a dark purplish colour.

[0037] The following preservatives were then added to said extract: 0.60% by weight of benzyl alcohol, 0.18% by weight of sodium benzoate and 0.12% by weight of potassium sorbate.

[0038] The glyceric extract thus obtained (also named as GExtract NdT) was used for the assays reported in the following Examples 4-6.

Example 2Preparation of the Composition in Gel for Topical Use Containing the Glyceric Extract of Pomace of Vitis vinifera of the Nero Di Troia Vine

[0039] The gel for topical use is obtained by diluting the GExtract NdT obtained in Example 1 with water in different proportions depending on the concentration of the compositions to be prepared 1:1, 1:2 or 1:4.

[0040] Subsequently to the dilution, xanthan gum is added, little by little, between 1 gram and 2 grams for every 100 grams of liquid mixture prepared and is mixed until the desired density is obtained. Subsequently, the gel obtained is left to rest for 24-48 hours.

Example 3Prevention of Oxidative Stress Damage Due to Sun Exposure

[0041] The efficacy of the composition obtained in Example 2 alone or in comparison with other compositions containing other Vitis vinifera pomace extracts is evaluated by carrying out one or more of the following tests/assays.

TABLE-US-00001 Measured Assay Type property Note/Reference ABTS.sup.+ Chemical- Nonspecific Spectophotometric test based on the scavenging physical total antioxidant interaction between antioxidant species activity and radical cation ABTS.sup.+ generated from potassium persulfate (PP) and ABTS [2,20-azino-bis (3- ethylbenzothiazoline-6-sulfonic acid)]). Refs. a) Re, R. et al. Antioxidant activity applying an improved ABTS radical cation decolorization assay. Free Radic. Biol. Med. 1999, 26, 1231-1237; b) Int. J. Mol. Sci. 2020, 21, 1131. DPPH Chemical- Nonspecific Spectrophotometric test based on the scavenging physical total antioxidant interaction between antioxidant species activity and DPPH [2,2-diphenyl-1-(2,4,6- trinitrophenyl)hydrazyl]. Ref. Sagar B. Kedare et al. Genesis and development of DPPH method of antioxidant assay. J Food Sci. Technol. 2011 August; 48(4): 412-422. FRAP assay Chemical- Nonspecific Colorimetric test which measures the physical total antioxidant antioxidant potential by reducing Fe(III) activity to Fe(II). Ref. Benzie et al. The ferric reducing ability of plasma (FRAP) as a measure of antioxidant power: the FRAP assay. Anal. Biochem. 1996 Jul. 15; 239(1): 70-6. ORAC assay Chemical- Nonspecific Spectrophotometri test (oxygen radical physical total antioxidant absorbance capacity assay) based on the activity interaction between antioxidant and peroxide radical scavenger in the presence of a fluorescent indicator). Ref. Ou, B. et al. Development and validation of an improved oxygen radical absorbance capacity assay using fluorescein as the fluorescent probe. Journal of Agricultural and Food Chemistry, 49, 4619-4626 MTT assay In vitro Neuroprotective Spectrophotometric test associated with (cell) activity vs Hypo the cellular viability after exposure to E22 cell line oxidative stress from H.sub.2O.sub.2. (rat Ref. Chiavaroli A. et al. Phenolic hypothalamus) Characterization and Neuropro tective Properties of Grape Pomace Extracts. Molecules 2021, 26, 6216. CAA cellular In vitro Antioxidant Spectrophotometric activity that antioxidant (cell) activity in measures the capacity to prevent the assay cancer cell line formation of dichlorofluorescein from 2,2-Azobis(2-amidinopropane) dihydrochloride (ABAP) generated from radicals in human hepatocellular carcinoma cells (HepG2). Ref. Kelly L Wolfe et al. Cellular antioxidant activity (CAA) assay for assessing antioxidants, foods, and dietary supplements. J Agric Food Chem. 2007 Oct. 31; 55(22): 8896-907. Nitric oxide In vitro Antioxidant Colorimetric test which measures the assay (cell) activity in capacity to prevent the release of NO in cancer cell line rat macrophage cells (RAW 264.7) stimulated by lipopolysaccharides. Ref. Nathan S. Bryan et al. Methods to Detect Nitric Oxide and its Metabolites in Biological Samples. Free Radic Biol Med. 2007 Sep. 1; 43(5): 645-657. Screening In vitro Proliferative Cancer biomarkers reduction (annexin V, agents for (cell) activity vs human leukocyte antigen-DR; E- melanoma melanoma cadherin, N-cadherin) in melanocytes prevention cancer cell lines subjected to physical (UV) and chemical oxidative stress. Refs. a) E. Elmore et al. Development and characteristics of a human cell assay for screening agents for melanoma prevention. Melanoma Research 2007, 17: 42-50. b) G. K. Couto. The Melding of Drug Screening Platforms for Melanoma. Front Oncol. 2019; 9: 512.

Example 4Effect GExtract NdT on the Cellular Viability: In Vitro Study with the HaCaT Cells

[0042] The aim of this study was to evaluate in vitro the effect of GExtract NdT (obtained in Example 1) on the viability of the human keratinocyte cell line HaCaT. The evaluation was performed on cells exposed to GExtract NdT at a dilution of 2% in complete medium for 24 hours.

[0043] The cellular viability was measured by MTS colorimetric essay through the absorbance measurement obtained from the conversion of a bioreduced tertazolium compound from viable cells into a coloured formazan soluble in the culture medium.

Results

[0044] The HaCaT cells were exposed for 24 hours to 2% of GExtract NdT of and Triton-X 100 and subsequently the viability was evaluated by MTS assay. The results are reported in Table 1.

[0045] In the HaCaT cells exposed for 24 hours at increasing concentrations of Triton-X 100 (used as positive control) a marked negative effect on cell viability was recorded already at the lowest tested concentration. The surfactant lysed all the cells starting from the concentration of 0.01%. The treatment with GExtract NdT had no effect on the cell viability at the concentration of 2%.

TABLE-US-00002 TABLE 1 GExtract NdT and Triton-X 100 effect on the HaCat cells. The results are expressed as percentage normalized on the control group. Test Item % (v/v) % CTR % STD CTR 100 3.3 GExtract NdT 2 95.26 4.52 TRITON-X 100 0.001 84.54 0.001 67.19 0.005 52.098 0.01 0 0.05 0 0.5 0

Conclusions

[0046] In the study no bacterial contaminations were found in the treatment chamber, while a concentration-dependent reduction in the number of viable cells was recorded in cells treated with the surfactant Triton-X (used here as a cell lysing treatment). The study can therefore be considered valid.

[0047] No statistically significant effects were observed in the cells treated with GExtract NdT for 24 hours at 2% v/v concentration.

[0048] From the data obtained using the in vitro test described herein and under those experimental conditions, we can conclude that: GExtract NdT did not lead to a statistically significant viability reduction in HaCaT cells exposed for 24 hours to a concentration of 2% v/v.

Example 5Antioxidant Efficacy of GExtract NdT in an In Vitro Study with the HaCat Cells

[0049] The aim of this study was to evaluate in vitro the antioxidant effect of GExtract NdT (obtained in Example 1) on of the human keratinocyte cell line HaCaT. The evaluation was carried out on cells exposed to a 2% dilution of the formulation for 24 hours and subsequently treated with menadione (as an oxidizing stimulus).

[0050] The antioxidant activity was quantified as the reduction of Reactive Oxygen Species (ROS) induced by exposure to menadione alone, by dichlorofluorescein diacetate assay.

Results

[0051] Pre-treating the cells with GExtract NdT at 2% v/v concentration resulted to be protective against both the stimuli: in fact, a reduction of about 20% and of about 5%, respectively, was registered with regard to menadione at 50 l and 100 l. The results of the exposure to menadione after pre-treatment with GExtract NdT are reported in Table 2

TABLE-US-00003 TABLE 2 Antioxidant effect of GExtract NdT at the concentration of 2% v/v. The results are expressed as relative units of fluorescence. With GExtract NdT Without GExtract NdT Statistical Menadione Standard Standard % significance (M) Average Dev. Average Dev. Reduction (p value) 50 3131.00 50.91 2755.67 134.84 11.99 <0.001 100 3430.00 46.67 3240.20 51.29 5.53 <0.001

Conclusions

[0052] The study can be considered valid, because no bacterial contamination was found in the treatment chamber and an increased fluorescence, concentration-dependent, was recorded in the cells treated with menadione (here used as an oxidative treatment).

[0053] With regard to antioxidant efficacy, a positive effect was found in cells pre-treated with GExtract NdT. Said effect was marked in pre-treatment with GExtract NdT at 2% v/v, with a percentage reduction of oxidative stress of about 12 and 5%, respectively, against menadione at 50 to 100 M.

[0054] From the results obtained using the in vitro described herein and in those experimental conditions, we can conclude that: GExtract NdT led to a statistically significant reduction in levels of reactive oxygen species (ROS) after 24 hours from treatment with the non-cytotoxic concentration tested in menadione-stimulated human keratinocytes.

[0055] In the light of what emerged in our experimental conditions, the antioxidant activity of GExtract NdT is confirmed.

Example 6Comparative Study on the Flavonoids and Terpenoids Content of Four Grapevine Varieties Extracts

[0056] The glyceric extracts obtained from four different varieties of vines (Nero di Troia [NdT], Primitivo, Aglianico and Falanghina) were prepared using the same protocol as described in Example 1, and stored at 20 C. until the start of the analysis cycle. An exactly weighed amount of each extract was analysed through MS-ESI-QTOF spectroscopy to define the relative profiles of the most representative substances in these matrices, such as polyphenols, terpenes and the main metabolites.

[0057] The results are also reported in the following Tables A to D and in the 3D pie charts reported in FIGS. 1 and 2. In particular, the following tables report the name of some of the compounds tentatively identified by the exact monoisotopic mass formula (ChemCalc).

TABLE-US-00004 TABLE A Exp m/z Calc m/z Example of a compound of Pomace Formula [M + H].sup.+ [M + H].sup.+ natural origin (class) References NdT C.sub.20H.sub.31O.sub.3 320.237 320.235 20-hydroxyarachidonate PubChem CID 40490645 (fatty acid) C.sub.22H.sub.34O.sub.3 321.237 321.242 Isocupressic acid D. Tsimogiannis and V. o(Ginkgolic Acid (C13:0) Oreopoulou Polyphenols in (terpenoid acid) Plants. 2019 Elsevier C.sub.20H.sub.36O.sub.5 357.259 357.2641 Eunicellanetetrol Dictionary of Natural Products (tricyclic terpene) Supplement 1-1995 C.sub.22H.sub.32O.sub.4 361.241 361.2408 Macrophorin A T. Sassa et al. Agric. Biol. (terpene cyclohexenone Chem.,47(I), 187~189, 1983 epox) C.sub.19H.sub.36O.sub.7 377.2582 377.2539 Myrsinionoside D PubChemCID10384912 (cyclohexylbutan glycoside) C.sub.23H.sub.40O.sub.4 381.290 381.2998 Persin (fatty acid) PubChemCID5283266 C.sub.22H.sub.33O.sub.6 393.241 393.2277 Mangromicin E4 T. Nakashima et al. The Journal (cyclopentadecane skeleton of Antibiotics (2014) 67, 533- tetrahydrofuran unit) 539 C.sub.29H.sub.24O.sub.5 453.1676 453.1701 Chamuvaritin (flavanone) PubChem CID100418

TABLE-US-00005 TABLE B Exp m/z Calc m/z Example of a compound of Pomace Formula [M + H].sup.+ [M + H].sup.+ natural origin (class) References Primitivo C.sub.8H.sub.10O.sub.6 203.0542 203.0555 Succinyl-acetoacetate Metabolism Vitis vinifera (wine grape). KEGG- T01084: 100244395 C.sub.11H.sub.18O.sub.7 263.1120 263.1130 Succinyl-D-diginose K. Ishii et al. The Journal Of Antibiotics April 1983 C.sub.22H.sub.34O.sub.3 321.237 321.2429 Isocupressic acid D. Tsimogianniset al o(Ginkgolic Acid (C13:0) Polyphenols in Plants. (terpenoid acid) 2019 Elsevier C.sub.17H.sub.17O.sub.10 381.082 381.0801 5-O-b-D-glucopyranosyl-8 Boeira et al. Cincia Rural, hydroxypsoralen v.52, n.9, 2022 C.sub.23H.sub.40O.sub.4 381.3007 381.2998 Persin (fatty acid) PubChemCID5283266 C.sub.26H.sub.36O.sub.3 397.274 397.2742 Strongylophorine-24 M. Birringer et al. (meroterpenoids) RSC Adv., 2018, 8, 4803 C.sub.27H.sub.48O.sub.5 453.3723 452.3501 Chimerol or Bufol Dictionary of Marine (cholestane analogs) Natural Products C.sub.35H.sub.26O.sub.10 607.1600 607.1603 6-(2-hydroxy-3-methyl-3- Al-Shagdari et al. Natural butenyl)-amentoflavone Product Communications Vol. 8 (9) 2013

TABLE-US-00006 TABLE C Exp m/z Calc m/z Example of a compound of Pomace Formula [M].sup.+/[M + H].sup.+ [M].sup.+/[M + H].sup.+ natural origin (class) references Aglianico C.sub.17H.sub.34O.sub.5 318.222 318.2406 Aleuritic acid methyl ester G. Tedeschi et al. (fatty acid) ACS Sustainable Chem. Eng. 2018, 6, 11, 14955-14966 318.2042 Seprilose (carbohydrate) SEPRILOSE(ncats.io) C.sub.20H.sub.31O.sub.3 320.234 320.235 20-hydroxyarachidonate PubChem (fatty acid) CID40490645 C.sub.20H.sub.32O.sub.3 321.240 321.242 Isocupressic acid R. Nicoletti et al. o(Ginkgolic Acid (C13:0) Agriculture 2015, terpenoid acid) 5(4), 918-970; C.sub.22H.sub.40O.sub.6 401.288 401.2902 Chalmicrin T. Fex, (mannitol ether Phytochemistry, of monocyclofarnesol) Volume 21, Issue 2, 1982, 367-369 C.sub.29H.sub.44O.sub.7 505.3194 505.3165 Capitasterone Lei Fang et al. (ecdysteroids) Molecules 2017, 22, 1310; C.sub.33H.sub.52O.sub.9 593.3723 593.3689 Nuatigenin-3-beta-D-gluco Gunstone, Frank D., pyranoside or Agavoside A John L. Harwood, (saponine and Albert J. monosaccharides) Dijkstra (2007). The lipid CD-ROM. CRC Press

TABLE-US-00007 TABLE D Exp m/z Calc m/z Example of a compound Pomace Formula [M].sup.+/[M + H].sup.+ [M].sup.+/[M + H].sup.+ of natural origin (class) references Falanghina C.sub.20H.sub.31O.sub.3 320.238 320.235 20-hydroxyarachidonate Ting Zheng et al. Front. (fatty acid, triterpenoid) Nutr. 2021, 8: 715528 C.sub.17H.sub.17O.sub.10 381.082 381.0801 5-O-b-D-glucopyranosyl-8 Boeira et al. Ciencia hydroxypsoralen Rural, v.52, n.9, 2022 (furanocoumarins) C.sub.22H.sub.28O.sub.10 453.1737 453.1760 Floribundal Wang C, et al. (Iridoids, cyclopentane Molecules. 2020 Jan. pyran monoterpenes) 10; 25(2): 287. C.sub.38H.sub.48O.sub.2 537.3621 537.3732 C38-iso- L. Ngamwonglumlert, Norcanthaxanthin Encyclopedia of Food (keto-carotenoid) Chemistry, 2019 C.sub.37H.sub.66O.sub.9 655.4784 655.4785 Salzmanolin Emerson F. Queiroz et (Acetogenins, polyketide) al. J. Nat. Prod. 2003, 66, 755-758

[0058] The results are also reported in the FIGS. 3-6, as images of the MS scans in positive ion mode of the samples. In most cases it was possible to associate a representative structure to the most intense m/z signals (see legend in each mass spectrum) by the exact monoisotopic mass conversion in elemental formula (ChemCalc), as thoughtful entry in available databases.

[0059] A preliminary assessment allowed the inventors to detect significant differences among the signal patterns of the three extracts from red vines and the one from white vine (Falanghina).

[0060] The number and molecular diversity appear much more contained in the red samples with a prevalence of fatty acid derivatives compared to fermentation metabolites, as a clue of a greater stabilization of the red-derived pomaces.

Conclusions

[0061] For some time now, the literature has given a plethora of papers, with numerous citations, aimed at defining and comparing the composition of pomaces by different grape varieties. Nevertheless, only a few works are aimed at the determination of lipids with the exclusion of some contributions inherent the antioxidant properties or as useful nutrients in dietary regimens (Ivana Dimic' et al., Antioxidants 2020, 9, 568, Mariana Spinei et al., Foods 2021, 10 (4), 867 and Yolanda Carmona-Jimnez et al., Molecules 2022, 27 (20), 6980).

[0062] To the best of our knowledge, there are no studies that quantify substances such as eicosapentaenamide (EPM), 24-hydroxyarachidonate (24-HAC) and ginkgolic acid (GKA), yet present with dominant signals in the samples of red vines derived pomaces, especially in the Nero di Troia (NDT) sample

[0063] In particular, while EPM and 24-HAC, derivatives of polyunsaturated acids, and GKA, a 6-tridecylsalicylic acid, have aroused interest for their therapeutic potential (Sebasti Parets et al., Frontiers in Cell and Developmental Biology, 8, 2020 and Cinzia Giordano et al., Int J Mol Sci 2020 Mar. 26; 21 (7): 2279), their possible use as surfactant-cleansing and emulsifying agent in cosmetics was not known.

[0064] Now, in light of our findings, we can affirm that pomace extracts, and in particular that of NdT, can be used as new and promising natural sources of polyunsaturated acid derivatives to modulate rheological properties in cosmetic production in a virtuous example of circular green economy.