ES2352403A1 - Extracts of saxifraga spathularis, process of obtaining and its cosmetic, pharmaceutical and/or food use as antioxidants/antirradicalarios/uv filters (ultraviolet). (Machine-translation by Google Translate, not legally binding) - Google Patents

Abstract

Extracts of saxifraga spathularis, obtaining procedure and its cosmetic, pharmaceutical and/or food use as antioxidants/antiradical/uv (ultraviolet) filters. Extracts obtained from leaves, roots, stems and flowers dried and/or frozen, milled and sieved, extraction with water, and/or alcohols, evaporation of this solvent, subsequent extraction with water and/or glycols of the dried extract and filtration; and its cosmetic, pharmaceutical and/or food use, for the care of the skin and/or hair, for example as antioxidants or radical inhibitors, as anti-aging agents or sunscreens. (Machine-translation by Google Translate, not legally binding)

Description

Extracts of Saxifraga spathularis , procedure for obtaining and its cosmetic, pharmaceutical and / or food use as antioxidants / anti-radicals / UV filters (ultraviolet).

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Object of the invention

The present invention falls within the technical field of plant extracts of Saxifraga spathularis , and its preparation for cosmetic, pharmaceutical and / or food use as antioxidants, anti-radicals and UV filters.

Background of the invention

At the beginning of the 20th century Mulliken investigated the photoluminic reactivity demonstrating that during the process of peroxidation and photoexcitation formed an excited state of oxygen (singlet oxygen: 1 O 2). The development of these investigations led to the introduction of the term of reactive oxygen species (ERO) and oxygen free radicals (RLO) in the field of biology and to the demonstration that you are radical species were responsible for the toxic effects of oxygen

Therefore, since ERO / RLO are species which are generated continuously as utilization products O2 cell, in aerobic organisms have been developed biological strategies to deactivate them, such as the system Enzymatic superoxide dismutase, glutathione peroxidase or catalase

Often, these basic mechanisms of defense are not enough to eliminate all species endogenously generated radicals (cellular respiration and other physiological processes) or those of exogenous origin (radiation, environmental pollution, drugs, toxic) that affect the environment in the organism The loss of balance between the level of prooxidation and the antioxidant tone of an organ or tissue can produce cellular damage and, secondarily, condition the appearance of disease states. Oxidative stress is defined as the cell damage situation that results when the generation and / or exogenous incidence of RLO exceeds the capacity of different physiological mechanisms of the organism to prevent or intercept its accumulation.

In addition to enzymatic defense mechanisms throughout biological evolution, organisms have developed the ability to synthesize molecules with antioxidant activity: ubiquinone, glutathione, ceruloplasmin, transferin and uric acid in mammals; and compounds such as ascorbic acid (vitamin C),? -tocopherol (vitamin E),? -carotene (pro-vitamin A), various flavonoids and other phenolic compounds in the case of vegetables. Thus, the relationship between the phenolic content of plant extracts and their antioxidant activity is well known [ YS Velioglu et al., Journal of Agricultural and Food Chemistry ; 1998 , 46 (10), 4113-4117 ].

Natural antioxidants in addition to protecting to the organism from damage caused by free radicals, responsible for degenerative diseases such as cancer, arteriosclerosis, arthritis and neurodegenerative processes and of aging; they may have antibactericidal activity, antiviral, antimutagenic, antiulcer or anticarlogenic.

In addition, if natural antioxidants absorb ultraviolet radiation can be used as sunscreens [EPO781544B1]. UV (ultraviolet) radiation can be classified in UV-C (wavelength less than 280 nm); UV-B (wavelength between 280 nm and 320 nm) and UV-A (wavelength between 320 nm and 400 nm). From these ultraviolet radiation, the most lethal is radiation UV-C, although most of it is absorbed by ozone Therefore, those that may have the greatest influence on the skin are UV-A radiation and UV-B

Saxifraga spathularis ( Plumbaginaceae ) is an endemic plant in the northwest of the Iberian Peninsula (Spain and Portugal) and Ireland. Its internal use as an anti-inflammatory and analgesic for veterinary treatments has been reported (E. Blanco et al ., El Caurel, plants and their inhabitants. Ethnobotanical study of the Sierra del Caurel (Lugo): the importance of plants for our ancestors; Fundación Caixa Galicia, A Coruña, 1996 , 203) and its topical use for the treatment of boils (MP González-Hernández et al ., A proceedings of the XXVI International Horticultural Congress, Toronto, 2002 , 63-75).

Description of the invention

The present invention describes extracts of the leaves, flowers, roots and stems of Saxifraga spathularis its method of obtaining and its cosmetic, pharmaceutical and / or food use as antioxidants / anti-radical / UV filters (ultraviolet).

It describes, among other things, a new procedure for obtaining extracts of Saxifraga spathularis from leaves, flowers, roots and stems, which allows the use of harmless solvents, such as water, glycols and other alcohols, of low cost and operation safe to obtain a stable product, easy to handle, to add to different products and that can be used as an ingredient of the cosmetic industry. It also describes said extracts of Saxifraga spathularis obtained by the described procedure, characterized by presenting, among others, high phenolic content and relevant antioxidant, anti-radical and UV protection properties, which makes them suitable for skin care and / or hair, for example as antioxidants or radical inhibitors, as agents to combat aging or sunscreens. Extracts frequently have higher antioxidant activity than synthetic antioxidants such as BHT (butyrohydroxytoluene) or BHA (butyrohydroxyanisole).

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The practice of the present invention it implies:

Obtaining procedure

i) Preparation of the leaves, flowers, roots and stems of Saxifraga spathularis :

The dried or frozen leaves, flowers, roots and stems of ground and sifted Saxifraga spathularis are used . They are subjected to a grind, preferably in a blade mill, and screened, preferably at a particle size of 0.6 mm. approximately.

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ii) Extraction of leaves, flowers, roots and stems of Saxifraga spathularis with solvents :

Dried or frozen leaves ground and sifted undergo a solid-liquid extraction process in continuous with solvents.

Liquid: high solid ratios are used, preferably 30 g / g.

Water and / or solvent are used as solvent to extract hydroalcoholic and / or alcoholic solvents. Is chosen advantageously C 1 to C 4 alcohols and, preferably, ethanol or methanol Among them, a mixture is advantageously chosen 1: 1 ethanol / water.

The temperature of the extraction is that of reflux of the solvent or mixture of extraction solvents and is in the range of 60-120 ° C and preferably between 85-95 ° C.

The extraction time is in the range of 1 to 3 hours

The extractive process is carried out protected from light to avoid possible alterations.

The performance of the extractive process oscillates between 25% and 30% (g / g).

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iii) Obtaining dry extracts or aqueous extracts of Saxifraga spathularis :

The aqueous, alcoholic or hydroalcoholic extracts obtained are subjected to vacuum evaporation to remove the solvent and obtain the dried extracts of Saxifraga spathularis .

Water is removed by evaporation under vacuum, preferably by lyophilization.

If, in order to obtain the liquid extracts of stage iv), water or hydroglycols are used as extraction solvents, in some cases only the alcoholic solvent of stage ii) can be removed under vacuum and the water cannot be removed, obtaining aqueous extracts from Saxifraga spathularis .

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iv) Obtaining the liquid extracts of Saxifraga spathularis :

Dry extracts or aqueous extracts of Saxifraga spathularis obtained in step iii), are subjected to a new solid-liquid extractive process in continuous with solvents, preferably by maceration with stirring.

Liquid: high solid ratios are used, preferably 99 g / g.

Water and / or solvent is used as solvent to extract hydroglycolic and / or glycolic solutions. Is chosen advantageously C2 to C6 glycols. Among these glycols are prefers to use propylene glycol and butylene glycol. Among them, it advantageously choose a 1: 1 butylene glycol / water mixture.

The extraction temperature is the temperature ambient, approximately 20 ° C.

The extraction time is 1 to 7 days.

The extractive process is carried out protected from light to avoid possible alterations.

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v) Obtaining the filtered liquid extracts of Saxifraga spathularis :

The solids of the liquid extracts obtained in step iv) are separated by filtration, preferably by porous plate No. 3 or 4, or weight filter paper 73 g / m2, obtaining the filtered liquid extracts from Saxifraga spathularis

These extracts are stored refrigerated, preferably at 4 ° C, and protected from light to avoid possible alterations

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Characterization of filtered liquid extracts of Saxifraga spathularis

i) Determination of UV (ultraviolet) absorption of filtered liquid extracts of Saxifraga spathularis :

UV (ultraviolet) radiation is classified as UV-C (wavelength less than 280 nm); UV-B (wavelength between 280 nm and 320 nm) and UV-A (wavelength between 320 nm and 400 nm), by this, if a product absorbs radiation between 250 to 400 nm can be Used as sunscreen.

The absorbance (Abs) of the filtered liquid extracts of Saxifraga spathularis obtained according to the extraction procedure is measured, at the concentration of 100 ppm in distilled water between the wavelengths of 250 to 400 nm against a blank of distilled water. All have maximum absorption in the UV (ultraviolet) region.

Saxifraga spathularis extracts at the wavelength of 280 nm have Abs>0.700; at 320 nm they present Abs> 0.250 and at 400 nm they present Abs> 0.050.

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ii) Determination of the phenolic content of the filtered liquid extracts of Saxifraga spathularis :

Phenolic compounds have a high antioxidant activity [ YS Velioglu, op. cited ( 1998 )]. Therefore, the phenolic content of the liquid extracts of Saxifraga spathularis is determined according to the method of Folin Ciocalteu's [ A. Escarpa et al., Analytica Chimica Acta ; 2001 , 427 (1), 119-127 ]. The absorbance at 765 nm of a solution of 0.5 mL of the antioxidant extract, 3.75 mL of distilled water, 0.25 mL of Folin Ciocalteau's reagent (1: 1 with distilled water) and 0.5 mL of 10% Na 2 CO 3; after 1 hour at room temperature, against a white without extract. The phenolic content of the extract is determined by comparing the absorbance with a straight gallic acid standard (0-100 ppm).

The filtered liquid extracts of Saxifraga spathularis obtained according to the extraction procedure, have, according to the described procedure, a high phenolic content, between 20-35%.

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iii) Determination of the capture activity of the α, α-diphenyl-β-picrylhydracil radical (DPPH) and its equivalence in BHT and BHA of the filtered liquid extracts of Saxifraga spathularis :

To measure the capture activity of the α, α-diphenyl-β-picrylhydracil (DPPH) radical [ I. Parejo et al., Journal of Agricultural and Food Chemistry ; 2002 , 50, 6882-6890 ], the decrease in absorbance at 515 nm, after 16 minutes, of a solution of 2 mL of DPPH 6 -5 M in methanol and 50 is measured µL of the antioxidant extract. The percentage of inhibition is calculated according to the following formula:

PI (Percentage of Inhibition) PI (%): [(Abs t = 0 min-Abs t = 16 min) / Abs t = 0 min)] * 100

The IC 50 or concentration that inhibits is measured 50% of the DPPH radical.

The filtered liquid extracts of Saxifraga spathularis , obtained according to the extraction procedure, have an IC 50 between 300 to 350 ppm.

The IC 50 of the BHA, measured according to it The procedure is 240 ppm and the BHT is 2790 ppm.

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iv) Determination of the antioxidant or iron reduction activity (FRAP) and its equivalence in ascorbic acid or vitamin C of the filtered liquid extracts Saxifraga spathularis :

The reducing power of iron or antioxidant activity of the liquid extracts of Saxifraga spathularis is determined according to the FRAP method [ Benzie IFF et al. Anal Biochem 1996 ; 239: 70-76 ]. The ability to reduce the Fe (III) / tripyridyltriazine complex is measured and the reductive activities of the extracts are expressed as nM equivalents of ascorbic acid (AscAE), / g of dry extract. To 0.1 mL of the antioxidant extract is added 3 mL of the FRAP reagent is left at room temperature for 6 minutes. The absorbance at 593 nm is measured and the results are compared with a straight standard of ascorbic acid or vitamin C (0.1-1 mM).

The filtered liquid extracts of Saxifraga spathularis obtained according to the extraction procedure, have a reducing power of 100 mg equivalent to between 0.100 mM to 0.1005 mM in ascorbic acid or vitamin C.

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Examples Example 1 Preparation of the hydrobutylene glycol extract of the leaves of Saxifraga spathularis

The leaves frozen with liquid N 2 of Saxifraga spathularis , ground and sieved to particle size of approximately 0.6 mm, are subjected to a continuous solid-liquid extractive process, with a liquid: solid ratio of 30 g / g , with a 1: 1 ethanol / water mixture, at the reflux temperature of the solvent (85-95 ° C), for 3 hours and protected from light. The solvent of the extract obtained is evaporated to dryness in vacuo. The yield of extraction is 28% (g / g).

The dry extract of Saxifraga spathularis obtained is subjected to a new continuous solid-liquid extractive process, by maceration with stirring, with a liquid: solid ratio of 99 g / g, at room temperature and protected from light, for 7 days with a 1: 1 butylene glycol / water mixture. The liquid extract of Saxifraga spathularis obtained is filtered, using a porous plate of No. 3, and the final product, the filtered liquid extract of Saxifraga spathularis , is stored refrigerated at 4 ° C and protected from light to prevent its alteration.

- The absorbance of this extract was measured at 100 ppm concentration in distilled water at wavelengths 280 nm (Abs = 0.714), 320 nm (Abs = 0.271) and 400 nm (Abs = 0.053) in front of a white distilled water.

- Phenolic content according to the method of Folin Ciocalteu's [ A. Escarpa, op. cited ( 2001 )]. The absorbance at 765 nm of a solution of 0.5 mL of the antioxidant extract, 3.75 mL of distilled water, 0.25 mL of Folin Ciocalteau's reagent (1: 1 with distilled water) and 0.5 mL of 10% Na 2 CO 3; after 1 hour at room temperature, against a white without extract. The phenolic content of the extract is determined by comparing the absorbance with a straight gallic acid standard (0-100 ppm).

The extract obtained according to the procedure described in this example 1 has a phenolic content of 24%

- Activity of the radical α, α-diphenyl-β-picrylhydracil (DPPH?) [ I. Parejo, op. cited ( 2002 )]. The decrease in absorbance at 515 nm, after 16 minutes, of a solution of 2 mL of 10-5-5M DPPH 6 in methanol and 50 µL of the antioxidant extract is measured. The percentage of inhibition is calculated according to the following formula:

PI (Percentage of Inhibition) PI (%): [(Abs t = 0 min-Abs t = 16 min) / Abs t = 0 min)] * 100

IC50 or concentration that inhibits was measured 50% of the DPPH radical.

The extract obtained according to the procedure described in this example 1 has an IC 50 = 334 ppm. The IC 50 of the BHA, measured according to the same procedure, is 240 ppm and the BHT of 2790 ppm.

- Antioxidant or iron reduction activity (FRAP) [ IFF Benzie, op. cited ( 1996 )]. The ability to reduce the Fe (III) / tripyridyltriazine complex is measured and the reductive activities of the extracts are expressed as nM equivalents of ascorbic acid (AscAE), / g of dry extract. To 0.1 mL of the antioxidant extract is added 3 mL of the FRAP reagent is left at room temperature for 6 minutes. The absorbance at 593 nm is measured and the results are compared with a straight standard of ascorbic acid or vitamin C (0.1-1 mM).

The extract obtained according to the procedure described in this example 1, presents at a concentration of 100 ppm an iron reducing power equivalent to 0.126 mM in acid ascorbic or vitamin C.

Claims (17)

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1. Procedure for obtaining extracts of Saxifraga spathularis characterized in that it comprises the following stages: i) Drying and / or freezing, grinding and sieving of the leaves, stems, roots and flowers of Saxifraga spathularis . ii) Continuous solid-liquid extraction of the dried, ground, and dried leaves, stems, roots and flowers of Saxifraga spathularis with aqueous, alcoholic or hydroalcoholic solvents. iii) Evaporation to dryness of the solvent from the extraction of stage ii) to obtain the dry extracts of Saxifraga spathularis -, or evaporation of the alcoholic solvent of stage ii) to obtain the aqueous extracts of Saxifraga spathularis . iv) Continuous solid-liquid extraction of the dry or aqueous extracts of stage iii) with aqueous, alcoholic, glycolic or hydro-glycolic solvents to obtain the liquid extracts of Saxifraga spathularis . v) Filtration of the liquid extracts of stage iv) to obtain the filtered liquid extracts of Saxifraga spathularis and cold storage and absence of light.

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2. Method of obtaining extracts of Saxifraga spathularis , according to claim 1, step i), characterized in that the dried leaves ground in a blade mill are used, and they are screened at a particle size of 0.6 mm. approximately. 3. Procedure for obtaining extracts of Saxifraga spathularis , according to claim 1, step ii), characterized in that the process of solid-liquid extraction in continuous with solvents; high liquid: solid ratios are used, preferably 30 g / g; the solvent is water, or a C 1 to C 4 alcohol, preferably methanol or ethanol, or a mixture methanol / water, or ethanol / water; the extraction temperature is the reflux of the solvent or mixture of extraction solvents and is in the range of 60-120 ° C; the extraction time is in the range of 1-3 hours; and the extractive process is carried out protected from light. 4. Method for obtaining extracts of Saxifraga spathularis , according to claim 3, characterized in that the extraction solvent is a 1: 1 ethanol / water mixture; and the extraction temperature is the reflux of the solvent mixture and is in the range of 85-95 ° C. 5. Procedure for obtaining extracts of Saxifraga spathularis , according to claim 1, step iii), characterized in that the evaporation to dryness of the solvent to obtain the dried extracts of Saxifraga spathularis is carried out under vacuum and the water is removed, preferably , by lyophilization. 6. Procedure for obtaining extracts of Saxifraga spathularis , according to claim 1, step iii), characterized in that the evaporation of the alcoholic solvent to obtain the aqueous extracts of Saxifraga spathularis is carried out under vacuum. 7. Procedure for obtaining Saxifraga spathularis extracts, according to claim 1, step iv), characterized in that the continuous solid-liquid extraction process with solvents is carried out by maceration with stirring; high liquid: solid ratios are used, preferably 99 g / g; the solvent is water, or a C2 to C6 glycol, preferably propylene glycol or butylene glycol, or a propylene glycol / water mixture, or butylene glycol / water; the extraction temperature is room temperature, approximately 20 ° C; The extraction time is 1 to 7 days and is carried out protected from light. 8. Method for obtaining extracts of Saxifraga spathularis , according to claim 7, characterized in that the extraction solvent is a 1: 1 butylene glycol / water mixture. 9. Procedure for obtaining extracts of Saxifraga spathularis , according to claim 1, step v), characterized in that the liquid extracts obtained in step iv) are filtered, preferably by porous plate of No. 3 or 4, or filter paper of grammage 73 g / m2, obtaining filtered liquid extracts of Saxifraga spathularis are stored cold, preferably at 4 ° C, and protected from light. 10. Dry extracts of Saxifraga spathularis obtained according to claims 1 to 5. 11. Liquid extracts of Saxifraga spathularis obtained according to claims 1 to 9. 12. Filtered liquid extracts of Saxifraga spathularis according to claim 11, characterized in that they have ultraviolet absorption between the wavelengths of 250 nm to 400 nm; for having a high phenolic content; for its ability to inhibit the DPPH? radical; for its ability to inhibit the oxidation of the micellar β-carotene and linoleic acid system; for its ability to inhibit the radical ABTS +; for its antioxidant or iron reduction capacity.

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13. Filtered liquid extracts of Saxifraga spathularis according to claim 12, characterized by having a concentration of 100 ppm at wavelengths of 280 nm have Abs>0.700; at 320 nm they have Abs> 0.250 and at 400 nm they have Abs> 0.050, because they have a phenolic content between 20-35%; for presenting an IC 50 between 300 to 350 ppm of inhibition of the DPPH radical and for its antioxidant capacity or reduction of iron at 100 ppm equivalent to between 0.100 mM to 0.150 mM in ascorbic acid or vitamin C. 14. Filtered liquid extract of the Saxifraga spathularis leaves according to claim 13, characterized in that it has a concentration of 100 ppm at wavelengths of 280 nm (Abs = 0.714), 320 nm (Abs = 0.271) and 400 nm (Abs = 0.053); for having a phenolic content of 24%; for presenting an IC 50 = 334 ppm of inhibition of the DPPH radical and for its antioxidant capacity or reduction of iron equivalent to 0.126 mM in ascorbic acid or vitamin C. 15. Use of the extracts of Saxifraga spathularis , according to claims 10 to 14, as a cosmetic agent for skin and / or hair care, intended to act as an anti-aging and antioxidant agent, for preventing alterations derived from cellular oxidation or oxidative stress, and for acting as a radical inhibitor and protector against ionizing radiation and free radicals. 16. Use of the extracts of Saxifraga spathularis , according to claims 10 to 14, as a cosmetic agent for skin and / or hair care, intended to act as an absorber of UV (ultraviolet) radiation, which gives it properties of sunscreen 17. Use of the extracts of Saxifraga spathularis , according to claims 10 to 14, in the pharmaceutical and / or food industry as an antioxidant.