ES2580762B1 - Procedure for obtaining a leaf extract of trees or shrubs of the genus Quercus, extract obtained and its use - Google Patents

Abstract

Method for obtaining a leaf extract of trees or shrubs of the genus Quercus, extract obtained and its use. # The present invention relates to a method of obtaining a leaf extract of a tree or shrub of genus Quercus comprising a) crush the sheet, b) add to the crushing of the sheet obtained in step a) a solvent selected from the group consisting of water, C {sub, 1} -C {sub, 6} alcohol and mixtures thereof, c) stir the mixture of the crushed and the solvent obtaining two phases, and d) separating the two phases obtained in step c) and collecting the supernatant comprising the extract. It also refers to the extract of the leaf of a tree or shrub of the genus Quercus obtainable by said procedure and its use as an antioxidant.

Description

DESCRIPTION

Procedure for obtaining a leaf extract of trees or shrubs of the genus Quercus, extract obtained and its use.

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Field of the Invention

The present invention belongs to the field of obtaining natural antioxidants. Specifically, it refers to a procedure for obtaining a leaf extract of trees or shrubs of the Quercus genus, the extract obtained by that procedure and its use, in particular as an antioxidant.

Background of the invention

Lipid oxidation is a process that leads to a significant deterioration in the quality of food, resulting in a variety of degradation products that produce bad odors, unpleasant flavors, changes in rheological properties and formation of toxic compounds (Faustman, C ., Cassens, RG (1990) .The Biochemical Basis for Discoloration in Fresh Meat: A Review. Journal of Muscle Foods, 1, 217-243).

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Therefore, the prevention of lipid oxidation during food processing and storage is essential to maintain quality and safety (Buckley, DJ, Morrisey, PA and Gray, JI (1995). Influence of dietary vitamin E on the oxidative stability and quality of pig meat. Journal of Animal Science 73: 3122-3130).

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Oxidation in food can be controlled or minimized by the use of antioxidants, which are substances present in low concentrations with respect to the oxidizable substrate and which significantly retard its oxidation. Antioxidants can be classified according to their nature as synthetic and natural. Synthetics are derived from phenolic structures (Giese, J. (1996). Antioxidants: tools for preventing lipid oxidation. Food 30 Technology, 73-81) such as gallates (among which those of propyl, octyl and dodecyl), Butyl -hydroxy-anisole (BHA), Butyl-hydroxy-toluene (BHT) and Terbutylhydroxyquinone (TBHQ). However, due to concerns about their toxicological safety, it may be desirable to replace these conventional antioxidants with natural antioxidants (Formanek, Z., Kerry, JP, Higgins, FM, Buckley, DJ, Morrissey, PA and Farkas, J. ( 2001) Addition of synthetic and natural antioxidants to alpha-tocopheryl acetate supplemented beef patties: Effects of antioxidants and packaging on lipid oxidation. 5 Meat Science, 58: 337-341). Plants are a source of molecules capable of capturing free radicals, such as vitamins, terpenoids, phenolic compounds, lignins, tannins, flavonoids, alkaloids, coumarins and other metabolites rich in antioxidant activity. Naturally occurring antioxidants have been widely studied due to their health benefits and their added value. Recent research highlights the relationship between antioxidant activity and content in plant phenolic compounds that are being used in the fight against diseases such as cancer (Whei, Z. & Shiow, YW (2001) Antioxidant activity and phenolic compounds in selected herbs, Journal of Agricultural Food Chemistry, 49: 5165-5170). The use of plants, spices and herbs as potential sources of natural antioxidants in fresh and processed foods is becoming increasingly important in the food industry, presenting itself as an alternative to synthetic antioxidants.

While fruits are the products most commonly used as sources of antioxidants, other parts of plants, such as bark, leaves, peels and roots, can also be used for this purpose due to their content in compounds with antioxidant activity. Thus studies of antioxidants have been carried out on the green leaves of vegetables such as amaranth, spinach, guava leaves, blackberry leaves, red raspberry leaves, strawberry leaves (Wang SY, Lin HS (2000) Antioxidant Activity in Fruits and Leaves of Blackberry, Raspberry, and Strawberry Varies with Cultivar and Developmental Stage. Journal of 25 Agricultural Food Chemistry, 48: 140-146).

Plants of the genus Quercus are usually large trees, although shrubs are also included. There are permanent foliage, deciduous and marescent. The fruit is called acorn, it is solitary and of axial origin, with flat cotyledons. About 30 dominant elements of the tree landscape participate in many territories of its range in Europe, West Asia, North America and South America. Hybridization phenomena are frequent among their species, which usually also present a facility for vegetative regeneration due to root or strain outbreaks. In Spain, the predominant species of Quercus trees are cork oak (Quercus suber L.), holm oak (Quercus ilex L.) and oak (Quercus robur L.). Its main uses are to obtain acorn, wood and cork (cork oak). Cork is the suberified cellular tissue 5 with uses in various sectors (construction, decoration, wine bottling stoppers). The acorn is the main fruit of the Dehesa and is an important source of food for wildlife and domestic animals, being used to fatten pigs during the montanera or acorn season, which runs from the beginning of November until the end of February. 10

With all this acquires a special interest in obtaining an extract that encompasses all these characteristics, that is natural, that belongs to an industrialized crop, that is obtained from a part not used within the industry of the same, that presents antioxidant activity, which It can be applied to food and preserve food 15 in which oxidative deterioration is included.

Description of the figures

Figure 1: Effect of the incorporation of extracts (water, water: 50:50 v / v ethanol, ethanol) of 20 cork oak leaf (Quercus suber L.) on lipid oxidation - measured as TBA-RS (mg MDA / kg ) - of a cooked product made from chicken meat and stored refrigerated for 10 days. N = 5 determinations / lot; a, b, c: For the same days of storage, bars with different letters imply statistically significant differences (Tukey test, p <0.05). 25

Object of the invention

In a first aspect, the present invention relates to a method of obtaining a tree leaf or shrub extract of the genus Quercus (method of the invention), which comprises the following steps:

a) Crush the sheet,

b) Add to the crushing of the sheet obtained in step a) a solvent selected from the group consisting of water, C1-C6 alcohol and mixtures thereof,

c) Stir the mixture of the crushed and the solvent obtaining two phases, and

d) Separate the two phases obtained in step c) and collect the supernatant comprising the extract.

Likewise, in a second aspect, the present invention refers to an extract of a Quercus tree or shrub leaf leaf obtainable by the process according to the first aspect of the invention. 10

A third aspect of the invention relates to the use of the Quercus tree or bush leaf extract according to the second aspect of the invention as an antioxidant.

Finally, a fourth aspect of the invention relates to a food, cosmetic or pharmaceutical product comprising the Quercus tree or bush leaf extract according to the second aspect of the invention.

Description of the invention

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Based on the needs of the state of the art, the inventors of the present invention have developed a process for obtaining a Quercus tree or bush leaf extract with a high polyphenol content which comprises an extraction stage with a solvent The extract obtained with the process of the invention has a high activity as an antioxidant. 25

Thus, in a first aspect, the present invention relates to a method of obtaining a Quercus tree or shrub leaf extract, comprising the following steps:

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a) Crush the sheet,

b) Add to the crushing of the sheet obtained in step a) a solvent selected from the group consisting of water (acidified or not), C1-C6 alcohol and mixtures thereof,

c) Stir the mixture of the crushed and the solvent obtaining two phases,

d) Separate the two phases obtained in step c) and collect the supernatant comprising the extract.

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Thus, the first stage of the process of the invention consists in the crushing of the Quercus tree or shrub leaf. The leaf can be obtained by manual or mechanical collection of trees or shrubs belonging to the genus Quercus. The crushing of the sheet is carried out by manual or mechanical means until obtaining small particles, preferably between 0.1 and 10 mm. 10

In step b), the proportions used of the sheet or solvent can be modified in order to obtain extracts with a greater or lesser concentration and therefore with different antioxidant activity. Preferably, the ratio between the crushed sheet and the solvent (sheet: solvent) is between 1:50 (weight: volume) and 1: 2 (weight: volume). In a particular embodiment, the sheet: solvent ratio is 1:10 (g: mL).

The use of one or the other solvent influences the amount of compounds extracted as well as the type of compounds with antioxidant activity that is obtained due to their characteristics and their affinity for the solvent. In a preferred embodiment, in step b) the selected solvent is a mixture of water and a C1-C6 alcohol.

In the context of the present invention, the term "C1-C6 alcohol" refers to an alcohol that can have from one carbon to six carbons in a straight or branched chain, with at least one hydroxide function. 25

In a particular embodiment, in step b) the selected solvent is a mixture of distilled water and ethanol, where the water: ethanol ratio is between 20:80 and 80:20 (v: v), more preferably between 30 : 70 and 70:30 (v: v) and more preferably still 50:50 (v: v). The extraction carried out with a mixture of water and ethanol in a 50:50 ratio is that which results in an extract with a higher content of phenolic and flavonoid compounds.

The term "phenolic compounds" refers to aromatic compounds comprising at least two hydroxyphenolic groups in the molecule. Flavonoids, a heterogeneous group of plant polyphenols that share a benzopyran structure, are included in this group of compounds. The main flavonoids with antioxidant activity are (+) - 5 catechin, (-) - epicatechin, (-) - epigallocatechin and the corresponding esters of gallic acid, (-) - epicatechin gallate and (-) - epigallocatechin gallate.

The conditions of temperature, time and exposure to light in step c) can be adjusted in a range in which obtaining compounds with antioxidant activity is optimal avoiding degradation. Preferably, step c) is carried out at a temperature between 20 ° C and 28 ° C, preferably between 22 ° C and 26 ° C, for a time between 30 and 210 minutes, preferably between 100 and 150 minutes, and under conditions of reduced brightness, preferably darkness. In a particular embodiment, step c) is carried out in dark conditions, at a temperature of 25 ° C and for a period of 2 hours.

By stirring stage c) two phases are obtained from which the supernatant phase that separates from the solid phase is used. In step d), the separation of the two phases formed in step c) to collect the supernatant comprising the extract is carried out by decantation or centrifugation or any other method that allows the differentiation of the phases.

The supernatant collected in step d), which comprises the leaf extract, may comprise some rest of the crushing or impurities, whereby in a particular embodiment, the process of the invention comprises a step e) of filtration of the supernatant collected in stage d). Also, the supernatant collected in stage d) or filtered in stage e) can be concentrated by distillation or transformed by lyophilization or any other modification that allows obtaining an extract that suits the application needs. Thus, in another particular embodiment, the process of the invention comprises a stage f) of distillation of the supernatant obtained in step d) or filtered in step e). And in another particular embodiment, the supernatant collected in step d), filtered in step e) or concentrated in step f) is lyophilized (step g). Depending on the final extract, its conservation may be carried out under ambient conditions or it will require controlled temperature storage in an appropriate container. In a particular embodiment, the extract is preserved under dark and cooling conditions.

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A second aspect of the invention relates to a Quercus tree or shrub leaf extract obtainable by the process of the invention according to any one of the embodiments according to the first aspect of the invention. In a preferred embodiment the extract has a content in phenolic compounds comprised between 5.6 and 11.4 mg equivalents of gallic acid per gram of sample. Preferably between 6.5 and 10.8 mg 10 equivalents of gallic acid per gram of sample. In a preferred embodiment the extract of the invention has a content in flavonoid compounds comprised between 5.3 and 8.0 equivalents of catechin per gram of sample. Preferably between 6.4 and 7.9 mg equivalent of catechin per gram of sample.

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The extract of the present invention, obtainable by the process according to any one of the embodiments of the first aspect of the invention, is effective as an antioxidant agent, with important applications in the food, cosmetic and pharmaceutical industry. Thus, a third aspect of the invention relates to the use of the Quercus tree or bush leaf extract according to the second aspect of the invention as an antioxidant. twenty

In addition, a fourth aspect of the invention refers to a food, cosmetic or pharmaceutical product comprising the extract according to the second aspect of the invention.

The incorporation of the extract into a product is carried out in multiple ways, such as by addition within a formulation, by spraying on the product or any other method that allows the correct distribution of the extract and its antioxidant action. The concentration of extract to be used is determined by factors specific to the extract, such as antioxidant capacity, as well as those attributable to the product such as type of product, content and type of fat, concentration in anti-and pro-oxidants, etc. Likewise, the technological processes that can be carried out after the addition of the extract as well as the product conservation characteristics must be taken into account.

Examples

Specific examples of embodiments of the invention that serve to illustrate the invention are detailed below. 5

In particular, it is described the obtaining of three extracts from the cork oak leaf (Quercus suber L.) and its characterization by means of total phenolic content and its flavonoid content.

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Example 1. Obtaining the extract of cork oak leaf (Quercus suber L.) by using solvent mixture.

To obtain the extract, it was split from cork leaves manually collected from the trees. The leaves were shredded by crushing in a conventional chopper. 15 The extraction of compounds with antioxidant properties was carried out by using two solvents and mixing both. Thus, distilled water, ethanol and a mixture of water: ethanol in 50:50 ratio (vol: vol) were used, which gave rise to three different extracts. Based on results obtained in previous experiences, a 1:10 ratio (g: mL) of sheet and solvent was used. The solvent and sheet mixtures were kept under stirring for two hours at a controlled temperature of 25 ° C under reduced light conditions. Once the stirring time elapsed, the phases were separated by centrifugation, obtaining two perfectly differentiated phases. The supernatant phrase was filtered through Whatman No. 54 paper filter and kept in an opaque container at refrigeration temperature. 25

Example 2. Characterization of three extracts of cork oak leaf (Quercus suber L.) by quantifying its total phenolic content.

The quantification of total phenols of the cork oak leaf extracts (Quercus suber 30 L.) was performed following the procedure described by Singleton et al. (1999). 50uL of each of the extracts were deposited in a microtiter plate well to which 20uL of Folin-Cicalteau reagent and 50uL of 20% sodium carbonate (w / v) were added. The mixture was incubated for one hour at room temperature, after which the absorbance at 765 nm was measured against a blank where the sample was replaced by distilled water. The quantification of the measurements carried out was performed against a standard curve of gallic acid. The results were expressed as equivalents of gallic acid and are reflected in Table 1.

Table 1. Phenolic compounds content (mg Eq gallic acid / g sample) of extracts (water, water: 50:50 v / v ethanol, ethanol) of cork oak leaf (Quercus suber L.)

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 Extraction solvent

 Phenolic compounds

 Water

 6.49 b ± 0.52

 Water: 50:50 v / v ethanol

 10.76 to ± 0.63

 Ethanol

 6.12 b ± 0.52

 p

 <0.001

N = 8 determinations

a, b: In the same column, means with different letters imply statistically significant differences (Tukey test, p <0.05)

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Example 3. Characterization of three extracts of cork oak leaf (Quercus suber L.) by quantifying its flavonoid content.

The quantification of the flavonoid content of extracts obtained from cork oak leaf (Quercus suber L.) was carried out following the method proposed by Zhishen et al., 20 (1999). This procedure is based on the formation of chelates between AlCl3 and flavonoids in the sample, which have a pink color. Thus, 400uL of sample was deposited in a test tube to which 60uL of 10% NaNO2, 60uL of 20% AlCl3 and 400uL of 1M NaOH were added. After stirring its absorbance was measured at 510 nm. The quantification of the measurements performed was performed against a catechin standard curve. The results were expressed in mg of equivalent catechin per gram of sample and are reflected in Table 2.

Table 2. Flavonoid content (mg Eq. Catechin / g sample) of extracts (water, water: 50:50 v / v ethanol, ethanol) of cork oak leaf (Quercus suber L.)

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 Extraction solvent

 Total flavonoids

 Water

 5.40 b ± 0.10

 Water: 50:50 v / v ethanol

 7.85 to ± 0.12

 Ethanol

 6.35 ab ± 0.10

 p

 <0.001

N = 8 determinations

a, b: In the same column, means with different letters imply statistically significant differences (Tukey test, p <0.05)

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The antioxidant capacity evaluation of cork oak leaf extracts (Quercus suber L.) was performed following two synthetic radical reduction methodology, FRAP method and ABTS method, as shown in examples 4 and 5. In these tests It demonstrates the antioxidant power of the sample by changes in the color of a solution containing a radical that can be reduced. In both cases, the response obtained is extrapolated to a Trolox calibration curve, so the results are expressed in TEAC (antioxidant activity equivalent to Trolox).

Example 4. Evaluation of the antioxidant capacity of three extracts of cork oak leaf (Quercus suber L.) using the ABTS radical. twenty

The method proposed by Re et al. (1999). Thus, the ABTS radical was obtained from the equal part mixing of a solution of ABTS / nM and 2.45nM potassium persulfate. This solution was kept at room temperature for 16 hours in dark conditions for the generation of the radial. The solution was diluted with ethanol to obtain an absorbance of 0.7 (± 0.1) at 734 nm. 245uL of the solution with the ABTS radical was deposited in a well of a microtiter plate and its absorbency was measured at 734nm. Then 5uL of the extract to be tested was added and, after one minute, the measurement was repeated at 734 nm. The results obtained faced a Trolox calibration curve. The results were expressed as mg TEAC / g sample and are reflected in table 3.

Table 3. Antioxidant capacity measured by the ABTS method (mg TEAC / g of sample) of 5 extracts (water, water: 50:50 v / v ethanol, ethanol) of cork oak leaf (Quercus suber L.)

 Extraction solvent

 ABTS

 Water

 32.34 b ± 1.40

 Water: 50:50 v / v ethanol

 51.14 to ± 4.72

 Ethanol

 16.94 c ± 0.79

 p

 <0.001

N = 8 determinations

a, b, c: In the same column, means with different letters imply statistically significant differences (Tukey's test, p <0.05)

Example 5. Evaluation of the antioxidant capacity of three extracts of cork oak leaf (Quercus suber L.) using the FRAP method.

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This method proposed by Benzie and Strain (1996), determines the capacity of ferric reduction that has a sample at low pH, transforming the tripyridyltriazine complex (TPTZ) with iron (III) to its ferrous form. Thus, the FRAP reagent was prepared from 2.5ml of a 10mM TPTZ solution together with 2.5mL of a 20mM FeCl3-6H2O solution and 25 mL of 0.3mM acetate buffer and pH 3.6. A 593nm absorbance reading of 20 200mL of the FRAP reagent was made to which 7uL of sample was subsequently added, with absorbency measurements taken after 4 and 30 minutes. The results obtained faced a Trolox calibration curve. The results were expressed as mg TEAC / g sample and are reflected in table 4.

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Table 4. Antioxidant capacity measured by the FRAP method (4min and 30min) (mg TEAC / g sample) of extracts (water, water: 50:50 v / v ethanol, ethanol) of cork oak leaf (Quercus suber L.) .

 Extraction solvent

 FRAP 4min FRAP 30min

 Water

 17.66 b ± 3.10 22.68 b ± 3.34

 Water: 50:50 v / v ethanol

 32.08 to ± 7.09 48.95 to ± 8.00

 Ethanol

 14.41 b ± 2.64 20.20 b ± 2.94

 p

 <0.001 <0.001

N = 8 determinations

a, b: In the same column, means with different letters imply statistically significant differences (Tukey test, p <0.05) 5

The extract obtained following the provisions of the present invention has shown its effectiveness as an antioxidant agent in the incorporation on food products. The incorporation of the extract can be carried out in multiple ways such as by adding it into a formulation, by spraying on the product or any other method that allows the correct distribution of the extract and its antioxidant action. The concentration of extract to be used will be determined by factors specific to the extract (antioxidant capacity) as well as those attributable to the food product such as type of product, content and type of fat, concentration in anti and prooxidants, etc. Likewise, the technological processes that can be carried out after the addition of the extract as well as the preservation characteristics of the food product must be taken into account.

The incorporation of three extracts of cork oak leaf (Quercus suber L.), obtained by means of three solvents, on a chicken meat product, was effective against oxidative phenomena such as cooking. Example 6 shows the comparative study that evaluated the evolution of the content in TBA-RS in a chicken product with added extract versus a product without extract in its formulation.

Example 6. Evaluation of the antioxidant activity of extracts of cork oak leaf (Quercus suber L.) incorporated in the formulation of a cooked chicken product. 25

A chicken product was prepared from breasts obtained in a retail store. The formulation used contained 79% minced meat, 18% water, 1% salt and 2% of each of the extracts with respect to the final weight. The extracts of water, ethanol and water: ethanol (50:50 v / v) and a control without added extract were used. The mixture was distributed in plastic tubes with approximately 30g in each tube. The tubes were introduced in hot water until a temperature in the center of the mixture of 72oC was obtained and kept for 30min to obtain a cooked product. 5 Subsequently, the samples were cooled on ice and stored at 4 ° C for 8 days.

The quantification of oxidative processes was performed using the thiobarbituric acid index (TBA-RS) method described by Salih et al. (1987) that measures the content of certain secondary products derived from lipid oxidation such as malondialdehyde (MDA). Thus, 2g of sample was taken and homogenized with 7.5mL of perchloric acid 10 3.86%. 2mL of homogenate were mixed with 2mL of 0.02M TBA and the mixture was heated at 90 ° C for 30 minutes. Subsequently, an aliquot was taken to measure its absorbency at 532 nm in front of a target. The results obtained faced a calibration curve of 1,1,3,3-tetraethoxypropane (PET). The results were expressed as mg of MDA / Kg sample and are reflected in Figure 1. 15

Claims (15)

1. Procedure for obtaining a Quercus tree or shrub leaf extract comprising the following stages: to. Shred the blade, 5 b. Add to the crushing of the sheet obtained in step a) a solvent selected from the group consisting of water, C1-C6 alcohol and mixtures thereof, c. Stir the mixture of the crushed and the solvent obtaining two phases, and d. Separate the two phases obtained in step c) and collect the supernatant comprising the extract. 2. A method according to claim 1, comprising a step e) of filtration of the supernatant collected in step d). 3. The method according to claim 1 or 2, comprising a stage f) of distillation of the supernatant collected in step d) or of the filtrate obtained in step e). fifteen 4. Method according to any one of claims 1 to 3, comprising a step g) of lyophilization of the supernatant collected in step d) or of the filtrate obtained in step e) or of the distillate obtained in step f). 5. Method according to any one of claims 1 to 4, wherein, in step a), the sheet is crushed to obtain a particle size between 0.1 and 10 mm. 6. Method according to any one of claims 1 to 5, wherein the crushed ratio of sheet: solvent is between 1:50 (weight: volume) and 1: 2 (weight: volume). 7. Process according to any one of claims 1 to 6, wherein the solvent selected is a mixture of water and a C1-C6 alcohol. 8. Method according to claim 7, wherein the water: C1-C6 alcohol ratio is between 20:80 and 80:20 (v / v). 9. Process according to claim 7 or 8 wherein the solvent selected in step b) is a mixture of water and ethanol. 30 10. Method according to any one of claims 1-9, wherein step c) is carried out at between 20 and 28 °, for 30 and 210 minutes. 11. Extract of tree leaf or bush of genus Quercus obtainable by the method according to any one of claims 1-10. 12. Extract of a tree or shrub leaf of the genus Quercus, according to claim 11, characterized in that it has a total phenolic content between 5.6 and 11.4 mg equivalent of gallic acid / g of sample. 5 13. Tree or shrub leaf extract of the genus Quercus, according to claim 11 or 12, characterized in that it has a flavonoid content of between 5.3 and 8 mg equivalent of gallic acid / g of sample. 14. Use of the Quercus tree or bush leaf extract according to any of claims 11-13, as an antioxidant. 10 15. Food, cosmetic or pharmaceutical product comprising the Quercus tree or shrub leaf extract of claims 11-13.