RO122894B1 - Process for producing a grape seed extract in powder form with a high content of proanthocyanidins - Google Patents

Abstract

The invention relates to a process for producing a grape seed extract in powder form with a high content of proanthocyanidins, said extract being used in pharmaceutical, food and cosmetic fields. According to the invention, the process consists in treating the grapes with water and/or ethyl alcohol at a temperature of up to 120A C and a pressure of 1 atm, for 45 ... 60 min, thereafter the polyphenol extract in ethyl alcohol solution, rich in proanthocyanidins, is concentrated, centrifuged and filtered, there being obtained an extract which, afterwards, is purified and enriched by the passage on a reversed-phase chromatography columns.

Description

The invention relates to a process for obtaining grape seed from a powdered extract with a high content of proanthocyanidins. The process according to the invention can be applied by wine producing units, by units profiled on the exploitation of natural resources (residual vegetable masses), and the product obtained by this process can be used as an ingredient with antioxidant properties in the food, cosmetic and pharmaceutical industry or as a food supplement. .

Oligoproanthocyanidins (OPCs), molecules with high antioxidative properties, constitute a family of less colored flavanolic compounds, present in a large number of plant materials. Their chemical structure is made up of monomer units of flavanols, mainly simple or gallylated catechins:

Structure of a proanthocyanidin dimer

Epicatechina Catechina

Phenolic compounds are some of the most abundant substances present in grapes and wines after specific carbohydrates and organic acids. They are present in all the constituent parts of the grape (skin, pulp and seeds). The amount of extractable phenols from grapes is about 10% in pulp, 60-70% in seeds and 28-35% in skin. Grape seeds are the constituent part of vines, the richest in polyphenols, even after vinification. The phenolic fraction of grape seeds consists almost exclusively of flavanols, mainly catechins (catechin and epicatechin, simple and gallylated) and polymers.

RO 122894 Β1 thereof, also called procyanidins, respectively proanthocyanidins, when there are 1 different substituted monomer units in nucleus B. Mainly dimers (B1, B2, B3, B4, B5) and trimers (C1, C2) have been identified. Proanthocyanidins containing up to 10 monomer units in 3 molecules are called oligoproanthocyanidins (OPCs).

The beneficial effects of polyphenolylordinary grape seeds are due to their 5 antioxidant properties. The ability of this category of polyphenols to consume oxygen free radicals from the environment in which they are located is superior to other well-known antioxidants, such as vitamin C, E, beta-carotene. Being consumers of free radicals of oxygen, such as superoxides - superoxide anion, single oxygen and hydroxyl radicals, they inhibit the oxidation of low density lipoproteins (LDL), inhibit platelet aggregation, intervene in the synthesis and release of many substances that cause inflammation. , inhibits enzymes 11 that can damage the integrity and elasticity of capillaries, skin and other tissues. Due to their affinity for proteins, polyphenols can reduce the nutritional value of certain types of food 13 (seeds, sausages, flour).

For these reasons, the products in this category are recommended: 15

- people with cardiovascular risk;

- in the case of chronic or degenerative diseases, some types of cancer, cataracts, arthritis, heart disease17;

- against the process of aging, pancreatitis, liver disease, obesity; 19

- to lower blood pressure;

- in the case of the elderly - for those who consume polyunsaturated fatty acids, 21 those who were exposed to solar radiation and at high altitudes, those who were exposed to risks such as prolonged medications with synthetic polycyclic substances, smoking, alcoholism, pollution, 23 pesticides, passive smoking, etc.

Studies regarding the research and use of bioactive substances extracted from 25 grape seeds are 40 years old in Romania. The experiments that have been carried out have led to the establishment of methods of identification, extraction and purification of polyphenols. 27

At the international level, the study of the bioactive components found in the vine and in the products from it, represents a concern in the field of research starting with the first half of the 20th century. Starting from these studies, the foundations of an industry based on obtaining total and / or purified grape seed extracts were laid. 31 These extracts are used in particular in the food supplements, tonic drinks, food, cosmetics and pharmaceuticals industry. 33

If the first inventions were based on methods of extraction with organic solvents such as methanol and / or acetone and ethyl acetate (OSIM patent 99206/1990, 35 patent application US 5484594 / 1996-01-16, US patent application 5773262/1998 -06-30, patent application US 5912363 / 1999-06-15, patent 6544581 / 2003-04-08), lately due to 37 regulations imposed in the food, pharmaceutical and cosmetic industry it was necessary to find solutions for the replacement their alternative methods, ecological, extraction, 39 purification and concentration.

From the prior art we cite the Chinese patent CN1212320, which describes a process 41 for the extraction of proanthocyanidins from grape seeds, in which the plant material is subjected to water and / or alcohol extraction, the obtained liquid extract is then filtered, concentrated in 43 for solvent recovery, filtered again, and the obtained filtrate is absorbed on a macroporous resin over which an aqueous alcoholic solution is passed, the liquid obtained, which contains 45 proanthocyanidins, finally being dried.

R0 122894 Β1

The technical problem that the invention aims to solve is to provide a powder extract, which has a high content of polyphenols, and especially proanthocinidins, by a non-toxic, non-polluting and, simpler process, compared to known procedures which comprise several intermediate steps.

The process for obtaining grape seed from a powdered extract with a high polyphenol content, according to the invention, comprises:

a) obtaining a crude liquid extract by extracting the polyphenolic components from the grape seeds with water or a 5-50% solution of ethyl alcohol in water, at a temperature of maximum 120 ° C and a pressure of 1 atm, for 45-60 min, the volume ratio between the seeds and the liquid phase being between 1: 2 and 1: 3;

b) obtaining a total liquid extract by concentrating, centrifuging and filtering the crude liquid extract;

c) optionally, obtaining a total liquid extract enriched in monomeric proanthocyanidins by passing the total liquid extract on a chromatographic column of silica with C8 alkyl chains and eluting with a 50-96% ethyl alcohol solution;

d) obtaining a powder by lyophilizing the total liquid extract or the enriched liquid extract.

In order to remove the disadvantages of the known processes from the prior art and to increase the efficiency of the extraction technology, a new extraction process is proposed within the present invention which combines the classical method (hydroalcoholic solutions) with methods such as applying high temperatures and pressures, purifying only by adsorption on stationary phase with C8 hydrophobic groups and elution with hydroalcoholic solutions.

The process described allows the reuse of the alcoholic component in the extraction and / or elution mixture. Because at no stage of the technological process are toxic solvents used, the product is safe for the human body.

The preservation method was chosen by freeze-drying and freeze-drying preservation followed by drying at positive temperatures and pressures of 10 ' ² torr, called process and freeze-drying. In the biotechnology of natural extracts, lyophilization is the most efficient means of stabilizing bioactive components, providing long-term stability to the final product and high bioavailability.

Next, in the description of the product characteristics, the expression "total extract" will be used both for the total liquid extract of step b) of the process according to the invention and for the sprayed form thereof, and the expression "enriched extract" for both the liquid form. of the enriched extract according to step c), as well as for its pulverized form.

Some features of the product that can be achieved through this process are:

- an antioxidant activity both in the total extract and in the enriched extract, comparable with the antioxidant activity of the extracts obtained by processes with several intermediate steps;

- In the variant "total extract are present and trace elements, components existing in grapes, beneficial for the human body. They do not pose standardization problems when used in the cosmetic or food industry.

- the variant "total extract contains a carbohydrate component, and the variant" enriched extract does not contain this component;

- the small percentage of the carbohydrate component in the "total extract and its absence in the" enriched extract "confers thermodynamic properties that are economically convenient in the technological-freeze-drying (freeze-drying) stage. The values of the eutectic temperature, the melting temperature, the glass transition temperature, the devitrification temperature, allow a low energy consumption in the production of the two extracts in powder form;

RO 122894 Β1

- the antioxidant activity of the two variants of extract in liquid form is 1 the same as the antioxidant activity of the extract in lyophilized form;

- all operations with hydroalcoholic solutions are carried out under controlled conditions, 3 removing the risks of a negative impact on the environment.

The methods used to identify and measure active compounds as well as 5 antioxidant activity are methods accepted by organizations in the United States -National Nutrition Food Association (NNFA), Food and Drug Administration (FDA), Dietary 7 Supplement Health and Education Act (DSHEA) for phenolic compounds extracted from grape seeds and provided for in the latest European regulations. In Romania, they are imposed by 9 new EU legislation that became applicable from August 1, 2005. Thus, for the content in total polyphenols the accepted methods are Folin Ciocâlteu and the Porter Index, 11 for the dosing of monomers and oligomers in extract the accepted method is chromatography. In liquid phase (HPLC), for the microelement dosages the accepted methods are according to 13 Pharmacopoea 2005, for the dosage of the oxygen free radicals absorption capacity the accepted method is the ORAC (oxygen radicals absorbance capacity) method. 15

The figure shows the diagram of the technological flow for obtaining the total and enriched extract (in liquid form or in powder form) from the grape seeds resulted 17 following the vinification process of the white or black grapes.

In order to obtain an extract with relatively uniform quantitative and qualitative characteristics, 19 the raw material requires processing by mechanical methods in order to separate from the rest of the plant material resulting from the vinification process, as well as to eliminate the unwanted influence 21 of the impurities. After separation, the seeds are washed with water, sorted, then dried to a humidity of 10-15%. The storage is done under conditions that ensure the constant humidity and prevent the development of molds.

The process for obtaining the proanthocyanidins consists of performing a solid-liquid extraction in a hydroalcoholic solvent (aqueous solution of ethyl alcohol) of a concentration of 5-50% or in water, at a temperature of up to 120'C and a pressure of up to 120'C. at 1 atm. 27

The seed / liquid phase ratio is 1: 2 or 1: 3 (v / v).

This method is characterized by a high extraction efficiency. The rational choice of the treatment duration and temperature allows the thermal decomposition of the extracted constituents to be avoided or reduced. 31

The duration of these conditions is between 45 and 60 min.

After extraction, the liquid phase, rich in proanthocyanidins, also called crude liquid extract 33, is separated from the solid material, concentrated in vacuo, until the removal of two thirds of the initial volume and cleared (removal of plant residues, pectic substances 35). and other types of macromolecules) by centrifugation at 5000-7000 rpm. for 30-50 min, resulting in the total liquid extract with a pH between 4.0-4.5. 37

The total liquid extract can be purified / enriched by passing it on a reverse phase chromatography column. Alkyl chains were chosen as active groups for adsorption 39

C-8 (Lichroprep RP-8).

The adsorption concentration procedure comprises two steps: 41

- a step of selective fixation of small and medium molecular mass proanthocyanidins (monomers, dimers, trimers, oligomers) and elimination of polymers with high molecular masses 43 (tannins), salts, sugars;

- an elution step of the proanthocyanidins adsorbed with a solution of ethyl alcohol of 45 50-96%.

The determination of the volume of adsorbent material was calculated as follows: 47

Volume = π x (1/2 inside diameter) ² x layer length

RO 122894 Β1

The amount of total liquid extract passed through the column is between 1 and 3 adsorbent volumes, and the elution volume is between 1 and 2 adsorbent volumes.

The resultant eluate is decalcoholized by vacuum concentration or, optionally, by reverse osmosis.

The dry matter content is determined refractometrically.

Transfomation of the total liquid extract or the enriched liquid extract, in powder, is carried out by freezing, sublimation and drying at positive temperatures, under vacuum.

For a liquid extract with a concentration of the active substance (refractometrically controlled) optimal for carrying out the lyophilization process, ie having sufficiently high values of the glass transition temperature, the freezing temperature was between -50 ° C and -35'C, the duration of application of these temperatures being 3-4 h.

Sublimation was performed at negative temperatures between -40 ° C and O'C, pressure 10 ' ² torr, for about 10 h.

The secondary drying temperature can reach 40-50 ^ 0, the duration being between 5 and 10 h depending on the thickness of the product layer subjected to drying.

The resulting powder is stored in the refrigerator, in containers that prevent the action of humidity and air.

Example 1. Obtaining the total liquid extract from white grape seed by hydroalcoholic solution, and converting this liquid extract into powder.

To obtain the crude extract, a 1: 2 (v / v) mixture of seeds / hydroalcoholic solution (25% ethyl alcohol in water) was obtained by adding 21 solutions to 11 seeds, which was heated to a maximum temperature. 120 ° C for 45 minutes at a pressure of 1 atm.

After removal of the solid fraction, the liquid phase was concentrated and centrifuged to obtain the liquid form of the total polyphenolic extract.

The total polyphenolic extract in powder form was obtained by freezing the total polyphenolic extract in liquid form, at temperatures between -40 ° C and -50 ° C, sublimation up to the temperature of 0 ° C and pressure of 10 ³ torr, after which s -They applied positive temperatures up to 40 ° C at pressure values of 10 ' ² torr, for a duration of 4-8 hours.

The characteristics of the product, ie of the total polyphenolic extract, are:

Total polyphenols-method Folin Ciocâlteu-250 mg EAG / g

Total polyphenols-Porter test -197 units

Antioxidant activity - ORAC-5 test 233 U / g (1 unit ORAC = 1 pMol TroloxjTrolox = vitamin E)

Proanthocyanidin level: - oligomeric polyphenols: 83-86%;

- monomeric polyphenols: 14-17%.

The results of the toxicological experiments on animals as well as its pharmaceutical presentation (non-hygroscopic fine powder, with high stability under normal atmospheric conditions), recommend the total polyphenolic extract to be safe for use as:

a) ingredient in functional foods and compatible with technologies for making pastry and bakery foods, as well as in the fruit processing industry;

b) ingredient in cosmetic products.

Example 2. Enrichment in monomer and oligomeric proanthocyanidins of the total liquid extract obtained in example 1 and its powdering enrichment in active substance, simultaneously with the removal of salts, sugars and a significant part of the initial amount of tannins, was achieved by passing of a total liquid polyphenolic extract, obtained according to the process described in Example 1, by a silica column modified by C-8 alkyl chain grafting (LichroprepRP 8), 96% alcohol elution of the adsorbed and proanthocyanidinic monomers and oligomers and the concentration thereof thus obtained by desalting under vacuum.

RO 122894 Β1

The powder form of the enriched polyphenolic extract was obtained by lyophilizing 1 of the purified liquid extract. The parameters applied in the lyophilization process were specific to the concentration degree of the active substance, but within the limits described in example 1. 3

The characteristics of the product, that is, of the enriched polyphenolic extract, are:

Total polyphenols - Folin Ciocâlteu method 641 mg EAG / g 5

Total polyphenols-Porter test 394 units

Antioxidant activity-ORAC-26 test 237 U / g (1 unit ORAC = 1 pMol Trolox; 7 Trolox = vitamin E)

Proanthocyanidin level: - oligomeric polyphenols: 15-21%; 9

- monomeric polyphenols: 80-85%

Example 3. Obtaining by total extraction in water, from grape seeds of aibi (ex3.a) or 11 grapes (ex 3.b), the total liquid extract and turning this liquid extract into powder

To obtain the crude extract, a 1: 2 (v / v) mixture of seeds / water was made, 13 by adding 2 I of water to 1 I of seeds, which was heated to a maximum temperature of 120 ° C for 45 min at the pressure of 1 atm. 15

After removal of the solid fraction, the liquid phase was concentrated and centrifuged, thus obtaining the liquid form of the total polyphenolic extract. 17

The total polyphenolic extract in powder form was obtained by freezing the total polyphenolic extract in liquid form, at temperatures between -40 ° C and -50 ° C, sublimation 19 up to 0 ° C and pressure of 10 ' ³ torr, after which were applied positive temperatures up to 40 ° C at pressure values of 10 ² torr, for a duration of between 4-8 h. 21

The characteristics of the product, ie of the total polyphenolic extract, are:

3. a) White grape seed extract: 23

Total polyphenols-Folin Ciocâlteu-182 mg EAG / g method

Antioxidant activity - ORAC-2055 U / g test (1 unit ORAC = 1 uMol Trolox; 25 Trolox = vitamin E)

Proanthocyanidin level: - oligomeric polyphenols: 81.33%; 27

- monomeric polyphenols: 16.84%.

3. b) Black grape seed extract: 29

Total polyphenols-method Folin Ciocâlteu-401 mg EAG / g

Antioxidant activity - ORAC-3272 U / g test (1 unit ORAC = 1 uMol Trolox; Trolox31 = vitamin E)

Proanthocyanidin level: - oligomeric polyphenols: 79.04%; 33

- monomeric polyphenols: 20.957%).

Claims (6)

1 Claim 3 Process for obtaining an extract in the form of powder with a high content of proanthocyanidins, from grape seeds, characterized in that it comprises: 5 a) obtaining a crude liquid extract, by extracting the polyphenolic components from the grape seeds with water or with a solution of 5-50% ethyl alcohol in water, at a temperature 7 of maximum 120'C and a pressure of 1 atm, for 45-60 min, the ratio in volumes between the seeds and the liquid phase being between 1: 2 and 1: 3; 9 b) obtaining a total liquid extract by concentrating, centrifuging and filtering the crude liquid extract; C) optionally, obtaining a total liquid extract enriched in monomeric proanthocyanidins by passing the total liquid extract on a chromatographic column of silica with 13 C8 alkyl chains and eluting with a 50-96% ethyl alcohol solution; d) obtaining a powder by lyophilizing the total liquid extract or the enriched liquid extract 15.