RU2314118C1 - Method for preparing extracts possessing antioxidant activity from cultivated grape compound fruit racemes - Google Patents

Abstract

FIELD: pharmaceutical industry and technology.

SUBSTANCE: invention relates to a method for preparing agent possessing antioxidant activity, in particular, to a method for preparing extract possessing antioxidant activity from cultivated grape (Vitis vinifera L.) compound fruit racemes. Matured cultivated grape compound fruits are dried and milled under the definite conditions, the raw is extracted twice with ethyl alcohol at room temperature in the definite ratio raw to extractant. Then the prepared extract id evaporated, condensed extract is homogenized repeatedly and dried. Then for separation of lipophilic compounds extract is treated with a benzine solvent of sort "Nefras" at heating to boiling and at stirring, and for separation of water-soluble component extract is treated with water under definite conditions, suspension is filtered and the obtained deposit is dried. Above described method provides preparing extract showing the enhanced content of polyphenols.

EFFECT: improved preparing method.

3 cl, 3 tbl

Description

The invention relates to the pharmaceutical industry and relates to a method for producing an extract with a high content of the main active biologically active substances (polyphenols) from axial brushes (ridges) of fruit grape cultivar (Vitis vinifera L.).

In patent RU No. 1072309 describes a method of obtaining funds with anti-alcohol action. The final product is a thick extract (comb of grapes) containing pectin (20 mg / g), organic acids (18 mg / g), free amino acids (20 mg / g), catechins (120 mg / g), leukrantocyanes (250 mg / g), flavonoids (80 mg / g), lignin (50 mg / g), phenolic acids (60 mg / g), aromatic acids (5 mg / g), alkyl ferruates (10 mg / g), lignans (5 mg / d) glycosides of lignans (5 mg / g). The product reliably inhibits the formation of physical dependence on alcohol.

It is also known (patent RU No. 2228761) a means of reducing the effect of alcohol, and a method for its production. The product includes water-alcohol extracts of grape ridges, St. John's wort and rose hips. Product standardization in terms of grape crest extract is based on the content of aromatic aldehydes and unoxidized phenolic compounds.

There are a number of foreign patents describing the preparation of liquid aqueous-alcoholic extracts of grape ridges.

Closest to the proposed is a tool based on an extract from grape ridges, protected by patent RU No. 2229302, modifying the toxic effect of alcohol, and a method for its production.

This patent presents options for obtaining the final product is an extract of grape ridges. Several products are presented: aqueous extract, hydroalcoholic extract, dry extract (extractant not specified). All products can be raw materials for: medicines, biologically active food additives, components of non-alcoholic, low alcohol and strong alcoholic drinks. A method for producing extracts, various temperature conditions of extraction: 40 °, 35 ° C, room; various extractants: water, water-alcohol mixture; drying modes of extracts: below 40 °, 35 ° C at room temperature. The resulting dry, aqueous fraction of the extract is enriched in a fraction of aromatic aldehydes. Thus, several methods for producing final products are presented.

The extract contains, based on the minimum single dose, at least 6 mg of aromatic aldehydes and at least 370 mg of unoxidized compounds of phenolic nature.

Modern requirements for substances for the production of drugs, dietary supplements include, first of all, accurate, quantitative determination of active substances by the methods of their standardization, which determines the quality of the product. In the finished form, the dosage varies significantly for different types of product (drugs or dietary supplements).

The patent indicates the content in the extract based on the minimum single dose of aromatic aldehydes - 6 mg and unoxidized compounds of phenolic nature - not less than 370 mg, but it is not indicated for which product these doses are determined.

To assess the correct choice of the method of extract production, it is necessary to indicate the content of the selected parameters (in this case, aromatic aldehydes and unoxidized phenolic compounds) in each mode. Secondly, these BAS groups are far from homogeneous and contain a wide variety of specific substances. One-time norms for individual groups of both aldehydes and phenolic compounds differ significantly. That is, these norms can not be a criterion for both a drug (LS) and a biologically active additive (BAA). In addition, the most important, active and generally accepted fractions for this product are polyphenolic compounds.

Thus, in the patent there are no clear characteristics of the products obtained using different modes of extraction and quantitative characteristics of biologically active substances (BAS) in the final products. Viticulture products are the richest sources of biologically active substances. In world practice, according to the proven properties, extracts based on them occupy one of the leading places. At the same time, extracts of wine, juice, peel, grape pulp seeds are actively used. Grape comb extract, registered for the first time as “Complex of grape comb polyphenols, (CPHG)” (first registered by Parapharm in 1996, last re-registration - 2005, State Registration Certificate No. 77.99.23.3.U.7831.7.05) refers to priority Russian developments and is gaining more and more popularity in recent years. Extract of grape fruit brushes (crests) contains a whole complex of polyphenolic compounds that have been studied previously [Eremina A.V., Reshetnyak V.Yu., Vezirishvili M.O. Determination of the polyphenolic composition of the dry extract of grape ridges by HPLC. Chemical Pharmaceutical Journal. - 2004. - T. 38, No. 3. - S.26-28; Eremina A.V., Vezirishvili M.O., Reshetnyak V.Yu. Standardization of polyphenolic compounds of dry extract of grape ridges, X1 Russian National Congress "Man and Medicine". - M: Medicine. - 2004. - S.785].

Grape comb extract has the following qualitative composition of biologically active substances:

Table. one
The results of the study of polyphenolic substances in a dry extract of grape ridges by high performance liquid chromatography NAME OF PCO HOLDING TIME RSO, MIN COMPONENTS OF THE STUDY SAMPLE IDENTIFIED
AT % Apigenin 92.58 - Hyperoside 26.94 + Hyperoside 2.97 Hesperidin 33.25 + Hesperedin 0.66 Gall 6,202 + Gall acid acid 67.27 Quercetin 70.12 - Coffee shop 9,205 + Coffee shop acid acid 0.34 Luteolin 85.06 - Luteolin- 23.73 + Luteolin-7- 7-glycoside glycoside 23.23 Routine 28.13 - Routine 1.23 Epicatechin 72.54 - RNO - 10 Unidentified substances substance 1.57; Unidentified substance 1.58

Initially, prior to the registration of “KPGV”, studies were conducted using a liquid water-alcohol extract, which showed the possibility of attributing the products of grape ridges to effective agents that increase the body's resistance to a number of negative environmental factors, including alcohol abuse. It is shown that the results of these effects are the formation of an increased number of free radicals that have a destructive effect on the whole organism, and primarily on the liver [TN Lopatkina Alcoholic liver disease. New honey. Journal.-1995-No. 1.s.16-18; Khazanov A.I. On the issue of alcoholic liver damage. Grew up. honey. lead - 1998. - No. 1 p. 540-44 .; Lankin V.Z. and others. Bioantioxidants are universal medicines. Sat materials sympos. - “Antioxidants in the prevention and complex therapy of free radical pathologies, M. 2002]

Among the most active natural remedies that have antioxidant effects are polyphenolic fractions from viticulture products, in particular extracts from the brushes of grape fertility Kushnerova N.F. The antioxidant effect of the food supplement "Korrd-K" in alcohol intoxication // V International Conference "Bioantioxidant", Moscow, 1998: Sat. thesis. doc. - M., 1998. - P.312; [Kushnerova N.F. et al. Plant complexes of polyphenols - essential nutrition components when living in ecologically unfavorable regions ”, V International Congress“ Act. problems creating new leks. drugs of natural origin. " - SPb. - 2001. - p. 413-445]. Hepatoprotective effect is largely associated with free radical processes. Earlier studies have shown that grape fruit brush extracts have alkoprotective, hepatoprotective and adaptogenic effects / G. Bezdetko. Adaptogenic effect of caprim // Valeology: Diagnostics, means and practice of ensuring health. - Vladivostok: Dalnauka. - 1995. - issue 2. - S.178-197; Kushnerova N.F. The anti-alcohol effect of the food supplement "Korrd-K" // V Russian National Congress "Man and Medicine", Moscow, April. 1998: Sat thesis. doc. - M., 1998. - P.108; Gordeychuk T.N. The plant polyphenolic complex from grape ridges is a promising stress protector // VI International Congress "Actual Problems of Creating New Medicines of Natural Origin", - St. Petersburg, 2002. - S, 558-561; Lee S.E. The effect of caprim on the indicators of physiological regeneration of the liver and kidneys in conditions of chronic alcoholization // Valeology: Diagnostics, means and practice of ensuring health. - Vladivostok: Dalnauka. - 1995. - issue 2. - S.157-162].

In clinical and experimental studies, the effectiveness of using drugs based on extracts of grape brushes to prevent the negative effects of alcohol on the body has been proved [I. Brekhman et al. Some results of experimental studies and polygon tests of the anti-alcohol (antidependant) action of caprim. Valeology. Diagnostics, means and practice of ensuring health. - Vladivostok: Dalnauka. - 1995, - issue 2, - S.140-149].

Thus, previous studies and the experience of Russian scientific practice have shown that extracts from viticulture products, including extracts from brushes of grape fruit (crests), in particular polyphenol fractions, have antioxidant, hepatoprotective, alkoprotective and adaptogenic effects.

On the basis of “KPVV” dietary supplement “Korda-Parafarm” is registered, which is declared as an additional source of flavonoids, antioxidants and reduces the toxic effect of alcohol on the body.

Thus, in connection with the proven biological activity, the need for a highly effective extract of grape ridges is constantly increasing.

The objective of the present invention is to develop a method for producing from the crests of grapes an extract with a high (up to 98%) content of polyphenolic compounds. The advantage of this method is that it represents a real alternative to expensive methods of purification of extracts from viticulture products using column chromatography on polymer sorbents.

To solve this problem, a method for producing an agent having antioxidant activity is proposed, characterized in that the ripe fruit fruit grape (Vitis vinifera L.) is dried at a temperature of 50 ° C until a moisture content of 8-10% is reached, crushed to a particle size of 2-5 mm, then the raw material is extracted twice with a 55% aqueous solution of ethanol at room temperature at a ratio of raw materials-extractant 1: 1, then the extract obtained is evaporated at 60-70 ° C and a pressure of 0.7 bar, the resulting condensed moisture extract is 60-70% repeatedly homogenized they are dried at 35-40 ° C and dried, then the extract is treated with Nefras gasoline to separate lipophilic compounds by heating to boiling and stirring, and then the extract is treated with water at a ratio of 1: 0.5 and water temperature up to + to separate water-soluble components 5 ° C with stirring, then the suspension is filtered and the precipitate is dried, the content of the sum of polyphenols in the target product is 98.66%.

In this case, the condensed extract is homogenized 4 times for 30 minutes, and drying is carried out in spray dryers at a temperature inside the dryer of 175 ° C, outside 89-95 ° C for 1-2 hours.

The method is as follows. The grapes of cultivated grape (Vitis vinifera L.) harvested after reaching maturity are separated from the berries on comb-crushers. Thus obtained grape fruit brushes are placed in drying chambers and dried at a temperature of 50 ° C until a moisture content of 8-10% is reached. Dried raw materials are crushed in feed crushers to a particle size of 2-5 mm. It was determined that this particular particle size of the grape-fruit brushes allows to obtain the maximum yield of polyphenolic compounds, since the larger particles impede the extraction process, and the smaller ones form a paste-like mass with an extractant, which exerts hydraulic resistance, as a result a large amount of ballast substances. The obtained plant raw materials “brushes of cultivated grape fruit, crushed” are controlled by organoleptic and physico-chemical indicators for compliance with the standards and requirements of GOST 7208. In addition, to ensure the proper amount of biologically active substances in the final product, the content of the amount of polyphenols is determined in the feedstock (should be at least 5%).

The grape fruit brushes are subjected to extraction with a 55% aqueous solution of ethanol at room temperature in two cycles in extractors of any type suitable for these operations.

First cycle. The ratio of raw materials and extractant for the first extraction cycle: 10 kg of dry raw materials per 10 l of an aqueous solution of ethanol. The output after the first cycle is 7.5 liters. The resulting drain is transferred to a second extractor, which contains 10 kg of new raw materials. The volume of ethanol is adjusted to 10 liters. The extraction time is determined by intermediate analyzes.

Second cycle. 10 liters of 55% alcohol are poured into the first extractor on the raw materials left after the first cycle and the whole cycle is repeated. At the conclusion of the extraction process, both solutions (obtained after the 1st and 2nd cycles) merge. Then they are filtered through standard filters and merged into metal (or from titanium) containers, where they remain until the next process.

Evaporation is carried out in evaporators of the LUWA type for rotational evaporation (with a centrifuge) or in other domestic similar evaporators. Evaporation temperature 60-70 ° C, pressure 0.7 bar.

Evaporation is carried out until the humidity of the extract reaches 60-70%. A condensed intermediate extract is obtained. To obtain a homogeneous mass, the condensed extract is placed in a homogenization tank. Homogenization is carried out 4 times for 30 minutes. The temperature at which the process is 35-40 ° C. Drying is carried out by spraying in standard spray dryers such as NIRO or domestic counterparts. The temperature inside the dryer is 175 ° C, outside 89-95 ° C. Duration of drying is 1-2 hours.

table 2
INDICATORS OF QUALITY AND SAFETY OF THE READY EXTRACT: 3.1. The taste of bitterness Taste astringent with light bitterness 3.2. The smell of tannins Pronounced with a distinctive aroma 3.3. Appearance Homogeneous mass of brown color 3.4. Humidity 2.0-4.0% 3.5. Ash content <20.0% 3.6. The content of ash insoluble in N% <4.0 3.7. pH 6-7 3.8. Authenticity According to TU 9176-050 - 46865780-04 3.9. Mass fraction of heavy metals There 3.10 Radionuclides There 3.11 Microbiological indicators There

The extract thus obtained contains 30 ± 3% of polyphenolic compounds. These compounds determine the biological activity of the extract, being strong antioxidants. To improve the pharmacological activity of the extract, it is necessary to increase the content of polyphenolic compounds in the extract.

A patent search showed that one of the methods for cleaning ballast substances from extracts from various viticulture products: seeds, bagasse, fresh grape fruits (not ridges), characterized by a high content of polyphenolic compounds (proanthocyanidins), the monomers of which are catechins, is as follows. The raw material is extracted with 50% ethyl alcohol by maceration, dried and an extract containing 3% proanthocyanidins is obtained. Next, the extract is dissolved in 50% ethanol (1.5 kg of extract in 10 liters of solvent) is filtered through a glass filter and subjected to deep purification using column chromatography on sorbents: "SEPHADEX LH-20" or "DIAION HP20". The method uses a chromatographic column 32 cm long, 30 cm in diameter. Initially, the column with the extract is washed with 100 liters of water, and then with 100 liters of ethanol, as a result of 25 kg of raw material, 105 grams of the extract with a content of 97% polyphenols are obtained [Lipoprotein lowering agent, cholesterol lowering agent, and medicaments containing these agents respectively / Application No. 9518644, MKI A61K 35/78. Kodo Kazuo, Itakura, Hiroshige, Koda. Claim 11/27/1995. Publ. May 29, 1999 No. 6129940]. This method of purification of the extract is very expensive.

In addition, the extraction of polyphenolic compounds from grape products, for example grape seeds, can be carried out with purified water. The resulting extract was subjected to ultrafiltration through an FP-200 membrane, then the solution was passed through a column with a polymeric support XUS-43520.00 (divinylbenzene copolymer) and the extract was finally purified using reverse osmosis [Method for extraction of proanthocyanidins from plant material / Application No. 08919508. MKI C 07D 311/62. Karim Nafisi-Movaghar, Tomas T. Svanoe, William A. Sero. Stated August 29, 1997. Published 06/15/1999, No. 5912363]. This method requires the use of expensive equipment.

To achieve this goal, the obtained (primary) extract was subjected to research on its purification. The grape crest extract has a dark brown color, is hygroscopic (it picks up moisture in the air and clumps), it is partially soluble in 95% alcohol and in water, but it is completely soluble in 50-60% aqueous alcohol.

When several drops of a 3% solution of iron (III) chloride are added to the aqueous-alcoholic solution of the extract, a greenish solution is formed, which indicates the presence of substances of the catechin group in it.

UV spectra of an aqueous-alcoholic solution of the extract and the standard substance: (+) - catechin in the region of 250-350 nm practically coincide. That allows, when determining the amount of polyphenols, to use (+) - catechin as a standard sample and to recount it.

In the process of further research, methods were developed for quantitative determination of the sum of polyphenols in terms of (+) - catechin.

At the same time, important attention was paid to the quantitative assessment of the content of the polyphenolic fraction in the feedstock, intermediate, and final (purified) extract.

According to the analysis, the content of the sum of polyphenols is in

* raw materials - 10.65%,

* in the intermediate dry extract - 29.5%,

which correlates with the yield of extractives (31%).

Development of a method for increasing the content of the polyphenolic fraction of the initial extract

In order to enrich the extract, attempts were made to clean it of related substances by using various domestic sorbents (silica gel, alumina (III), polyamide, etc.). It was found that the bulk of the polyphenols contained in the extract irreversibly precipitated on sorbents.

Based on the above studies, the extract was purified by column chromatography. As the sorbent used "SEPHADEX LH-20", which allows you to separate high-molecular substances, being essentially a "molecular sieve".

When passing the extract of grape ridges in the form of an aqueous-alcoholic solution through "SEPHADEX LH-20", a product was obtained (after evaporating the eluate to a dry residue) with a total polyphenol content of 33.70%, which indicates the low efficiency of using this sorbent for this type grape extract.

Further experiments showed that the main components of the extract (polyphenols) are insoluble in chloroform and other non-polar solvents, and also limitedly soluble in water at low temperatures.

In this regard, the isolation of polyphenols from the initial extract was carried out as follows.

Department of lipophilic compounds

The use of chloroform for the purification of plant extracts due to its high toxicity creates a number of difficulties in its use. The main one is the regulation and strict control of the residual amounts of this solvent in the final product (purified extract). An alternative to chloroform is gasoline of the NEFRAS brand, widely used in the food industry for the extraction production of vegetable oils, which is similar in its lipophilic properties to chloroform.

10.0 g of the extract was placed in a flask with a capacity of 50 ml, filled with 30 ml of NEFRAS, the reflux condenser was connected and heated to boiling under continuous stirring on a magnetic stirrer for 15 minutes. After cooling, the contents of the flask were filtered under vacuum through a No. 2 porous glass filter (pore 41), the filter residue was washed with 20 ml of gasoline, and then dried in air. After distilling off the solvent, 0.2266 g of a residue containing chlorophyll and other lipophilic substances was obtained. The yield of lipid residue is 2.266%.

Separation of water soluble components

The lipid-free extract was placed in a 50 ml flask and filled with 25 ml of water cooled to + 5 ° C. The contents of the flask were stirred using a magnetic stirrer for 20 minutes, then the flask with the contents was placed in a refrigerator and kept at a temperature of about + 5 ° C for 30 minutes. After that, the contents of the flask (suspension) were filtered through a paper filter under vacuum. The filter cake was washed with 10 ml of water cooled to + 5 ° C and dried first in air and then in a desiccator over anhydrous calcium chloride.

Received 2.30 g of dry residue. The yield from the start of the process was approximately 68%. The loss of basic substances at the stage of lipid separation was about 2%, and at the stage of washing with water about 30%.

The content of the amount of polyphenols in the final extract is 98.66%.

Thus, the proposed method, which increases the content of the main active fractions (polyphenols) of the extract of grape ridges, allows to obtain a highly effective extract for the pharmaceutical production of biologically active food additives, drugs, as well as for the needs of the cosmetic and food industries.

One of the most important properties of extracts from viticulture products, including extracts of viticulture ridges, is an antioxidant effect. Free radical processes are the dominant factors in damage to the cellular structures of the liver, cardiovascular and central nervous systems, and others. The harm done to the body by alcohol abuse is largely associated with an increase in free radicals. The study of the antioxidant activity of the initial and purified extracts used in the production of dietary supplements "Korda-Parapharm" was carried out using in vitro enzyme biotest systems developed in VILAR and patented in the Russian Federation (patents No. 218190, 218191, 218192). The results of the effect of extracts of brushes of unplaned and peeled grape fertility brushes are presented in table 3.

Table 3 The effect of dry grape extracts on the rate of glutathione reductase (GR) and catalase (CAT) reactions in vitro Experience option GR rate μmol / min per mg protein Percentage Relative (%) CAT rate μmol / min per mg protein Percentage Relative (%) Control (no additives added) 9.22 ± 0.10 100.0 ± 1.08 19.16 ± 1.05 100.0 ± 1.52 Extract 1: 5 mcg / ml 9.21 ± 0.41 99.9 ± 4.45 30.99 ± 1.09 49.9 ± 3.52 10 mcg / ml 9.43 ± 0.39 100.2 ± 4.14 28.74 ± 0.74 46.2 ± 3.35 15 mcg / ml 9.49 ± 0.27 100.8 ± 2.57 22.07 ± 0.97 35.5 ± 4.39 Extract 2: 5 mcg / ml 9.43 ± 0.47 102.3 ± 4.98 45.68 ± 0.89 73.5 ± 1.95 10 mcg / ml 9.60 ± 0.43 104.1 ± 4.48 44.69 ± 0.57 71.9 ± 1.28 15 mcg / ml 10.57 ± 0.19 114.6 ± 1.80 43.96 ± 0.96 70.7 ± 2.18

From table 3 it can be seen that extracts 1 and 2 increase the rates of glutathione reductase and catalase reactions, which indicates their antioxidant activity. A quantitative assessment shows the presence of a significantly greater antioxidant activity in extract 2. Thus, the purified grape fertility brush extract, which has a high polyphenol content (up to 98%), has a more pronounced antioxidant activity.

Claims (3)

1. A method of obtaining an extract from brushes of fruit grapes of a cultural grape with antioxidant activity, characterized in that the mature fruit grape of grape cultivars (Vitis vinifera L.) is dried at a temperature of 50 ° C until a moisture content of 8-10% is reached, crushed to a particle size of 2-5 mm, then the raw material is extracted twice with a 55% aqueous solution of ethanol at room temperature with a raw material-extractant ratio of 1: 1, then the extract obtained is evaporated at 60-70 ° C and a pressure of 0.7 bar, the resulting condensed moisture extract is 60-70% repeatedly homoe They are insulated at 35–40 ° С and dried; then, to separate lipophilic compounds, the extract is treated with Nefras gasoline by heating to boiling and stirring, and then, to separate water-soluble components, the extract is treated with water at a ratio of 1: 0.5 and water temperature up to 5 ° C with stirring, then the suspension is filtered and the precipitate is dried, the content of the sum of polyphenols in the target product is 98.66%. 2. The method according to claim 1, where the condensed extract is homogenized 4 times for 30 minutes 3. The method according to claim 1, where the drying is carried out in spray dryers at a temperature inside the dryer 175 ° C, outside 89-95 ° C for 1-2 hours