SI21460A - Process for preparation of concentrated extracts based on rosemary acid and their use - Google Patents

Abstract

The procedure comprises the following: a) grinding of dried plant material from the mint family (Labiatae) in inert atmosphere; b) water or organic solvent extraction of ground material obtained under a) and/or marc obtained after extraction of oil-soluble antioxidants from plants of the mint family (Labiatae); c) evaporation of organic solvent; d) concentration of the product obtained by traditional extraction, by ion exchange chromatography; e) extraction of rosemary acid by liquid-liquid extraction; f) concentration of product by elution chromatography; g) concentration of product based on rosemary acid by application of high pressure gases, using a cosolvent optionally. Products are applicable in antioxidative and antimicrobial agents.

Description

Technical field of the invention

The present invention relates to a process for the preparation of concentrated extracts of rosemary acid from plants of the mouth (Labiaiae) family and to their use in antioxidant and antimicrobial agents.

The state of the art

Labiatae are one or more perennial herbs, semi-shrubs or shrubs. The family of around 3500 plants is widespread in all climates, especially in the Mediterranean and Asia. Some of the most famous plants of this family are: rosemary, sage, thyme, peppermint, lavender, oregano and lemon balm.

High levels of isoprenoid substances in the form of monoterpenes (essential oils), sesquiterpenes, diterpenes (bitterness) and triterpenes are observed in the lips. In addition, the lips also contain many phenolic compounds.

Plants of the oral family are known for their antioxidant activity, which is especially true of rosemary and sage. Essential oil causes the undesirable odor and taste of antioxidant extracts, while at the same time some components of the essential oil act pro-oxidatively, so it is desirable to keep the essential oil content of these extracts as low as possible.

Rosemary extract contains a variety of active components such as carnosic acid, camosole, methyl-kamosate and rosin acid,

Rosemary acid is a caffeic acid depside and α-hydroxyhydro caffeic acid. It is one of the most famous esters of coffee acid, found mainly in the plants of Lamiaceae and Boriginaceae. It plays a role in the prevention of bacterial infections in plants. The content of rosemary acid in plants is between 0.07 and 0.84%.

Formula I: Structure of Rosemary Acid.

Rosemary acid is in the form of a white powder with a melting point T _m = 174 ° C and an absorption maximum at λ _ιηΗΧ = 288, 326 nm. It is well soluble in water. Rosemary acid has antioxidant properties, prevents lipid oxidation and destroys cell membranes. In addition to its antioxidant activity, it also has anti-inflammatory, anti-viral and antibacterial properties.

Plant phenols are a characteristic group of secondary metabolites that have a range of potential uses in medicine. Plants of the oral family are an important source of pharmaceutical ingredients of the phenolic type. The therapeutic use of these components has been known for many centuries in many cultures of the world. Many articles mention the use of phenolic compounds for various medical purposes. Examples are shown in Table 1.

Table 1: Examples of the use of plants of the oral Labiatae family in medicine.

PLANTS METABOLIT APPLICATION Hyptis verticillata rosemary acid in the stomach and intestines problems lavender (Lavandula spp.) rosemary acid anti-inflammatory action Railway (Lithospermum rosemary acid, anti-inflammatory and erythorhizon) lithosperic acid antigonadotropic action oregano (Origanum vulgare) galangin and rosemary acid antimutagenic action orthosiphon (Ortosiphon ari status) rosemary acid diuretic and anti-inflammatory operation Rosemary (Rosmarinus officinalis) rosemary acid anti-inflammatory action sage (Salvia multiorrhiza) salvianolic acid against wounds, especially on the duodenum and stomach thyme (Thymus vulgaris) timol antioxidant and antiquarian action

At the same time, cholecretic, antiseptic, astringent and hypoglycemic activity, use in bacterial infections and urinary tract inflammation have been mentioned in the literature. Native cultures used rosemary acid for skin infections.

Numerous patents protect the use of rosemary acid in the food, cosmetic and pharmaceutical industries. The use of rosemary acid for the stabilization of meat and meat products is described in US Patent 6099879, which provides stabilization of meat and meat products with rosemary extract containing camosolic acid, camosole and rosemary acid prior to exposure to ionizing radiation, which promotes aroma and prolongs life. US 6020009 describes the stabilization of rosemary acid meat flavor, and CA 2353536 states the use of rosemary acid to stabilize the oxidation of unstable materials (catalysts, colorants, flavorings, proteins, cell cultures, bioactive components, biological extracts and enzymes). The stabilization of the substance subjected to oxidation with rosemary acid derivatives is covered by patent W00039248.

Patent type (DE10028638, US6383543, US4857325, US6274124 and US6231877) states the use of rosemary acid for the oxidative stabilization of cosmetic preparations, for the treatment and prevention of inflammatory processes on the skin (FR 2652001), for anti-aging and drying of the skin (JP 9067251, for US 53935261, and for US 53935261) treatment of viral skin disorders where rosemary acid is used as a virucide (CA 2079867). Rosemary acid-based extract or mixture thereof, used for the treatment of peptic ulcer (DE 3247610, CN 1153778), for the prevention of immune reactions in transplanted organs, for the prevention and / or diagnosis of rejection of transplanted organs, autoimmune and inflammatory diseases (US 6140363) .

US 4329361 discloses the use of rosemary acid for the treatment of inflammatory diseases and US 4358442 and IE 802588 for arteriosclerosis and for the stimulation of the immune system.

Several patents (US 4012531, US 5908650, and CA 2269288) describe the use of rosemary acid to stabilize dyes (carotenoids, anthocyanins, chlorophyll), fruit juices, and powdered red beet extract, and US patent 6306450 describes citrate stabilization, key components of citrus and citrus juices.

A technical problem

None of the patents known to us mentions the antimicrobial activity of rosemary acid. action on the following micro-organisms: Bacillus cereus, Staphylococcus epidermidis and Escherichia coli and Aspergillus niger, Penicillium cyclopium, Pityrosporum ovale and Saccharomyces cerevisiae. At the same time, none of the cited patents addresses the possibility of using rosemary acid to stabilize fat emulsions.

In general, the prior art processes for the isolation of rosemary acid by conventional extraction of plant material at different temperatures are carried out. Extraction solvents are alcohols, water and their solutions. The procedures are described in the patents

CN 1153778, US 4012531, US 6383543, and JP 9067251). Higher concentrations of rosemary acid in the extracts are obtained by chromatographic procedures on different carriers using conventional solvents (US 5908650). Patent DE 3247610 describes the production of rosemary acid from cell cultures in an aqueous medium.

Our patent Sl 9600104 deals with the isolation of natural antioxidant components (camosole, camosolic acid, rosemary, rosemary defenol and rosemary quinone) from the lips using supercritical fluids. Rosemary acid isolation is not mentioned in this patent.

Description of solution to a technical problem with implementation examples

The subject of the invention is a new process for the preparation of concentrated extracts of rosemary acid from plants of the mouth (Labiatae) family and their use in antioxidant and antimicrobial agents. We can also use as a starting material the pomace obtained from the extraction of oil-soluble antioxidants from plants of the mouth (Labiatae) family.

The process of the present invention comprises the following steps.

a) Grinding of dried plant material from plants of the Labiatae family in an inert atmosphere. Extraction efficiency depends on the granulation of the starting material, because the finer the material, the larger the contact surface with the solvent. The preferred granulation of the material is 0.1-5 mm.

b) Extraction with water or. organic solvents (alcohols, esters, ethers, aldehydes, ketones, hydrocarbons, acids, bases and their solutions and mixtures) in the case of a) ground material and / or marc obtained from the extraction of oil-soluble antioxidants from plants of the Labiatae family. The solvent / extraction ratio is 1/10. The temperature is between 20 ° C and the boiling point of the solvent or. mixtures, extraction times are between 0.5 and 24 hours. By extraction with water or. organic solvents are given a crude product containing a low concentration of rosemary acid, so further concentration is recommended.

c) Evaporation of the organic solvent.

d) Concentration of the product obtained by conventional extraction with ion exchange chromatography.

XAD 1180, XAD-16, XAD1600, IRA 900 LL, DIAION SA 11 A, DIAION PA 308, XAD-2 and DIAION IIP-20 ion exchangers are used as stationary phase. alcohols (ethanol, methanol) and their mixtures with water are the mobile phase.

e) Rosemary acid extraction by liquid-liquid extraction.

After concentration by ion-exchange chromatography, further concentration of the rosemary acid extract is carried out by liquid-liquid extraction, using esters (such as ethyl acetate, butyl acetate, etc.) or ethers (such as diethyl ether, isopropyl ether, etc.) as the organic phase. ).

f) Concentration of the product with elution chromatography.

The concentrated rosemary acid-based product obtained after the liquid-liquid extraction was further concentrated using elution chromatography. Estimates (such as ethyl acetate, butyl acetate), ethers (such as petroleum ether) and mixtures thereof are used as the mobile phase for elution chromatography, and silica gel or alumina are used as the stationary phase.

g) Concentration of rosemary acid-based product using high pressure gases. The rosemary acid-based dried product obtained after liquid-liquid extraction is further concentrated using high pressure gases. The following gases are used as high pressure extraction media: CO2, hydrocarbons having 1-4 carbon atoms, freons or mixtures thereof. The pressures of concentrating the crude product with gases are from 10 to 500 bar and temperatures from 0-80 ° C. High-pressure extraction gas consumption is between 1 and 500 kg of gas / kg of extracted substance. In concentrating the high-pressure extraction of the rosemary acid-based crude product, a co-solvent may be used, which may be water, alcohol, ester, ether, aldehyde, ketone, hydrocarbon, acid, base and their solutions.

-7When carrying out a variant of the process for the preparation of rosemary acid concentrate, where only the pomace obtained from the extraction of the oil-soluble antioxidants of plants of the oral family is used as the starting material, omit step a), that is, the grinding of the raw material.

It is a further object of the invention to use the products of the above process for the preparation of agents for slowing the oxidation of fat in an emulsion. Concentrations of 0.02-1% of the products in the agents are preferred.

It is a further object of the invention to use the products of the above process for the preparation of antimicrobial agents for the protection of the products against the following microorganisms: Bacillus cereus, Staphylococcus epidermidis and Escherichia coli and Aspergillus niger, Penicillium cyclopium, Pityrosporum ovalere and Saccharome ovalere and Saccharomia ovale and Saccharomia ovale and Saccharomia ovale and Saccharomia ovale and Saccharomia ovale and Saccharomia ovale and Saccharomia ovalere and Saccharomia ovale and Saccharomia ovale and Saccharomia ovale and Saccharomia ovale.

It is a further object of the invention to use the products of the above process mixed with other components (such as camosolic acid, ursolic acid and oleanolic acid) for the preparation of agents with synergistic antioxidant and antimicrobial action.

The new process involves the use of supercritical fluids to concentrate with conventional extraction of the obtained rosemary extract based on rosemary acid. The product does not contain solvent residues, has improved flow properties, increases the content of the active component, and removes waxes and aromas that cause unwanted odor and reduce its activity.

EXAMPLE 1

Grinding

The dried leaves of rosemary, sage, lemon balm, oregano, thyme or any other plant of the Labiatae family are ground in an inert atmosphere. As a raw material, pomace can also be used after extraction of oil-soluble antioxidants. 100 g of drug (median grain = 310 pm, most commonly represented grain = 98 pm) extracted with

1000 ml of water, organic solvent or mixture at 20 ° C for 2 hours. The extraction mixture was stirred with a magnetic stirrer (n = 5000min ^-i ). The marc was filtered off and the solution was evaporated on a rotary evaporator at 50 ° C and 150 mbar to dryness or at 20% initial volume to obtain a product with a higher concentration of rosemary acid. The results of conventional rosemary extraction are shown in Table 2.

Table 2: Isolation of rosemary active ingredients using conventional solvents

Method and operating conditions Solvent % RK in the extract Profit extraction% CLASSICAL EXTRACTION Particle size = 0.315-0.500 mm 0.9% RC - Input mat. acetone 0 1,990 th most common 10,400 10 2,276 th most common 18.33 20 3,472 th most common 18.30 35 4,930 th most common 17.73 50 6,106 th most common 17.30 70 5,297 th most common 17.53 96 2,765 th most common 10,33 ethanol 0 1,990 th most common 10.40 10 2,059 th most common 17,43 20 2,788 th most common 17,67 35 3,293 th most common 17.70 50 5,101 th most common 16.13 70 4,435 th most common 16.93 96 2,536 th most common 12.33 ethyl acetate ' 0 1,990 th most common 10.40 10 2,206 th most common 23.60 20 1,865 th most common 17.50 35 0,521 th most common 18,70 50 0.284 21,93 70 0,163 20.33 96 0.030 13,00 methanol 0 1,990 th most common 10.40 10 1,374 th most common 16.10 20 1,474 th most common 18,00 35 2,563 th most common 17,47 50 4,536 th most common 16.77 70 5,324 th most common 16.57 96 2,205 th most common 16.63

The partially evaporated extract was acidified with phosphoric acid at pH = 2-3 and concentrated by ion-exchange chromatography. The following ion exchangers XAD 1180, XAD-16, XAD-1600, IRA 900 LL, D1AION SA 11 A, DIAION PA 308, XAD-2 and DIAION HP-20 were used as stationary phase and 0.25% (w as eluent). / w ^=: w / w) NaOH in 85% methanol. The concentration of rosemary acid in the product using different ion exchangers is shown in Table 3. Evaporate the eluate to 10% of the initial volume on a rotary evaporator at 50 ° C and 150 mbar. The resulting solution was acidified to pH = 2 and extracted 3 times with 50 ml of organic solvent each. The organic phases are combined and evaporated to a viscous liquid on a rotary evaporator at 40 ° C and a pressure of 150 mbar. The crude product obtained is concentrated by elution chromatography on silica gel as a stationary phase. A mixture of organic solvents is used as the eluent. The crude product obtained is concentrated using supercritical fluids under the conditions set out below. The product treated with supercritical fluids is free of the undesirable odor of ethereal oil, contains no organic solvent residues and has improved flow properties.

Table 3: Concentration of rosemary active substances by ion exchange chromatography

Method and operating conditions The carrier RK (%) in the product Ionic-edit evaluation XAD 16 27,656 XAD2 33,875 th most common AMBERLITE XAD 1180 27,693 chromatography AMBERLITE XAD 1600 25,132 RK concentration in the inlet solution = 0.300% AMBERLITE IRA 900 LL 20,450 th most common DIAION SA11A 17,719 th most common DIAION PA 308 31,708 th most common DIAION HP 20 21,450 th most common

Extraction with supercritical fluids (carbon dioxide, propane and dimethyl ether)

Fill the autoclave with a material and pump a certain amount of solvent per gram of starting material (50, 100 or 150 g) using a high-pressure pump. The extract is trapped in a glass tube (trap) where, by reducing the working pressure to normal, all the solvent is evaporated and the solvent analyzed is dropped. The extraction residue remaining in the autoclave is also analyzed.

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Using a variety of co-solvents in supercritical fluid extraction, we tried to obtain a product with the highest content of rosemary acid.

Extraction with CO?

Extraction of the rosemary extract based on carbon dioxide with carbon dioxide at pressures of 100 to 300 bar and a temperature of 30 ° C was carried out. Rosemic acid itself was not present in the extract itself, mainly fats and colorants were extracted, and the content of rosemary acid increased in the extraction residue. The addition of the co-solvent to carbon dioxide did not increase the content of rosemary acid in the extraction residue relative to the use of pure carbon dioxide. The largest increase in the content of rosemary acid in the extraction residue was obtained by extraction with pure carbon dioxide, without a boiler, at a pressure of 300 bar and at a temperature of 30 ° C. The results of high pressure carbon dioxide extraction are shown in Table 4.

Table 4: Results of high pressure carbon dioxide extraction.

Extraction conditions Solvent % RK v extract % RK in ext. the rest 300 bar, 30 ° C 30.58% RC - Input Mat. co ₂ - 39,00 300 bar, 30 ° C 4.01% RC - Input Mat. CO ₂ + ethyl acetate: petroleum ether = 6: 4 - 6.37 300 bar, 30 ° C 30.58% RC - Input Mat. CO ₂ + acetone - 30.50 300 bar, 30 ° C 33,9% RC - input mat. CO ₂ + petrol ether - 37,80 300 bar, 30 ° C 33,9% RC - input mat. CO ₂ + ethyl acetate - 36,30

Concentration of rosemary active ingredients using propane

Extraction of rosemary with different content of rosemary acid was concentrated on the extraction apparatus using propane. The extraction was carried out at pressures of 60 and 100 bar and a temperature of 30 ° C. Propane flow was between 4 and 40ml / min. After done

-1111 extraction The content of active components in the extract and the extraction residue were measured.

Table 5: Concentration of rosemary active ingredients using propane

% KK % K % RK Input material 19,805 th most common , 14,943 2,229 th most common P-100 bar, T = 30 ° C extract 48,424 th most common 1,624 th most common - the rest 7,601 th most common 1,783 th most common 2,159 th most common P = 60 bar, T = 30 ° C extract 90,555 th most common 2,560 th most common the rest 21,772 th most common 12,824 th most common 2.380

Concentration of rosemary active ingredients using dimethyl ether

On a dynamic apparatus, rosemary extract with different rosemary acid content at pressures of 100 to 200 bar and temperatures of 30, 60 and 80 ° C was concentrated using dimethyl ether. The dimethyl ether flow rate was between 4 and 40 ml / min. After extraction was carried out, the content of the active components in the extract and the extraction residue were measured.

Table 6: Concentration of rosemary active ingredients using dimethyl ether operating parameters / I

Method and operating conditions % RK in the extract P (bar) 30 ° C 60 ° C 80 ° C Supercritical extraction with DME 13% RC - input mat. 50 g DME / g mat. 100 150 200 15,844 th most common 9,739 th most common 13,928 th most common 9,739 th most common 8,372 th most common 8.63 - 100 g DME / g mat 100 150 200 4,341 th most common 11,566 th most common 6,987 th most common 8,592 th most common 9,720 7,926 th most common 10,206 th most common 150 g DME / g mat 100 150 200 10,612 th most common 12,848 th most common 6,117 th most common 10,267 th most common 12,147 th most common 6,307 th most common 13,088 th most common

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Table 7: Concentration of rosemary active ingredients using dimethyl ether operating parameters / II

Method and operating conditions % RK P (bar) extract ext. the rest nek extract ext. the rest nek 30 ° C 60 ° C Supercritical extraction with DME 8.72% RK Input Mat. 90 g DME / g mat. 100 150 200 18,58 14,12 19.30 8.37 8.65 19.30 13.07 10,33 15.70 8.17 8.21 7.98 100 g DME / g mat 200 18,94 8.53 - 150 g DME / g mat 200 19,20 8.16 - 200 g DME / g mat 200 20.03 8.12 - 100 g DME / g mat + 1 ml ethyl acetate / min 200 12,37 - - 100 g DME / g mat + 2 ml ethyl acetate / min 200 14.14 - -

Supercritical fluid chromatography

The essence of the research was to prepare a baseline or to determine the conditions for supercritical preparative chromatography. The process of separation of individual components was optimized by changing the process parameters: temperature, pressure, flow and boiler and the detector wavelength.

The process can be optimized by separating an RK sample containing 300 mg of 35% rosemary acid in 50 ml of acetonitrile. The operating conditions of the chromatography were a pressure of 180 bar and a temperature of 65 ° C. Rosemary acid separated from the other impurities only in the presence of cotopil, which in our case was acetonitrile, at a flow rate of 0.2 ml / min, the stationary phase was RP 18.

The antioxidant efficiency of the emulsion was determined in the β-carotene-linolenic acid system, which was added to the products obtained by the above procedures. The method is based on heat-induced oxidation of β-carotene-linolenic acid emulsion.

To determine the antimicrobial activity of rosemary acid on non-pathogenic and pathogenic microorganisms, we used the method of microbial growth on

-1313 agar. Tests were conducted on bacteria or Bacillus cereus. Staphylococcus epidermidis and Escherichia coli, and fungi Aspergillus niger, Penici l Hum cyclopium, Pityrosporum ovale and Saccharomyces cerevisiae. The radial growth of mold on PDA agar containing 1 ml of alcoholic solution of the active substance was measured daily. Because ethanol itself has antimicrobial activity, a PDA plate with 1 ml of 96% ethanol was taken as an alcohol control. At the same time, control with alcohol-free agar and active substance was prepared. The plates were incubated for 10-17 days at 28 ° C and in the dark.

EXAMPLE 2

The dried leaves of rosemary, sage, lemon balm, oregano, thyme or any other plant of the Labiatae family are ground in an inert atmosphere. Extract 100 g of the drug with 1000 ml of 35% acetone at 20 ° C for 2 h. The extraction mixture was mixed with a magnetic stirrer (n = 5000mi ¹ ). The marc were filtered off and the solution was evaporated to dryness on a rotary evaporator at 50 ° C and a pressure of 150 mbar. 14 g of the extract are obtained which is determined by the antioxidant efficiency of the emulsion. The obtained extract 25.7% inhibits the oxidation of linoleic acid.

EXAMPLE 3

We worked in analogy to Example 2, except that instead of antioxidant efficiency, the fungicidal action on Aspergillus niger was determined in the extract emulsion. The radial growth of mold on PDA agar containing 1 ml of alcoholic solution of the extract was measured daily. Because ethanol itself has antimicrobial activity, a PDA plate with 1 ml of 96% ethanol was taken as an alcohol control. At the same time, control with alcohol-free agar and active substance was prepared. The plates were incubated for 10-17 days at 28 ° C and in the dark.

The results of measuring the fungicidal properties of rosemary acid (sample 1) on Aspergillus niger are shown in Table 13. The extract at a concentration of 0.5% 44.4% inhibits the occurrence of said mold, and at a concentration of 1% 50.93%.

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EXAMPLE 4

We worked analogously to Example 2, except to determine the fungicidal action on Penicillium cyclopium.

The results of measuring the fungicidal properties of rosemary acid (sample 1) on Penicillium cyclopium are shown in Table 14. The extract at a concentration of 0.5% 5.94% and at a concentration of 1% 26.64% inhibits the appearance of said mold.

EXAMPLE 5

We worked analogously to Example 2 except that instead of 35% acetone, 5-20% acetic acid was used as the solvent. We obtained 12.6 g of the extract, which was determined by its fungicidal action on Aspergillus niger.

The results of measuring the fungicidal properties of rosemary acid (sample 2) on the fungus Aspergillus niger are shown in Table 13. The extract at a concentration of 0.5% 41.04% inhibits the appearance of said mold, and at a concentration of 1% 56.72%.

EXAMPLE 6

We worked analogously to Example 5, except that instead of the fungicidal action of the extract, we determined the antioxidant efficiency in the emulsion. The obtained extract 46.4% inhibits the oxidation of linoleic acid.

EXAMPLE 7

We worked analogously to Example 5 except that the extract was determined to have a fungicidal action on Penicillium cyclopium.

The results of measuring the fungicidal properties of rosemary acid (sample 2) on Penicillium cyclopium are shown in Table 14. The extract at a concentration of 0.5% 6.15%, and at a concentration of 1% 100% inhibits the appearance of the said mold.

EXAMPLE 8

The dried leaves of rosemary, sage, lemon balm, oregano, thyme or any other plant of the Labiatae family are ground in an inert atmosphere. 100 g of drug (median grain = 310 pm, most commonly represented grain = 98 pm) was extracted with 1000 ml of 35% acetone at 20 ^c C for 2 h. The extraction mixture is mixed with magnetic

-1515 agitator (n-SOOOmer ¹ ). The marc were filtered off and the solution was evaporated on a rotary evaporator at 50 ° C and a pressure of 150 mbar at 20% of the initial volume. The extract was acidified with phosphoric acid to a pH ^of 2-3, and concentrated by ion-exchange chromatography. The following ion exchanger XAD 1180, ΧΑΠΙ 6. XAD-1600, IRA 900 LE, DIAION SA 11 A, DIAION PA 308, XAD-2 and DIAION HP-20 were used as stationary phase, and the eluent was 0.25% (w / w = wt / wt) NaOH in 85% methanol. Evaporate the eluate to 10% of the initial volume on the rotary evaporator at 50 ° C and a pressure of 150 mbar. The resulting solution was acidified to pH = 2 and extracted 3 times with 50 ml of ethyl acetate each. The organic phases are combined and evaporated on a rotary evaporator at 40 ° C and 150 mbar. 0.5 g of the extract was obtained with a concentration of rosemary acid of 35%, which was determined by its antioxidant efficiency in the emulsion. The resulting extract 28.5% inhibits the oxidation of linoleic acid.

EXAMPLE 9

We worked analogously to Example 8, instead of antioxidant efficacy in the emulsion, the product was determined to have a fungicidal action on Aspergillus niger.

The results of measuring the fungicidal properties of rosemary acid (sample 3) on the fungus Aspergillus niger are shown in Table 13. The extract at a concentration of 0.5% 63.62%, and at a concentration of 1% 66.23% inhibits the appearance of said mold.

EXAMPLE 10

We worked analogously to Example 8, except that the product was determined to have a fungicidal action on Penicillium cyclopium.

The results of measuring the fungicidal properties of rosemary acid (sample 3) on Penicillium cyclopium are shown in Table 14. The extract at a concentration of 0.5% 60.66%, and at a concentration of 1% 100% inhibits the appearance of said mold.

EXAMPLE

We worked in analogy to Example 1, except that the crude product obtained was further concentrated using supercritical fluids under the conditions listed in Tables 3, 4, 5 and 6. Approx. 0.4 g of purified extract with the concentration of rosemary acid 43% which we are

-1616 determined its antioxidant effectiveness in the emulsion. The obtained extract, 44.42%, inhibits the oxidation of linoleic acid.

EXAMPLE 12

We worked in analogy to Example 11, except that instead of antioxidant efficiency in the emulsion, the extract was determined to have a fungicidal action on the fungus Aspergillus niger.

The results of measuring the fungicidal property of rosemary acid (sample 4) on the fungus Aspergillus niger are shown in Table 13. The extract at a concentration of 0.5% 57.65%, and at a concentration of 1% 74.25% inhibits the appearance of said mold.

EXAMPLE 13

We worked analogously to Example 11, except to determine the fungicidal action on Penicillium cyclopium.

The results of measuring the fungicidal properties of rosemary acid (sample 4) on Penicillium cyclopium are shown in Table 14. The extract at a concentration of 0.5% 85.25%, and at a concentration of 1% 100% inhibits the appearance of said mold.

EXAMPLE 14

We worked in analogy to Example 1 except that the combined organic phases were evaporated to liquid viscous on a rotary evaporator at a temperature of 40 ° C and a pressure of 150 mbar after the liquid-liquid extraction. The crude product obtained is concentrated by flash chromatography on silica gel. A solvent mixture of 1: 1 ethyl acetate and petroleum ether is used as the eluent. The product was dried on a rotary evaporator and lyophilized.

We got approx. 0.2 g of purified extract with a concentration of rosemary acid approx. 70% of which antioxidant efficacy was determined in the emulsion. The obtained extract 25.84% inhibits the oxidation of linoleic acid.

EXAMPLE 15

We worked in analogy to Example 14, except that instead of antioxidant efficiency, the fungicidal action on Aspergillus niger was determined in the extract emulsion. Results of measurement of the fungicidal property of rosemary acid (sample 5) on Aspergillus

-1717 niger are shown in Table 13. The extract at a concentration of 0.5% 20.15%, and at a concentration of 1% 34.51% inhibits the appearance of the said mold.

EXAMPLE 16

We worked analogously to Example 14 except that instead of Aspergillus niger, fungicidal activity was determined on Penicillium cyclopium.

The results of measuring the fungicidal properties of rosemary acid (sample 5) on Penicillium cyclopium are shown in Table 14. The extract at concentrations of 0.5% and 1% 100% inhibits the appearance of the said mold.

EXAMPLE 17

We acted analogously to Example 1, except that we did not lyophilize the product. We got approx. 0.3 g of purified extract with a concentration of rosemary acid approx. 50% of which antibacterial activity was determined against Bacillus cereus, Escherichia coli or Staphylococcus epidermidis.

The results of measuring the antimicrobial activity of 50% rosemary acid are shown in Table 8. The results are calculated according to control, where only nutrient broth (without rosemary acid) was incubated with the addition of Bacillus cereus, Escherichia coli or Staphylococcus epidermidis. For comparison, Table 9 shows the antimicrobial activity of 100% rosemary acid. The results show that 100% rosemary acid is less active than 50%. It is concluded that admixtures in 50% rosemary acid synergistically affect its activity. Table 10 shows the effective doses that inhibit the reproduction of microorganisms by 50%.

EXAMPLE 18

We acted analogously to Example 1 except that fungicidal activity was determined on Pityrosporum ovale and Saccharomyces cerevisiae.

The results of measuring the fungicidal activity of 50% rosemary acid are shown in Table 11. The results are calculated from the control, where only nutrient broth (without rosemary acid) was incubated with the addition of the culture of Piivrosporum ovale and Saccharomyces cerevisiae. Table 12 shows the effective doses that inhibit the reproduction of these microorganisms by 50%.

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EXAMPLE 19

In Examples 7-25, the pomace can also be used as a feedstock for extraction after pre-day extraction of oil antioxidants.

Table 8: Results of measurement of antimicrobial activity of 50% rosemary acid.

BACTERIA CONCENTRATION % BACTERIOCIDITY Bacillus cereus 0.1 g / l 21 + 6 0.2 g / l 43 ± 11 Staphylococcus epidermidis 0.4 g / l 18 ± 7 0.6 g / l 58 + 27 Escherichia coli 6 g / l 58 ± 18

Table 9; Results of measurement of antimicrobial activity of 100% rosemary acid.

BACTERIA CONCENTRATION % ERIOCIDITY BACT Bacillus cereus 1.5 g / l 0 7 g / l 90.1 Staphylococcus epidermidis 3 g / l 0 7 g / l 94 Escherichia coli 7 g / l 21.9 9 g / l 87,1

Table 10: Effective dose (ED50) of rosemary acid (50% and 100%), which inhibits the reproduction of microorganisms by 50%. In the case of 50% rosemary acid, the results are given as the mean + standard deviation of 3-6 measurements.

BACTERIA ED ₅₀ (g / l) 50% Rosemary Acid 100% Rosemary Acid Bacillus cereus 0.24 ± 0.05 4.5 Staphylococcus epidermidis 0.57 ± 0.07 5.1 Escherichia coli 5.2 ± 1.8 7.8

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Table 11: Results of measurement of fungicidal action of 50% rosemary acid.

Fungus CONCENTRATION % FUNGICIDITY Pityrosporum ovale 14 g / l 28 ± 19 15 g / l 66 ± 26 Saccharomyces cerevisiae 3 mg / l 91.2 ± 6.2

Table 12: Effective dose (ED50) of rosemary acid (50% and 100%), which inhibits the reproduction of microorganisms by 50%. In the case of 50% rosemary acid, the results are given as the mean ± standard deviation of 3-6 measurements.

Fungus ed ₅ "(g / l) 50% Rosemary Acid 100% Rosemary Acid Pityrosporum ovale 14.6 + 1.2 / Saccharomyces cerevisiae 2.55 ± 0, ll /

Table 13: Results of measurement of fungicidal activity of rosemary acid on Aspergillus niger

SAMPLE CONCENTRATION % FUNGICIDITY SAMPLE 1 0.5% 44,4 1% 50.93 SAMPLE 2 0.5% 41.04 1% 56.72 SAMPLE 3 0.5% 63.62 1% 66,23 SAMPLE 4 0.5% 57.65

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1% 74.25 SAMPLE 5 0.5% 20.15 1% 34.51 SAMPLE 6 0.5% 17,12 1% 31.13 SAMPLE 7 0.5% 47.83 1% 72.15 SAMPLE 8 0.5% 42.15 1% 71.35

Table 14: Results of measurement of fungicidal activity of rosemary acid on Pemcillium cyclopium.

SAMPLE CONCENTRATION % FUNGICIDITY SAMPLE 1 0.5% 5.94 1% 26.64 SAMPLE 2 0.5% 6.15 1% 100 SAMPLE 3 0.5% 60.66 1% 100 SAMPLE 4 0.5% 85.25 1% 100 SAMPLE 5 0.5% 100 1% 100 SAMPLE 6 0.5% 67.1 1% 82,3 SAMPLE 7 0.5% 71,12 1% 100 SAMPLE 8 0.5% 77.15 1% 100

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EXAMPLE 20

The dried leaves of rosemary, sage, lemon balm, oregano, thyme or any other plant of the Labnatae family are ground in an inert atmosphere. 100 g of drug (median grain = 310 pm, most commonly represented grain = 98 μηι) was extracted with 1000 ml of 35% acetone at 20 ° C for 2 h. The extraction mixture was mixed with a magnetic stirrer (n ^ SOOOmin ' ¹ ). The marc were filtered off and the solution was evaporated on a rotary evaporator at 50 ° C and a pressure of 150 mbar. The obtained crude product was concentrated using propane under the conditions listed in Table 3. We obtained ca. 0.2 g of purified extract with a rosemary acid concentration of 2.38%, which was determined to have fungicidal action on Aspergillus niger.

The results of measuring the fungicidal properties of rosemary acid (sample 6) on Aspergillus niger are shown in Table 13. The extract at a concentration of 0.5% 17.12%> inhibits the occurrence of said mold at a concentration of 1%.

EXAMPLE 21

We operate analogously to Example 20, except that instead of Aspergillus niger, fungicidal activity is determined on Penicillium cyclopium.

The results of changing the fungicidal properties of rosemary acid (sample 6) to Penicillium cyclopium are shown in Table 14. The extract at a concentration of 0.5% 67.1%, and at a concentration of 1% 82.3% inhibits the appearance of said mold.

EXAMPLE 22

The dried leaves of rosemary, sage, lemon balm, oregano, thyme or any other plant of the Labiatae 'family are ground in an inert atmosphere. 100 g of drug (median grain = 310 pm, most commonly represented grain = 98 pm) was extracted with 1000 ml of 35% acetone at 20 ° C for 2 h. The extraction mixture was mixed with a magnetic stirrer (n ^ OOOmin * ¹ ). The marc were filtered off and the solution was evaporated on a rotary evaporator at 50 ° C and a pressure of 150 mbar at 20% of the initial volume. The extract was acidified with phosphoric acid at pH = 2-3 and concentrated by ion-exchange chromatography. The following ion exchangers XAD 1180, XAD16, XAD-1600, IRA 900 LL, DIAION SA 11 A, DIAION PA 308, XAD-2 and DIAION HP-20 were used as stationary phase and 0.25% (w / w as eluent) = w / w = w / w) NaOH in 85%

-2222 methanol. The eluate was evaporated on a rotary evaporator at a temperature of 50 ⁱⁿ C and a pressure of 150 mbar. The crude product obtained was concentrated using DME under the conditions indicated in Tables 5 and 6. Approx. 0.1 g of purified extract with a concentration of rosemary acid approx. 18%, determined by the fungicidal action on Aspergillus niger.

The results of measuring the fungicidal property of rosemary acid (sample 7) on the fungus Aspergillus niger are shown in Table 13. The extract at a concentration of 0.5% 47.83%, and at a concentration of 1% 72.15% inhibits the appearance of said mold.

EXAMPLE 23

We acted analogously to Example 22 except to determine the fungicidal action on Penicillium cyclopium.

The results of measuring the fungicidal properties of rosemary acid (sample 7) on Penicillium cyclopium are shown in Table 14. The extract at a concentration of 0.5% 71.12%, and at a concentration of 1%, 100% inhibits the appearance of said mold.

EXAMPLE 24

We operate analogously to Example 1, except that the crude product obtained is concentrated using supercritical fluid chromatography with propane as the mobile phase and acetonitrile as the co-solvent. An extract with a concentration of rosemary acid of about 62% was obtained, which was determined by its fungicidal action on Aspergillus niger.

The results of measuring the fungicidal properties of rosemary acid (sample 8) on Aspergillus niger are shown in Table 13. The extract at a concentration of 0.5% 42.15%, and at a concentration of 1% 71.35% inhibits the appearance of said mold.

EXAMPLE 25

We acted analogously to Example 24 except that the fungicidal action of the extract was determined on Penicillium cyclopium.

The results of measuring the fungicidal properties of rosemary acid (sample 8) on Peniciilium cyclopium are shown in Table 14. The extract at a concentration of 0.5% 77.15%, and at a concentration of 1% 100% inhibits the appearance of said mold.

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EXAMPLE 26

Rosemary acid extract approx. 50% is mixed with an extract containing 60% camosolic acid in a 1: 1 ratio. The mixture was determined to have a fungicidal action on Aspergillus niger. The radial growth of mold on PDA agar containing 1 ml of alcoholic solution of the extract was measured daily. Because ethanol itself has antimicrobial activity, a PDA plate with 1 ml of 96% ethanol was taken as an alcohol control. At the same time, control with alcohol-free agar and active substance was prepared. The plates were incubated for 10-17 days at 28 ° C and in the dark.

The extract at a concentration of 1% 82% inhibits the appearance of said mold.

EXAMPLE 27

Rosemary acid extract approx. 50% is mixed with an extract containing 60% camosolic acid in a 1: 1 ratio. The mixture was determined to have a fungicidal action on Penicittium cyclopium. The radial growth of mold on PDA agar containing 1 ml of alcoholic solution of the extract was measured daily. Because ethanol itself has antimicrobial activity, a PDA plate with 1 ml of 96% ethanol was taken as an alcohol control. At the same time, control with alcohol-free agar and active substance was prepared. The plates were incubated for 10-17 days at 28 ° C and in the dark.

The extract at a concentration of 1% 100% inhibits the appearance of said mold.

EXAMPLE 28

Rosemary acid extract approx. 50% is mixed with an extract containing 50% ursolic acid and 10% oleanolic acid in a 1: 1 ratio. The mixture was determined to have a fungicidal action on Aspergillus niger. The radial growth of mold on PDA agar containing 1 ml of alcoholic solution of the extract was measured daily. Because ethanol itself has antimicrobial activity, a PDA plate with 1 ml of 96% ethanol was taken as an alcohol control. At the same time, control with alcohol-free agar and active substance was prepared. The plates were incubated for 10-17 days at 28 ° C and in the dark. Extract at a concentration of 1% 85% inhibits the appearance of said mold.

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EXAMPLE 28

Rosemary acid extract approx. 50% is mixed with an extract containing 50% ursolic acid and 10% oleanolic acid in a 1: 1 ratio. The mixture was determined to have a fungicidal action on Penicillium cyclopium. The radial growth of mold on PDA agar containing 1 ml of alcoholic solution of the extract was measured daily. Because ethanol itself has antimicrobial activity, a PDA plate with 1 ml of 96% ethanol was taken as an alcohol control. At the same time, control with alcohol-free agar and active substance was prepared. The plates were incubated for 10-17 days at 28 ° C and in the dark. The extract at a concentration of 1% 100% inhibits the appearance of said mold.

Claims (14)

1. A process for the preparation of concentrated extracts of rosemary acid from plants of the Labiatae family, characterized in that it comprises the following steps: a) grinding of dried plant material from plants of the mouthpiece family (Lubiatae) in an inert atmosphere; b) extraction with water or organic solvent at a) the milled material and / or marc obtained from the extraction of oil-soluble antioxidants from plants of the Labiatae family, at a solvent / extraction ratio of 1/10, a temperature between 20 ° C and the boiling point of the solvent, respectively. mixtures, for a time between 0.5 and 24 hours; c) evaporation of the organic solvent; d) concentrating the product obtained by conventional extraction by ion exchange chromatography; e) extraction of rosemary acid by liquid-liquid extraction using esters or ethers as the organic phase; f) concentrating the product by elution chromatography using esters, ethers and mixtures thereof as the mobile phase and silica gel or aluminum oxide as the stationary phase; g) concentrating the rosemary-based product using gases at high pressures of 10 to 500 bar, at a temperature of 0-80 ° C, optionally using a boiler. Process according to Claim 1a), characterized in that the granulation of the milled material is 0.1-5 mm. Process according to claim 1b), characterized in that the preferred solvents for extraction are alcohols, esters, ethers, aldehydes, ketones, hydrocarbons, acids, bases and their solutions and mixtures. -2626 Process according to claim ld), characterized in that XAD 1180, XAD-16, XAD-1600, 1RA 900 LL, D1A1ON SA 11 A, D1A1ON PA 308, XAD-2 and DIA1ON HP- ion exchangers are stationary phase. 20 and, as the mobile phase, alcohols, preferably ethanol, methanol, and mixtures thereof with water. 5. A process according to claim 1), characterized in that, in the liquid-liquid extraction, ethyl acetate, butyl acetate, diethyl ether, isopropyl ether is used as the organic phase. Process according to claim 1f), characterized in that ethyl acetate, butyl acetate, light petroleum and mixtures thereof are used as the mobile phase. Process according to claim 1g), characterized in that CO2, hydrocarbons having 1-4 carbon atoms, freons or mixtures thereof are used as high pressure extraction media. 8. A process according to claim 1g), characterized in that the gas consumption of the high-pressure extraction is between 1 and 500 kg of gas / kg of extracted substance. 8. A process according to claim 1g), characterized in that the high pressure extraction boiler is selected from water, alcohols, esters, ethers, aldehydes, ketones, hydrocarbons, acids, bases and their solutions. Use of concentrated extracts of rosemary acid from plants of the labiatae family obtained according to claim 1 for the preparation of agents for slowing the oxidation of fat in emulsions. Use of concentrated extracts of rosemary acid from plants of the oral family (Labiatae) obtained according to claim 1, for the preparation of agents for antimicrobial protection of products against the following microorganisms: Bacillus cereus, Staphylococcus epidermidis and Escherichia coli fungi, Aspergumium cyclerium niger fungi, niger fungus, Niger fungi ovals and Saccharomyces cerevisiae. -2727 Use of concentrated rosemary acid extracts from plants of the Labiatae family obtained according to claim 1, in combination with other components such as camosolic acid, ursolic acid and oleanolic acid for the preparation of agents with synergistic antioxidant and antimicrobial action. 12. Means of retardation of fat oxidation in emulsions containing concentrated extracts of rosemary acid from plants of the mouth (Labiatae) family obtained according to claim 1. 13. Antimicrobial agents for the protection of products against the following micro-organisms: Bacillus cereus, Staphylococcus epidermidis and Escherichia coli, Aspergillus niger, Penicillium cyclopium, Pityrosporum ovale and Saccharomyces cerevisia according to claim 1. 14. Synergistically antioxidant and antimicrobial agents containing concentrated extracts of rosemary acid from Labiatae plants obtained according to claim 1, in combination with other components such as camosolic acid, ursolic acid and oleanolic acid.