RU2557953C2 - Method for measuring anthocyanes in crude drugs - Google Patents

Abstract

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to chemical-pharmaceutical industry, and can be used in drug quality control centres and analytical laboratories when measuring anthocyanes in such crude drugs, as bilberry, black chokeberry, black currant, etc. The presented method for measuring anthocyanes involves recovering anthocyanes from crude drugs in alcohol water mixtures containing 0.5-1.0% muriatic acid with the ethanol concentration of less than 70% and measuring anthocyanes quantitatively by optical density in 95% ethanol containing 0.5% ammonium as compared to a reference solution at analytical wavelength of peak absorption within the range of 611-630 nm with using a reference solution of cyanidine-3-O-glucoside or with using its specific absorption in 95% ethanol containing 0.5% ammonium.

EFFECT: higher sensitivity and result reproducibility, as well as shortening the analysis procedure as compared to its prototype.

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Description

The invention relates to the pharmaceutical industry and can be used in drug quality control centers and analytical laboratories when analyzing anthocyanins in medicinal plant materials: common blueberries, chokeberry, blackcurrant, etc.

The current quality control system requires continuous improvement of approaches to standardization of biologically active compounds (BAS) using modern methods of analysis and relevant data on their properties, which allow differentially determining specific groups of BAS.

Anthocyanins are a widespread group of natural ALS with antioxidant, vasoprotective, anticoagulant properties, the ability to improve microcirculation, trophic retina, as well as in some in vitro studies showing antitumor activity (1,2).

Anthocyanins are flavonoids and, by their chemical nature, they are a glycosylated form of hydroxy- and methoxy substituted derivatives of 2-phenylchromene (anthocyanidins) (Fig. 1) (3). For anthocyanins, the ability to change their color depending on the pH of the medium is known. In an acidic environment (pH <3), they are present in solution in the form of red benzopyrilium salts. With increasing pH (4-5), the hydroxyl group joins in the second position with the formation of a colorless pseudo base. At pH 6-7, blue phenolates are formed. At pH greater than 8.0, the chromene cycle opens and the corresponding chalcon forms (4).

Known pH-differential method of quantitative determination, based on the ability of anthocyanins to change their color depending on the pH value. It is based on measuring the optical density of a solution at pH 1.0 and pH 4.5. The indicated pH values are created using buffer solutions (5). However, this method is characterized by high complexity, especially in the process of sample preparation at the stage of preparation of buffer solutions. In the course of reproducing this technique, we also noted a number of limitations, in particular, for some analyzed samples, the formation of an opalescent solution when adjusted to the mark with a buffer solution, possibly due to the replacement of the solvent. An overproduction of optical density may be associated with mild opalescence, which in the differential embodiment may result in an underestimation of the results.

Thus, the aim of the invention is to develop a new method for the quantitative determination of anthocyanins.

The technical result is to increase the sensitivity and reproducibility, reducing the duration and cost of the method for the quantitative determination of anthocyanins in medicinal plant materials.

The technical result is achieved by the fact that the method is carried out by extracting anthocyanins from medicinal plant raw materials with water-alcohol mixtures containing 0.5-1.0% hydrochloric acid with an ethyl alcohol concentration of at least 70% and determining the quantitative content of anthocyanins by optical density of 95% ethyl alcohol containing 0.5% ammonia against a background of a comparison solution at an analytical maximum absorption wavelength in the range of 611-630 nm using a solution of a standard sample of cyanidin-3-glucoside or using lzovaniem value of its specific absorption index of 95% ethanol containing 0.5% ammonia.

When analyzing known solutions in the scientific literature, no research results were found on the quantitative assessment of anthocyanins in our chosen direction and, accordingly, information on the influence of extraction and sample preparation parameters on the achievement of a technical solution.

It is possible to perform the proposed method using a standard sample of cyanidin-3-O-glucoside, which increases the accuracy of the analysis taking into account the specific conditions of the test, or in the absence of a sample using its specific absorption coefficient in 95% ethyl alcohol containing 0.5 % ammonia.

The method is as follows. Extract is obtained from anthocyanin-containing medicinal plant materials under optimal conditions. It is recommended to use water-alcohol mixtures with a concentration of ethyl alcohol of at least 70% and containing 0.5-1.0% hydrochloric acid. Two identical aliquots are taken and placed in two volumetric flasks, in one of which the volume of the solution is adjusted to the mark with 95% ethyl alcohol - a comparison solution, in the other - 95% ethyl alcohol containing 0.5% ammonia - the test solution. The solutions are mixed and the optical density of the obtained solutions is measured for 1 min in a cuvette with a working layer thickness of 10 mm at a wavelength corresponding to the maximum difference in optical density between the sample in the ammonia solution and the initial solution (Fig. 2).

In the case of using a standard sample, the optical density of cyanidin-3-O-glucoside is measured in parallel under the same conditions.

Preparation of a solution of cyanidin-3-O-glucoside: an exact sample of a standard sample of cyanidin-3-glucoside in an amount of about 0.02 g is placed in a 50 ml volumetric flask, dissolved in methyl alcohol containing 1% hydrochloric acid, adjusted to the mark with a solvent and stirred, 5 ml of the resulting solution was placed in a 25 ml flask, adjusted with 95% ethyl alcohol containing 1% hydrochloric acid, mixed. 2 ml of the resulting solution was placed in 25 ml volumetric flasks, in one of them the volume was adjusted to the mark with 95% ethyl alcohol, in the other with a 0.5% solution of ammonia in 95% ethyl alcohol.

The optical density is measured for 1 min after preparation of the solution at an analytical wavelength in the range of 611-630 nm, depending on the wavelength of the absorption maximum of the test object.

When using a standard sample, the calculation of the results of the quantitative determination of the sum of anthocyanins in terms of cyanidin-3-O-glucoside is carried out according to the formula:

Where:

And _x is the optical density of the test solution;

A _st is the optical density of a solution of a standard sample;

m _x - exact sample weight of the analyzed sample;

m _{st is an} exact weighed portion of cyanidin-3-O-glucoside;

and _x is an aliquot of the analyzed solution;

V ₁ - volumetric volumetric flask for dilution of extraction from plant materials;

V ₂ - the amount of extraction from medicinal plant materials.

In the absence of a standard sample is calculated taking into account the specific absorption rate of cyanidin-3-O-glucoside:

Where:

And _x is the optical density of the test solution;

m _x - exact sample weight of the analyzed sample;

and _x is an aliquot of the analyzed solution;

V ₁ - volumetric volumetric flask for dilution of extraction from plant materials;

V ₂ - the amount of extraction from medicinal plant materials; 250 is the specific absorption rate of cyanidin-3-O-glucoside. The proposed method is illustrated by the following examples.

Example 1. Determination of anthocyanins in the fruits of common blueberries fresh Vaccinium myrtillus L., Heather family - Ericaceae, using a solution of a standard sample of cyanidin-3-O-glucoside.

About 1.0 raw material (accurately weighed) is placed in a conical flask with a thin section with a capacity of 100 ml, 50 ml of 95% ethyl alcohol containing 1% hydrochloric acid are added. The flask is closed with a stopper and weighed on a scale balance accurate to ± 0.01 g. The flask is attached to a reflux condenser and heated in a boiling water bath (moderate boiling) for 30 minutes. Then close the same stopper, cool to room temperature. Then the flask is again weighed and make up the missing extractant to the original mass. The extract is filtered through a paper filter and the contents are mixed.

5 ml of the resulting solution is placed in a volumetric flask with a capacity of 25 ml, adjusted with a 0.5% solution of ammonia in 95% ethyl alcohol to the mark. As a comparison solution, use a solution prepared under the same conditions, but brought to the mark with 95% ethyl alcohol. The optical density is measured for 1 min after preparation of the solution at an analytical wavelength of 623 nm.

The quantitative content is calculated by the formula:

Where:

And _x = 0.7382 is the optical density of the test solution;

A _st = 0.1808 is the optical density of a solution of a standard sample;

m _x = 1.2263 - an exact sample of the analyzed sample;

m _st = 0,0226 - an exact sample of cyanidin-3-O-glucoside;

and _x = 5 - an aliquot of the analyzed solution;

V ₁ = 25 - volumetric volumetric flask for dilution of extraction from plant materials;

V ₂ = 50 - the volume of extraction from medicinal plant materials.

The test was carried out in several replicates. All data were statistically processed. The content of anthocyanins was 0.60 ± 0.02%. The relative error of the method was ± 3.33%. In the quantitative determination of anthocyanins using pH differential spectrophotometry, the content of anthocyanins in terms of cyanidin-3-O-glucoside was 0.61 ± 0.03%, the relative error of the method was ± 4.92%.

Example 2. Determination of anthocyanins in the fruits of common blueberries fresh Vaccinium myrtillus L., the Heather family - Ericaceae, using the specific absorption coefficient of cyanidin-3-O-glucoside.

The determination is carried out analogously to example 1. Next, the content is calculated by the formula:

Where:

And _x = 0.7382 is the optical density of the test solution;

m _x = 1.2263 - an exact sample of the analyzed sample;

and _x = 5 - an aliquot of the analyzed solution;

V ₁ = 25 - volumetric volumetric flask for dilution of extraction from plant materials;

V ₂ = 50 - the volume of extraction from medicinal plant materials;

250 is the specific absorption rate of cyanidin-3-O-glucoside.

The test was carried out in several replicates. All data were statistically processed. The content of anthocyanins was 0.60 ± 0.02%.

Example 3. Determination of anthocyanins in the fruits of blueberries ordinary air-dried Vaccinium myrtillus L., Heather family - Ericaceae, using a solution of a standard sample of cyanidin-3-O-glucoside.

About 1.0 raw material (accurately weighed) is placed in a conical flask with a thin section with a capacity of 100 ml, 50 ml of 95% ethyl alcohol containing 1% hydrochloric acid are added, 5 ml of the resulting solution is placed in a volumetric flask with a capacity of 25 ml, adjusted with 0.5% a solution of ammonia in 95% ethyl alcohol to the mark (test solution A). Next, the process is carried out in accordance with example 1. The optical density is measured within 1 min after preparation of the solution at an analytical wavelength of 627 nm. The calculation is carried out on absolutely dry raw materials.

Calculated by the following formula:

Where:

And _x = 0.381 is the optical density of the test solution;

A _st = 0.1808 is the optical density of a solution of a standard sample;

m _x = 1.2340 - an exact sample of the analyzed sample;

m _st = 0,0226 - an exact sample of cyanidin-3-O-glucoside;

and _x = 5 - an aliquot of the analyzed solution;

V ₁ = 25 - volumetric volumetric flask for dilution of extraction from plant materials;

V ₂ = 50 - the volume of extraction from medicinal plant materials;

W - moisture content of raw materials,%.

The test was carried out in several replicates. All data were statistically processed. The content of anthocyanins was 0.325 ± 0.009%. The relative error of the method was ± 2.77%. In the quantitative determination of anthocyanins using pH differential spectrophotometry, the content of anthocyanins in terms of cyanidin-3-O-glucoside was 0.312 ± 0.123%, the relative error of the method was ± 3.85%.

Example 4. Determination of anthocyanins in the fruits of chokeberry fresh Aronia melanocarpa (Michx.) Elliott., Family Rosaceae - Rosaceae, using a solution of a standard sample of cyanidin-3-O-glucoside.

About 1.0 raw material (accurately weighed) is placed in a conical flask with a thin section with a capacity of 100 ml, 50 ml of 95% ethyl alcohol containing 1% hydrochloric acid are added. The flask is closed with a stopper and weighed on a scale balance with an accuracy of ± 0.01 g. Next, the process is repeated in accordance with example 1.

2 ml of the resulting solution was placed in a volumetric flask with a capacity of 25 ml, adjusted with a 0.5% solution of ammonia in 95% ethyl alcohol to the mark. As a comparison solution, use a solution prepared under the same conditions, but brought to the mark with 95% ethyl alcohol. The optical density is measured directly within 1 min after preparation of the solution at an analytical wavelength of 617 nm.

Where:

And _x = 0.1362 is the optical density of the test solution;

A _st = 0.1808 is the optical density of a solution of a standard sample;

m _x = 1,0256 - an exact sample of the analyzed sample;

m _st = 0,0226 - an exact sample of cyanidin-3-O-glucoside;

and _x = 2 - an aliquot of the analyzed solution;

V ₁ = 25 - volumetric volumetric flask for dilution of extraction from plant materials;

V ₂ - 50 - the volume of extraction from medicinal plant materials.

The test was carried out in several replicates. All data were statistically processed. The content of anthocyanins was 0.332 ± 0.010%. The relative error of the method was ± 2.92%. In the quantitative determination of anthocyanins using pH differential spectrophotometry, the content of anthocyanins in terms of cyanidin-3-O-glucoside was 0.342 ± 0.011%, the relative error of the method was ± 3.31% (Fig. 2).

Example 5. Determination of anthocyanins in the fruits of chokeberry fresh Aronia melanocarpa (Michx.) Elliott., Rosaceae family - Rosaceae, using the specific absorption coefficient of cyanidin-3-O-glucoside.

The process is carried out analogously to example 4. Next, calculate the content of anthocyanins using the values of the specific absorption index:

Where:

And _x = 0.1362 is the optical density of the test solution;

m _x = 1,0256 - an exact sample of the analyzed sample;

and _x = 2 - an aliquot of the analyzed solution;

V ₁ = 25 - volumetric volumetric flask for dilution of extraction from plant materials;

V ₂ = 50 - the volume of extraction from medicinal plant materials;

250 is the specific absorption rate of cyanidin-3-O-glucoside.

The test was carried out in several replicates. All data were statistically processed. The content of anthocyanins was 0.332 ± 0.010%.

Example 6. Determination of anthocyanins in the flowers of a blue cornflower Centaurea cyanus L., Compositae family, using a solution of a standard sample of cyanidin-3-O-glucoside.

About 1.0 raw material (accurately weighed) is placed in a conical flask with a thin section with a capacity of 100 ml, 50 ml of 95% ethyl alcohol containing 1% hydrochloric acid are added. Next, the process is carried out in accordance with example 4. The optical density is measured for 1 min after preparation of the test solution at an analytical wavelength of 622 nm. The calculation is carried out in terms of absolutely dry raw materials according to the formula:

Where:

And _x = 0.2748 is the optical density of the test solution;

A _st = 0.1808 is the optical density of a solution of a standard sample;

m _x = 0.9662 - exact sample weight of the analyzed sample;

m _st = 0,0226 - an exact sample of cyanidin-3-O-glucoside;

and _x = 2 - an aliquot of the analyzed solution;

V ₁ = 25 - volumetric volumetric flask for dilution of extraction from plant materials;

V ₂ = 50 - the volume of extraction from medicinal plant materials;

W - moisture content of raw materials,%.

The test was carried out in several replicates. All data were statistically processed. The content of anthocyanins was 0.79 ± 0.02%. The relative error of the method was ± 2.67%. In the quantitative determination of anthocyanins using pH differential spectrophotometry, the content of anthocyanins in terms of cyanidin-3-O-glucoside was 0.61 ± 0.03%, the relative error of the method was ± 4.92%.

Example 7. Determination of anthocyanins in the fruits of black currant air-dried Ribes nigrum L., Gooseberry family - Grossulariaceae, using a solution of a standard sample of cyanidin-3-O-glucoside.

About 1.0 raw material (accurately weighed) is placed in a conical flask with a thin section with a capacity of 100 ml, 50 ml of 95% ethyl alcohol containing 1% hydrochloric acid are added. Next, the process is carried out in accordance with example 1. The optical density is measured within 1 min after preparation of the analyzed solution at an analytical wavelength of 626 nm. The calculation is carried out in terms of absolutely dry raw materials according to the formula:

Where:

And _x = 0.1632 is the optical density of the test solution;

A _st = 0.1808 is the optical density of a solution of a standard sample;

m _x = 1,1702 - an exact sample of the analyzed sample;

m _st = 0,0226 - an exact sample of cyanidin-3-O-glucoside;

and _x = 5 - an aliquot of the analyzed solution;

V ₁ = 25 - volumetric volumetric flask for dilution of extraction from plant materials;

V ₂ = 50 - the volume of extraction from medicinal plant materials;

W - moisture content of raw materials,%.

The test was carried out in several replicates, all data were statistically processed. The content of anthocyanins was 0.155 ± 0.004%, the relative error of the method ± 2.58%. In the quantitative determination of anthocyanins using pH differential spectrophotometry, the content of anthocyanins in terms of cyanidin-3-O-glucoside was 0.065 ± 0.004%, the relative error of the method was ± 6.15%.

Thus, the proposed method for quantitative determination of the amount of anthocyanins in medicinal plant materials was developed for the first time for the analysis of this group of ALS and has the following advantages (Fig. 3).

1. Almost does not take into account the contribution of other compounds (based on the known ability for anthocyanins to stain blue in an alkaline environment).

2. It is characterized by lower time and financial costs when performing analysis in comparison with the prototype.

3. It is characterized by a smaller error of a single method definition for the considered examples.

INFORMATION SOURCES:

1. Cyanidin 3-Glucoside and Peonidin 3-Glucoside Inhibit Tumor Cell Growth and Induce Apoptosis In Vitro and Suppress Tumor Growth In Vivo / Pei-Ni Chen et al. // Nutrition And Cancer. - 2005. - Vol. 53, No. 2. - P. 232-243.

2. Kowalczyk, E., Krzesinski P., Kura M., Szmigiel B., Blaszczyk J. Anthocyanins in medicine. - Pol. J. Pharmacol. - 2003. - Vol. 55. - P. 699-702.

3. Kurkin V.A. Pharmacognosy: Textbook for students of pharmaceutical universities / Ed. 2nd, rev. and additional - Samara: LLC “Etching”, GOU VPO “SamGMU”, 2007. - 1239 p.

4. Rein, M. Copigmentation reactions and color stability of berry anthocyanins: Academic dissertation / Maarit Rein. - Helsinki. - 2005 .-- 88 + 34 pp.

5. Giusti, M.M. Characterization and measurement of anthocyanins by UV-visible spectroscopy. Unit F1.2 / M.M. Giusti, R.E. Wrolstad, S.J. Schwartz (Ed.). -Current Protocols in Food Analytical Chemistry. " - John Wiley & Sons, Inc. New York, NY. Pp. F1.2.1-F1.2.13.

Claims (1)

A method for the quantitative determination of anthocyanins in medicinal plant materials using spectrophotometry, characterized in that the method is carried out by extracting anthocyanins from medicinal plant materials with water-alcohol mixtures containing 0.5-1.0% hydrochloric acid, with ethyl alcohol concentration not less than 70% and determining the quantitative content of anthocyanins by optical density in 95% ethyl alcohol containing 0.5% ammonia, against the background of a comparison solution at an analytical wavelength of maximum absorption in dia azone 611-630 nm using the reference solution cyanidin-3-O-glucoside, or using the value of its specific absorption index of 95% ethanol containing 0.5% ammonia.

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