Method for measuring anthocyanes in crude drugs

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.

3 dwg, 7 ex

 

The invention relates to chemical-pharmaceutical industry and can be used in the centers of drug quality control and analytical laboratories in the analysis of anthocyanins in medicinal plant material: the fruits of bilberry, chokeberry Aronia, black currant, etc.

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

Anthocyanins are a widespread group of natural BASS with antioxidant, vasoprotective, anticoagulation properties, ability to improve microcirculation and trophism of the retina, as well as in some in vitro studies showed antitumor activity (1,2).

Anthocyanins belong to the flavonoid and chemical nature are glycosylated form of hydroxy - and methoxsalen derivatives of 2-phenylpropene (anthocyanidins) (Fig. 1) (3). For anthocyanins is known for the ability to change colour depending on the pH of the medium. In an acidic environment (pH<3) they are present in the solution in the form benzopirilievyh salts red�and. With increasing pH (4-5) is joining the hydroxyl group in the second position with the formation of colorless pseudomonotone. At pH 6-7 are formed phenolate blue. At pH above 8.0 the unlocking promenevogo cycle is formed and the corresponding Halcon(4).

Known pH-differential method of quantitative determination, based on the ability of anthocyanins to change its color depending on the pH. It is based on the measurement of the optical density of the solution at pH 1.0 and pH 4.5. These pH values are created by using buffer solutions (5). However, this method is characterized by high complexity, especially in the process of sample preparation in the preparation stage of the buffer solutions. Also, we had noted during the playback this technique a number of limitations, particularly for some of the analyzed samples education opalescense solution if brought up to the mark with buffer solution, probably due to solvent exchange. With slight opalescence may be due to the overestimation of optical density in the differential embodiment, may lead to underestimation of the results.

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

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

The technical result is achieved in that the method is carried out by extraction of anthocyanins from medicinal vegetative raw material of water-alcohol mixtures containing 0.5 to 1.0% hydrochloric acid with concentration of ethyl alcohol of not less than 70% and the quantification of anthocyanins by optical density in 95% ethanol containing 0.5% of ammonia, on the background of the reference solution at the analytical wavelength of maximum absorption in the range 611-630 nm using a solution of the standard sample cianids-3-glucoside or using the values of its specific absorbance in 95% ethanol containing 0.5% of ammonia.

In the analysis of the known solutions in the scientific literature found no research results on the quantification of anthocyanins in the chosen direction and, accordingly, information on the influence of extraction parameters, sample preparation to achieve a technical solution.

Perhaps the performance of the proposed method with the use of the standard sample cianids-3-O-glucoside, which increases the accuracy of analysis based on specific test conditions, or in the absence of the sample using the estimated �ormula its specific absorbance in 95% ethyl alcohol, containing 0.5% of ammonia.

The method is as follows. Obtain an extract from anthocyanograms medicinal plants in optimal conditions. It is recommended to use water-alcohol mixtures with concentration of ethyl alcohol of not less than 70% and containing 0.5 to 1.0% hydrochloric acid. Take two equal aliquots and placed in two volumetric flasks, one of which is the volume of the solution was adjusted to the mark with 95% ethanol - solution comparison, in the other 95% ethyl alcohol containing 0.5% of ammonia, - the test solution. Solutions and stirred for 1 min measure the optical density of the obtained solutions in a cell with a thickness of working layer 10 mm at a wavelength corresponding to the maximum difference of optical density between the sample in ammonia solution and the original solution (Fig. 2).

In the case of the standard sample measured parallel to the optical density cianids-3-O-glucoside in the same terms.

Preparation of solution cianids-3-O-glucoside: an accurately weighed sample standard sample cyanidin-3-glucoside in an amount of about 0.02 grams is placed in a volumetric flask of 50 ml, dissolved in methyl alcohol containing 1% hydrochloric acid, adjusted with solvent to the mark and mix 5 ml of the resulting solution is placed in a flask of 25 ml, adjusted 95% ethyl alcohol, with�orgasim 1% hydrochloric acid, mix. 2 ml of the resulting solution is placed in a volumetric flask with a capacity of 25 ml, in one volume was adjusted up to the mark with 95% ethyl alcohol, the other with 0.5% solution of ammonia in 95% ethyl alcohol.

Measure the optical density for 1 min after preparation of the solution in analytical wavelength in the range 611-630 nm depending on the wavelength of maximum absorption of the object.

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

where:

Andx- optical density of test solution;

Astoptical density of the solution of the standard sample;

mx- a precisely weighed test sample;

mst-a precisely weighed cyanidin-3-O-glucoside;

andxthe aliquot of the test solution;

V1- volume of the volumetric flask for dilution of extraction from vegetable raw materials;

V2- the volume of extract from medicinal plant raw material.

When no standard sample is calculated based on the specific absorption index cianids-3-O-glucoside:

where:

Andx- optical density of test solution;

mx- a precisely weighed analyzed�about the sample;

andxthe aliquot of the test solution;

V1- volume of the volumetric flask for dilution of extraction from vegetable raw materials;

V2- the volume of extraction of medicinal plants; 250 - specific absorption coefficient cianids-3-O-glucoside. The proposed method is illustrated by the following examples.

Example 1. Determination of anthocyanins in fruits of fresh bilberry Vaccinium myrtillus L., Heath family - Ericaceae, using a solution of the standard sample cianids-3-O-glucoside.

About 1.0 raw materials (a precisely weighed) placed in a conical flask with a ground joint with a capacity of 100 ml, add 50 ml of 95% ethanol containing 1% hydrochloric acid. The flask is closed with a stopper and weighed on terimah scales with an accuracy of ±0.01 g. the Flask is attached to the reverse refrigerator and heated in a boiling water bath (moderate boiling) for 30 min. Then close the same tube, cooled to room temperature. Then the flask is weighed again and fill the missing extractant to the initial weight. The extract was filtered through paper filter and the contents stirred.

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

Expect quantitative content by the formula:

where:

Andx=0,7382 - optical density of test solution;

Ast=0,1808 optical density of the solution of the standard sample;

mx=1,2263 - a precisely weighed test sample;

mst=0,0226 - a precisely weighed cianids-3-O-glucoside;

andx=5 - aliquot analyzed solution;

V1=25 - volume of the volumetric flask for dilution of extraction from vegetable raw materials;

V2=50 - volume of extract from medicinal plant raw material.

The test has been carried out in several replications. 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 the pH-differential spectrophotometry, the content of anthocyanins in terms of cyanidin-3-O-glucoside was 0.61±0.03%, and the relative error of the method±4,92%.

Example 2. Determination of anthocyanins in fruits of fresh bilberry Vaccinium myrtillus L., Heath family - Ericaceae, and the value of the specific absorbance cianids-3-O-glucoside.

About�the definition is carried out analogously to example 1. Further, the content calculated by the formula:

where:

Andx=0,7382 - optical density of test solution;

mx=1,2263 - a precisely weighed test sample;

andx=5 - aliquot analyzed solution;

V1=25 - volume of the volumetric flask for dilution of extraction from vegetable raw materials;

V2=50 - volume of extract from medicinal plant raw materials;

250 is the specific absorption coefficient cianids-3-O-glucoside.

The test was carried out in several replications. All data were statistically processed. The content of anthocyanins was 0.60±0,02%.

Example 3. Determination of anthocyanins in fruits of bilberry dry air Vaccinium myrtillus L., Heath family - Ericaceae, using a solution of the standard sample cianids-3-O-glucoside.

About 1.0 raw materials (a precisely weighed) placed in a conical flask with a ground joint with a capacity of 100 ml, add 50 ml of 95% ethanol containing 1% hydrochloric acid, 5 ml of the resulting solution is placed in a volumetric flask with a capacity of 25 ml, adjusted with 0.5% ammonia solution (95% ethyl alcohol to the mark and mix (test solution a). Further the process is carried out in accordance with example 1. The optical density measured for 1 min after preparation of the solution at the analytical wavelength of 627 nm. Calculation of wire�tons of dry raw material.

Calculated by the following formula:

where:

Andx=0,381 - optical density of test solution;

Ast=0,1808 optical density of the solution of the standard sample;

mx=1,2340 - a precisely weighed test sample;

mst=0,0226 - a precisely weighed cianids-3-O-glucoside;

andx=5 - aliquot analyzed solution;

V1=25 - volume of the volumetric flask for dilution of extraction from vegetable raw materials;

V2=50 - volume of extract from medicinal plant raw materials;

W - moisture content of raw materials, %.

The test has been carried out in several replications. All data were statistically processed. The content of anthocyanins amounted 0,325±0,009%. The relative error of the method was ±2,77%. In the quantitative determination of anthocyanins using the pH-differential spectrophotometry, the content of anthocyanins in terms of cyanidin-3-O-glucoside accounted 0,312±0,123%, relative error of the method ±3,85%.

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

About 1.0 raw materials (a precisely weighed) placed in a conical flask with a ground joint with a capacity of 100 ml, add 50 ml of 95% ethanol containing 1% of chlorotoluron�th acid. The flask is closed with a stopper and weighed on terimah scales with an accuracy of ±0.01 g. Then the process is repeated in accordance with example 1.

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

where:

Andx=0,1362 - optical density of test solution;

Ast=0,1808 optical density of the solution of the standard sample;

mx=1,0256 - a precisely weighed test sample;

mst=0,0226 - a precisely weighed cianids-3-O-glucoside;

andx=2 - aliquot analyzed solution;

V1=25 - volume of the volumetric flask for dilution of extraction from vegetable raw materials;

V2- 50 - volume of extract from medicinal plant raw material.

The test has been carried out in several replications. All data were statistically processed. The content of anthocyanins amounted 0,332±0,010%. The relative error of the method was ±2,92%. In the quantitative determination of anthocyanins using the pH-differential spectrophotometry contents�tion of anthocyanins in terms of cyanidin-3-O-glucoside accounted 0,342 ±0,011%, the relative error of the method ±3,31% (Fig. 2).

Example 5. Determination of anthocyanins in the fruits of fresh chokeberry Aronia melanocarpa (Michx.) Elliott, family Rosaceae - Rosaceae, and the value of the specific absorbance cianids-3-O-glucoside.

The process is carried out analogously to example 4. Then calculate the content of anthocyanins and the value of the specific absorbance:

where:

Andx=0,1362 - optical density of test solution;

mx=1,0256 - a precisely weighed test sample;

andx=2 - aliquot analyzed solution;

V1=25 - volume of the volumetric flask for dilution of extraction from vegetable raw materials;

V2=50 - volume of extract from medicinal plant raw materials;

250 is the specific absorption coefficient cianids-3-O-glucoside.

The test was carried out in several replications. All data were statistically processed. The content of anthocyanins amounted 0,332±0,010%.

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

About 1.0 raw materials (a precisely weighed) placed in a conical flask with a ground joint with a capacity of 100 ml, add 50 ml of 95% ethanol containing 1% chlorotetracycline. Further the process is carried out in accordance with example 4. The optical density measured for 1 min after preparation of the test solution at the analytical wavelength of 622 nm. The calculation is carried out in terms of absolutely dry raw material by the following formula:

where:

Andx=0,2748 - optical density of test solution;

Ast=0,1808 optical density of the solution of the standard sample;

mx=0,9662 - a precisely weighed test sample;

mst=0,0226 - a precisely weighed cianids-3-O-glucoside;

andx=2 - aliquot analyzed solution;

V1=25 - volume of the volumetric flask for dilution of extraction from vegetable raw materials;

V2=50 - volume of extract from medicinal plant raw materials;

W - moisture content of raw materials, %.

The test has been carried out in several replications. 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 the pH-differential spectrophotometry, the content of anthocyanins in terms of cyanidin-3-O-glucoside was 0.61±0.03%, and the relative error of the method ±4,92%.

Example 7. Determination of anthocyanins in fruits of black currant dry air Ribes nigrum L., the Gooseberry family - Grossulariaceae, using a solution with�andariego sample cianids-3-O-glucoside.

About 1.0 raw materials (a precisely weighed) placed in a conical flask with a ground joint with a capacity of 100 ml, add 50 ml of 95% ethanol containing 1% hydrochloric acid. Further the process is carried out in accordance with example 1. The optical density measured for 1 min after preparation of the test solution at the analytical wavelength of 626 nm. The calculation is carried out in terms of absolutely dry raw material by the following formula:

where:

Andx=0,1632 - optical density of test solution;

Ast=0,1808 optical density of the solution of the standard sample;

mx=1,1702 - a precisely weighed test sample;

mst=0,0226 - a precisely weighed cianids-3-O-glucoside;

andx=5 - aliquot analyzed solution;

V1=25 - volume of the volumetric flask for dilution of extraction from vegetable raw materials;

V2=50 - volume of extract from medicinal plant raw materials;

W - moisture content of raw materials, %.

The test has been carried out in several replications, all the data is aggregated. The content of anthocyanins amounted 0,155±0,004%, relative error of the method is ±2.58 per cent. In the quantitative determination of anthocyanins using the pH-differential spectrophotometry, the content of anthocyanins in terms of cyanidin-3-O-glucoside accounted 0,065±0,004%, relative error of the method is ±15 percent.

Thus, the method of quantitative determination of total anthocyanins in medicinal plant raw materials first developed for the analysis of this group BASS and has the following advantages (Fig. 3).

1. Virtually ignores the contribution of other compounds (based on the known ability of anthocyanins to be painted in blue colour in alkaline medium).

2. Has lower time and financial costs when performing analysis in comparison with the prototype.

3. Characterized by a lower error of a single determination method for the considered examples.

SOURCES of INFORMATION:

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., M. Kura, Szmigiel V., J. Blaszczyk Anthocyanins in medicine. - Pol. J. Pharmacol. - 2003. - Vol. 55. - P. 699-702.

3. Kurkin V. A. Pharmacognosy: a Textbook for students of pharmaceutical universities / Ed. 2nd, revised and enlarged. - Samara: LLC "Etching", GOU VPO "Samara state medical University", 2007. - S. 1239

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.

Method of quantitative determination of anthocyanins in medicinal plant raw materials with SP�trophotometric, characterized in that the method is carried out by extraction of anthocyanins from medicinal plant raw water-alcohol mixtures containing 0.5 to 1.0% hydrochloric acid, with the concentration of ethyl alcohol of not less than 70% and the quantification of anthocyanins by optical density in 95% ethanol containing 0.5% of ammonia, on the background of the reference solution at the analytical wavelength of maximum absorption in the range 611-630 nm using a solution of the standard sample cianids-3-O-glucoside or using the values of its specific absorbance in 95% ethanol containing 0.5% of ammonia.



 

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2 tbl

FIELD: medicine.

SUBSTANCE: method involves scanning pulse with glass plate placed on pulse measurement point. The glass plate bears biological test system containing composition of 0.1% aqueous amino acid solutions like tryptophan, aspartic acid, alanine, treonine, valine, serine, glycine and tyrosine taken in equal proportions, 0.5% aqueous solution of dopamine, 0.5% aqueous solution of histamine, 12% aqueous solution of magnesium sulfate taken in 3:3:13 proportion. The plate is hold on pulse for 1-2 min before and 10-15 min after giving the pharma- or parapharmaceutic under study to a patient. Then, the plate is dried at T=+18-20°C during 2-3 min and another glass plate is covered with biological test system surrounded with wall of the pharma- or parapharmaceutic under study arranged along periphery and dried at T=+18-20°C during 2-3 min. The biological test system structures formed on the plates are compared in polarized light with quartz compensator. Structural similarity being detected, identification of pharma- or parapharmaceutic introduced into human organism is to be recorded.

EFFECT: enhanced effectiveness in identifying biologically active substances introduced into human organism.

26 dwg

FIELD: medicine, medicinal toxicology, microbiology, biology.

SUBSTANCE: invention relates to a method for testing different immune preparations, for example, immunoglobulins. Method involves carrying out the combined culturing test-strain of microorganism with the corresponding immune preparation under standard conditions of the human embryo monolayer of cultured cells. Results are estimated by degree of the adhesion decrease value of test-strain of microorganism as compared with control value. Invention provides elevating rate in evaluation of an anti-infectious activity of immune preparations based on approaching conditions of evaluation to macroorganism conditions.

EFFECT: improved method of evaluation.

2 tbl, 2 ex

FIELD: analytical chemistry.

SUBSTANCE: invention relates to method for quantitative determination of thiotriazoline and pyracetam in complex drugs by high performance chromatography, wherein silicagel with grafted 3-(chlorodimethyl)-propyl-N-dodecylcarbamate having particle size of 5 mum is used as sorbent; and degassed 0.05 M aqueous solution of potassium dihydrophosphate is used as mobile phase. Mobile phase velocity is 1 ml/min, and column temperature is 30°C. Method of present invention makes it possible to determine content of two abovementioned active ingredients simultaneously.

EFFECT: simplified process of sample preparation.

3 ex, 3 tbl

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