RU2475724C2 - Method of quantitative determination of flavonoids in vegetable raw material by fluorimetric method - Google Patents

Abstract

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention describes method of quantitative determination of flavonoids in vegetable raw material, includes exhaustive extraction of vegetable raw material with ethyl alcohol, concentration, optimal for each type of raw material, obtaining alcohol extract, containing flavonoids, addition of aluminium chloride solution to extract, where fluorimetry of obtained solution is performed at excitation wavelengths and registration wavelengths which are individual for each particular compound of flavonoid nature, relative to alcohol aluminium chloride solution in comparison with standard solutions of individual flavonoids, processed in the same conditions as extraction from raw material.

EFFECT: invention ensures quantitative determination of individual flavonoids in vegetable raw material.

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Description

The invention relates to pharmacy, namely to pharmaceutical chemistry, and can be used for the quantitative determination of pharmacologically active substances - flavonoids in medicinal plant materials.

A known method for the quantitative determination of flavonoids by gravimetry, spectrophotometry, photocolorimetry (Natural flavonoids / D.Yu. Korulkin [et al.]. - Novosibirsk: Academic publishing house 'Theo, 2007. - 232 p.); polyphenolic compounds by the photometric method (SU 1215708, A61K 35/78, 1986); routine by applying a reaction with diazotized sulfanilamide, accompanied by the appearance of an orange color (SU 139473, 1961).

The prototype of the developed method is a method for determining riboflavin by the fluorimetric method (FS 42 7024-02 of March 21, 2002 "Riboflavin").

It consists in preparing the test solution by dissolving riboflavin in an aqueous solution of acetic acid. In a similar manner, a solution of a standard riboflavin sample is prepared. Measure the fluorescence intensity of the standard solution at 530 nm (excitation wavelength of about 444 nm). After the measurement, hydrosulfite is added to the sodium solution and the fluorescence intensity is measured. The difference between the two readings is equal to the fluorescence intensity of the standard sample.

Similarly measure the fluorescence intensity of the test sample. The content of riboflavin in the substance (%) is calculated by the formula.

The spectrophotometric technique previously used allows one to obtain information on the quantitative content of the total amount of flavonoids in plant materials in terms of the standard of an individual substance, which is rather arbitrary, the developed method is highly specific and allows to determine the content of individual flavonoids (rutin, quercetin) in various types of medicinal plant materials.

The objective of the invention is the development of a new, specific and highly sensitive method for controlling the quality of medicinal plant materials by the quantitative content of active substances - flavonoids.

The technical result of the invention is the ability to determine the content of individual flavonoids (rutin, quercetin) in various types of medicinal raw materials without prior separation.

The method for the quantitative determination of flavonoids in plant materials involves exhaustive extraction of plant materials with ethyl alcohol, the concentration optimal for each type of raw materials, obtaining alcoholic extracts containing flavonoids by adding aluminum chloride to the alcoholic solution, according to the invention, fluorimetry of the resulting solution is carried out at excitation wavelengths and lengths waves of registration, which are individual for each particular compound of flavonoid nature, relatively alcohol solution of aluminum chloride in comparison with standard solutions of individual flavonoids processed under the same conditions as extraction from raw materials.

The proposed method, unlike the available ones, allows obtaining information on the quantitative content in plant raw materials not of the sum of flavonoids in terms of the standard of an individual substance, which is rather arbitrary, but to determine the content of an individual flavonoid (rutin, quercetin), in the individual spectral maxima of each of which determination is made.

Example 1. Determination of the routine in the grass of Hypericum perforatum

An analytical sample of the raw material is crushed to the size of the particles passing through a sieve with holes with a diameter of 1 mm. About 1 g (accurately weighed) of the crushed raw material is placed in a flask with a thin section with a capacity of 150 ml, 30 ml of 50% alcohol are added. The flask was attached to a reflux condenser and heated in a boiling water bath for 30 minutes. The hot extract is filtered through cotton wool into a 100 ml volumetric flask so that the particles of the feed do not enter the filtrate. Cotton wool is placed in an extraction flask and 30 ml of 50% alcohol are added. The extraction is repeated twice more under the conditions described above, filtering the extraction into the same volumetric flask. After cooling, the extraction volume was adjusted with 50% alcohol to the mark and stirred (solution A).

In a volumetric flask with a capacity of 25.0 ml, 1.0 ml of solution A, 1.0 ml of a solution of aluminum chloride in 95% alcohol are placed and the volume of the solution with 95% alcohol is adjusted to the mark. After 40 minutes, measurements are taken on a fluorimeter at a wavelength of 426 nm for excitation, 512 nm for registration in a cuvette with a layer thickness of 10 mm.

The background solution is a solution consisting of 1.0 ml of an alcohol solution of aluminum chloride, adjusted to 25.0 ml with 95% ethyl alcohol.

As a standard solution, a solution is used consisting of 1.0 ml of a standard solution of rutin, 1 drop of diluted acetic acid, 1 ml of a 2% solution of aluminum chloride, brought 95% alcohol to the mark in a 25 ml volumetric flask.

Data on the content of rutin in the studied object in concentration units are read from the display of the device.

Example 2. The definition of the routine in the grass cyanosis blue

An analytical sample of raw materials (about 1.0 g), crushed to a particle size passing through a sieve with holes with a diameter of 1 mm, is placed in a 150 ml thin flask and extracted with 25 ml portions of ethyl alcohol twice in a boiling water bath with a reverse refrigerator for two hours. The extracts are filtered into a 100 ml flask through a pleated filter and adjusted to 70% with ethyl alcohol.

1.0 ml of the filtrate was taken, placed in a 25.0 ml volumetric flask, 1.0 ml of a 2% solution of aluminum chloride in 95% ethyl alcohol was added, adjusted to 95% with ethyl alcohol and fluorimetered after 40 minutes at the excitation wavelength 426 nm, registration 512 nm in a cell with a layer thickness of 10 mm

The background solution is a solution consisting of 1.0 ml of an alcohol solution of aluminum chloride, brought in a flask to 25.0 ml of ethyl alcohol 95% to the mark.

As a standard solution, use a solution consisting of 1.0 ml of a standard solution of rutin, 1 ml of a 2% solution of aluminum chloride, brought 95% alcohol to the mark in a 25 ml volumetric flask.

Data on the content of rutin in the studied object in concentration units are read from the display of the device.

Note. Preparation of a solution of the State standard sample (GSO) rutin: about 0.05 g (accurately weighed) GSO rutin, previously dried at a temperature of 130-135 ° C for 3 hours, dissolved in 85 ml of 95% alcohol in a 100 ml volumetric flask at heated in a water bath, cooled, quantitatively transferred to a volumetric flask with a capacity of 100 ml, bring the volume of the solution with the same alcohol to the mark and mix.

Example 3. The determination of quercetin in the grass of the mountaineer pepper

An analytical sample of the raw material is crushed to the size of the particles passing through a sieve with holes with a diameter of 1 mm. About 1 g (accurately weighed) of the raw material is placed in a flask with a thin section with a capacity of 150 ml, 30 ml of 90% alcohol containing 1% concentrated hydrochloric acid are added, the flask is attached to a reflux condenser and heated in a boiling water bath for 30 minutes. Then the flask was cooled to room temperature and filtered through a paper filter into a 100 ml volumetric flask. The extraction is repeated once more in the above manner, then 1 more time with 90% alcohol for 30 minutes. The extracts are filtered through the same filter into the same volumetric flask, washed with 90% alcohol and the filtrate volume adjusted with 90% alcohol to the mark (solution A).

In a volumetric flask with a capacity of 25 ml, add 2 ml of solution A, add 2 ml of a 1% solution of aluminum chloride in 95% alcohol and adjust the volume of the solution with 95% alcohol to the mark. After 20 minutes, measurements are taken on a fluorimeter at a wavelength of excitation of 460 nm, by registration of 495 nm in a cell with a layer thickness of 10 mm. The background solution is a solution consisting of 1.0 ml of an alcohol solution of aluminum chloride, adjusted to 25.0 ml with 95% ethyl alcohol. As a standard solution, a solution is used consisting of 1.0 ml of a standard solution of quercetin, 2 ml of a 1% solution of aluminum chloride, brought 95% alcohol to the mark in a 25 ml volumetric flask.

Data on the content of quercetin in the studied object in concentration units are read from the display of the device.

Note. Preparation of a solution of the State standard sample (GSO) of quercetin: about 0.05 g (accurately weighed) of GSO quercetin, previously dried at a temperature of 130-135 ° C for 3 hours, dissolved in 85 ml of 95% alcohol in a 100 ml volumetric flask at heated in a water bath, cooled, quantitatively transferred to a volumetric flask with a capacity of 100 ml, bring the volume of the solution with the same alcohol to the mark and mix.

The table shows the results of the determination of flavonoids of the proposed method in various plant materials.

The proposed method allows to obtain information on the quantitative content in plant materials, not the sum of flavonoids in terms of standard, but to determine the content of an individual flavonoid (rutin, quercetin), at specific maximums of determination of each of which a determination is made.

Table Plant object The content of flavonoids,% Hypericum perforatum herb 0.14 Routine Cyanosis grass blue 0,039 Routine Highlander Grass 0.0045 Quercetin

Claims (1)

The method for the quantitative determination of flavonoids in plant materials includes exhaustive extraction of plant materials with ethyl alcohol, the concentration optimal for each type of raw materials, production of alcoholic extracts containing flavonoids, adding to the extraction of an alcoholic solution of aluminum chloride, characterized in that the resulting solution is fluorimetric at excitation wavelengths and wavelengths for registration, which are individual for each particular compound of flavonoid nature, relatively alcohol solution of aluminum chloride in comparison with standard solutions of individual flavonoids processed under the same conditions as extraction from raw materials.