RU2554780C1 - Method for measuring flavonoid amount in choleretic tea № 3 - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: method is based on measuring a flavonoid amount by differential spectral photometry, calculated as Cinaroside at wave length 400 nm, extracting in water and alcohol and using 70% ethanol as an extraction fluid; the total flavonoid amount calculated as Cinaroside and an absolutely dry raw material in percentage (X) is calculated by formula.

EFFECT: method enables measuring the total flavonoid amount as biologically active components having a basic therapeutic action.

3 ex, 14 dwg, 2 tbl

Description

The invention relates to the pharmaceutical industry and can be used in drug quality control centers and analytical laboratories when analyzing flavonoids in “Cholagogue collection No. 3” (“Phytohepatol No. 3”).

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

"Cholagogue collection No. 3" is widely used in modern medicine in the complex treatment of diseases of the liver and biliary tract (2, 5, 6). The pharmacological effect of this drug collection is due to various groups of biologically active compounds. The composition of the collection is represented by chamomile flowers, peppermint leaves, marigold flowers, yarrow grass, tansy flowers. The designation in the chamomile, peppermint, yarrow and tansy of the two most important groups of active substances - essential oil and flavonoids allows us to talk about forecasting (8). It is known that the antispasmodic and anti-inflammatory effect of the drug is manifested due to the presence of essential oil, while the choleretic effect is due to the second group of biologically active compounds with flavonoids. The presence of calendula flowers in the collection enhances the anti-inflammatory effect of the drug collection (5, 6, 8). According to the regulatory documentation for "Cholagogue collection No. 3" quality is determined by the content of essential oil (1). Taking into account the fact that the components of the essential oil are practically insoluble in water upon receipt of the infusion, the level of the essential oil in "Cholagogue collection No. 3" does not allow us to evaluate the effectiveness of this drug. In our opinion, in the indicated collection it is advisable to determine the content of water-soluble active substances - flavonoids, since they determine the main pharmacological action, namely, the choleretic effect of the collection (9, 10).

Thus, the aim of the invention is to develop a method for the analysis of flavonoids in "Cholagogue collection No. 3".

The technical result of the proposed method is to improve the method of quantitative analysis "Cholagogue collection No. 3" by identifying flavonoids as biologically active compounds that determine the main pharmacological properties of the collection.

The technical result is achieved by the quantitative determination of the amount of flavonoids by differential spectrophotometry, in terms of cinaroside, at a wavelength of 400 nm, water-alcohol extraction and use of 70% ethyl alcohol as an extractant, while the content of the amount of flavonoids in terms of cinaroside and absolutely dry raw materials in percent (X) is calculated by the formula:

, $$X=\frac{D\ast m_0\ast \mathrm{thirty}\ast 10\ast 25\ast \mathrm{one\; hundred}\ast \mathrm{one\; hundred}}{D_0\ast m\ast 25\ast \mathrm{one}\ast 25\ast (\mathrm{one\; hundred}-W)}$$

,

where D is the optical density of the test solution;

D ₀ - the optical density of the solution of the state standard sample of cinaroside;

m is the mass of raw materials, g;

m ₀ is the mass of the state standard sample of cinaroside, g;

W is the loss in mass upon drying in percent;

in the absence of a standard sample of cinaroside, the theoretical value of the specific absorption rate of its alcohol solution is used - 350, and absolutely dry raw materials in percent (X) are calculated by the formula:

$$X=\frac{D\ast \mathrm{thirty}\ast 25\ast \mathrm{one\; hundred}}{m\ast 350\ast (\mathrm{one\; hundred}-W)}$$

where D is the optical density of the test solution;

m is the mass of raw materials, g;

спиртового раствора государственного стандартного образца цинарозида при 400 нм;350 - specific absorption rate $$\left(E_{\mathrm{one}\mathrm{from}m}^{\%}\right)$$

alcohol solution of the state standard sample of cynaroside at 400 nm;

W is the loss in mass of raw materials upon drying, in percent.

During the analysis, the optical density of the complex of flavonoids with aluminum chloride of the analyzed solution is measured against the background of the initial solution. In this case, a bathochromic shift of the long-wavelength band of flavonoids was observed, which is detected in the UV spectrum as the absorption maximum at a wavelength of 406 nm. This is confirmed by differential spectrophotometry: the absorption maximum at 406 nm (Fig. 1). A study of the UV spectra of the state standard sample of cinaroside showed (Fig. 2) that the solution of this standard in the presence of aluminum chloride has a close absorption maximum (400 nm). Therefore, cinaroside can be used in the analysis procedure as a standard sample.

We studied the spectral characteristics of water-alcohol extracts from plant materials that make up the Cholagogue collection No. 3 (Fig. 3), to determine the content of the sum of flavonoids in each of the components of the collection (Fig. 4).

Based on the spectral characteristics of the components of the collection and the model mixture (Fig. 1, 3), having determined the optimal extraction conditions for Cholagogue collection No. 3, we developed a method for determining the amount of flavonoids in "Cholagogue collection No. 3" by differential spectrophotometry in terms of cynaroside.

The method is implemented as follows.

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 of crushed raw materials (accurate weighed) is placed in a flask with a thin section with a capacity of 50 ml, 30 ml of 70% ethyl alcohol 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 90 minutes. Then the flask is closed with the same stopper, weighed again and make up the missing extractant to the original mass. The extract was filtered through a paper filter and cooled for 30 minutes. The test solution is prepared as follows: 1 ml of the obtained extract is placed in a volumetric flask with a capacity of 25 ml, 1 ml of 3% alcohol solution of aluminum chloride is added and the solution volume is adjusted to the mark with 95% ethyl alcohol (test solution A). As a comparison solution, a solution prepared under the same conditions, but without the addition of aluminum chloride (comparison solution A), is used. The measurement of optical density is carried out on a spectrophotometer at a wavelength of 400 nm. At the same time, the optical density of the solution of the state standard sample of cynaroside at a wavelength of 400 nm, prepared by analogy with the test solution (see note), is measured.

Note. Preparation of a solution of cynaroside standard sample. About 0.02 (accurately weighed) of cinaroside is placed in a volumetric flask with a capacity of 25 ml, dissolved in 15-20 ml of 70% ethyl alcohol when heated in a water bath. After cooling the contents of the flask to room temperature, bring the solution volume with 70% ethyl alcohol to the mark (solution A). 1 ml of cinaroside solution A is placed in a 25 ml volumetric flask, 1 ml of a 3% alcohol solution of aluminum chloride is added and the volume of the solution is 95% ethyl alcohol to the mark (test solution B). As a comparison solution, a solution is used which is prepared as follows: 10 ml of cinaroside solution A is placed in a 25 ml volumetric flask and the solution volume is adjusted to the mark with 95% ethanol (comparison solution B).

The content of the sum of flavonoids in terms of cinaroside and absolutely dry raw materials in percent (X) is calculated by the formula:

, $$X=\frac{D\ast m_0\ast \mathrm{thirty}\ast 10\ast 25\ast \mathrm{one\; hundred}\ast \mathrm{one\; hundred}}{D_0\ast m\ast 25\ast \mathrm{one}\ast 25\ast (\mathrm{one\; hundred}-W)}$$

,

where D is the optical density of the test solution;

D ₀ - the optical density of the solution of the state standard sample of cinaroside;

m is the mass of raw materials, g;

m ₀ is the mass of the state standard sample of cinaroside, g;

W is the loss in mass upon drying in percent;

in the absence of a standard sample of cinaroside, the theoretical value of the specific absorption rate of its alcohol solution is used - 350, and absolutely dry raw materials in percent (X) are calculated by the formula:

$$X=\frac{D\ast \mathrm{thirty}\ast 25\ast \mathrm{one\; hundred}}{m\ast 350\ast (\mathrm{one\; hundred}-W)}$$

where D is the optical density of the test solution;

m is the mass of raw materials, g;

спиртового раствора государственного стандартного образца цинарозида при 400 нм;350 - specific absorption rate $$\left(E_{\mathrm{one}\mathrm{from}m}^{\%}\right)$$

alcohol solution of the state standard sample of cynaroside at 400 nm;

W is the loss in mass of raw materials upon drying, in percent.

The proposed method is illustrated by the following examples.

Example 1

An analytical sample of raw materials (collection “Phytohepatol No. 3” (“Cholagogue collection No. 3”), OJSC Krasnogorsklexredstva, series 60912) is crushed to the size of particles passing through a sieve with holes with a diameter of 1 mm. About 1 g of crushed raw materials (accurate weighed) is placed in a flask with a thin section with a capacity of 50 ml, 30 ml of 70% ethyl alcohol 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 90 minutes. Then the flask is closed with the same stopper, weighed again and make up the missing extractant to the original mass. The extract was filtered through a paper filter and cooled for 30 minutes. The test solution is prepared as follows: 1 ml of the obtained extract is placed in a volumetric flask with a capacity of 25 ml, 1 ml of a 3% alcohol solution of aluminum chloride is added and the solution volume is adjusted to the mark with 95% ethyl alcohol (test solution A). As a comparison solution, a solution prepared under the same conditions, but without the addition of aluminum chloride (comparison solution A), is used. The measurement of optical density is carried out on a spectrophotometer at a wavelength of 400 nm. In parallel, the optical density of a solution of a standard sample of cinaroside at a wavelength of 400 nm, prepared by analogy with the test solution (see note), is measured.

The content of the sum of flavonoids in terms of cinaroside and absolutely dry raw materials in percent (X) is calculated by the formula:

$$X=\frac{0.413569\ast 0.0014\ast \mathrm{thirty}\ast \mathrm{one}\ast 25\ast \mathrm{one\; hundred}\ast \mathrm{one\; hundred}}{0.9562\ast \mathrm{0,9973}\ast 25\ast \mathrm{one}\ast 25\ast (\mathrm{one\; hundred}-10)}$$

The optical density D = 0.413569, the mass of the raw material m = 0.9973.

The content of the total flavonoid = 1.05%.

All results were statistically processed. The single error of the quantitative determination was ± 3.12%.

Example 2

They open 10 filter bags (collection “Phytohepatol No. 3” (“Cholagogue collection No. 3”), OJSC “Krasnogorsklexredstva”, filter bags, series 30213). About 1 g of crushed raw materials (accurate weighed) is placed in a flask with a thin section with a capacity of 50 ml. Next, the process is carried out in accordance with example 1.

The content of the sum of flavonoids in terms of cinaroside and absolutely dry raw materials in percent (X) is calculated by the formula:

$$X=\frac{0.597248\ast 0.0014\ast \mathrm{thirty}\ast \mathrm{one}\ast 25\ast \mathrm{one\; hundred}\ast \mathrm{one\; hundred}}{0.9562\ast \mathrm{1,0016}\ast 25\ast \mathrm{one}\ast 25\ast (\mathrm{one\; hundred}-10)}$$

The optical density D = 0.597248, the mass of the raw material m = 1.0016.

The content of the amount of flavonoids = 1.16%.

All results were statistically processed. A single error in the quantification was ± 3.28%.

Example 3

An analytical sample of the model mixture with the composition shown in Fig. 5, crushed to a particle size passing through a sieve with holes with a diameter of 1 mm About 1 g of crushed raw materials (accurate weighed) is placed in a flask with a thin section with a capacity of 50 ml. Next, the process is carried out in accordance with example 1.

The content of the sum of flavonoids in terms of cinaroside and absolutely dry raw materials in percent (X) is calculated by the formula:

$$X=\frac{0.526487\ast 0.0014\ast \mathrm{thirty}\ast \mathrm{one}\ast 25\ast \mathrm{one\; hundred}\ast \mathrm{one\; hundred}}{0.9562\ast \mathrm{0,9963}\ast 25\ast \mathrm{one}\ast 25\ast (\mathrm{one\; hundred}-10)}$$

The optical density D = 0.526487, the mass of the raw material m = 0.9963.

The content of the total flavonoid = 1.03%.

All results were statistically processed. A single error in the quantification was ± 3.19%.

Thus, the proposed method for the quantitative determination of the amount of flavonoids in "Cholagogue collection No. 3" was developed for the first time to analyze this group of biologically active compounds of the collection and has the following advantages over the methodology presented in the Pharmacopoeia article:

1. The definition is based on the content of water-soluble substances - flavonoids, instead of essential oils, the components of which are practically insoluble in water.

2. The specified method allows to evaluate the content of the sum of flavonoids as biologically active components that have the main therapeutic effect - choleretic effect.

3. The developed method is specific because it allows to exclude the contribution of other compounds (the ability of flavonoids to form complexes with aluminum chloride is taken into account).

INFORMATION SOURCES:

1. VFS 42-2558-95. Cholagogue collection No. 3 / Pharmacopoeia State Committee. Enter 07/09/1996. - M., 1996. - 12 p.

2. The state register of medicines. Official publication as of April 1, 2009: in 2 volumes T. 1. - M.: Medical Council Publishing House, 2009. - 1359 p.

3. The State Pharmacopoeia of the USSR. - Eleventh edition. - Vol. 1. - M .: Medicine, 1987. - 336 p.

4. The State Pharmacopoeia of the USSR. - Eleventh edition. - Vol. 2. - M .: Medicine, 1990. - 400 p.

5. Kurkin V.A. Fundamentals of herbal medicine: A manual for students of pharmaceutical universities. - Samara: Etching LLC, GOU VPO SamGMU of Roszdrav, 2009. - 963 p.

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

7. Kurkin V.A. Prospects for the creation and implementation of import-substituting herbal medicines / V.A. Kurkin, E.V. Avdeeva, V.B. Braslavsky, O.E. Pravdivtseva, A.V. Kurkina et al. // XVIII Russian National Congress "Man and Medicine": abstracts. - Moscow, 2011 .-- S. 507.

8. Kurkina A.V. Actual aspects of standardization of medicinal plants containing flavonoids / A.V. Kurkina // Materials of the Interregional Scientific Conference with international participation dedicated to the 70th anniversary of the Faculty of Pharmacy of the Siberian State Medical University. - Tomsk, 2011 .-- S. 86-90.

9. Kurkina A.V. New approaches to standardization of medicinal plant materials containing flavonoids. 1. Tansy ordinary / A.V. Kurkina // 65th Interregional Conference on Pharmacy and Pharmacology "Development, research and marketing of new pharmaceutical products": a collection of scientific papers. - Pyatigorsk, 2011. - Issue. 66. - S. 134-137.

10. Kurkina A.V. Flavonoids as a source of drugs based on raw materials of pharmacopeia plants / A.V. Kurkina // 3rd International Congress of Phytotherapists and Herbalists "Modern Problems of Herbal Medicine and Herbalism": proceedings of scientific papers. - M., 2013 .-- S. 111-114.

Claims (1)

The method for the quantitative determination of flavonoids in "Cholagogue collection No. 3" is carried out by differential spectrophotometry, calculated on cinaroside, at a wavelength of 400 nm, water-alcohol extraction and using 70% ethyl alcohol as extractant, while the content of the total flavonoids in terms of cinaroside and absolutely dry raw materials in percent (X) is calculated by the formula:

,
where D is the optical density of the test solution;
D _o - the optical density of the solution of the state standard sample of cinaroside;
m is the mass of raw materials, g;
m _o is the mass of the state standard sample of cinaroside, g;
W is the loss in mass upon drying in percent;
in the absence of a standard sample of cinaroside, the theoretical value of the specific absorption rate of its alcohol solution is used - 350, and absolutely dry raw materials in percent (X) are calculated by the formula:

where D is the optical density of the test solution;
m is the mass of raw materials, g;
350 - specific absorption rate

alcohol solution of the state standard sample of cynaroside at 400 nm;
W is the loss in mass of raw materials upon drying, in percent.

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