RU2752316C1 - Method for quantifying amount of flavonoids in leaves of ordinary lilac - Google Patents

Abstract

FIELD: chemical and pharmaceutical industry.SUBSTANCE: invention relates to the chemical and pharmaceutical industry and can be used in drug quality control centers and control and analytical laboratories for the quantitative determination of the amount of flavonoids in the leaves of ordinary lilac (Syringa vulgaris L.). A method is described for the quantitative determination of the amount of flavonoids in the leaves of ordinary lilac by obtaining an aqueous-alcohol extract from vegetable raw materials by extraction of 1 g of an exact sample of vegetable raw materials crushed to a particle size of 1 mm with 70% ethyl alcohol, followed by sample preparation and determination of optical density by differential spectrophotometry using a standard sample of rutin, and in its absence, using a theoretical specific absorption index, the extraction of crushed leaves of ordinary lilac is carried out for 45 minutes at a ratio of raw materials: extractant 1:50; quantitative determination of the amount of flavonoids in ordinary lilac leaves is carried out at a wavelength of 412 nm in terms of rutin and the content of the amount of flavonoids in terms of rutin and absolutely dry raw materials is calculated by the formulawhere x is the content of the sum of flavonoids in terms of rutin,%; D is the optical density of the test solution; Dois the optical density of the solution of the State Standard sample of rutin; m is the mass of raw materials, g; mois the mass of the State Standard sample of rutin, g; W is the mass loss during drying,%, in the absence of a standard sample of rutin, it is advisable to use the theoretical value of its specific absorption index equal to 240:where x is the content of the amount of flavonoids in terms of rutin,%; D is the optical density of the test solution; m is the mass of raw materials, g; mois the mass of the State Standard sample of rutin, g; 240 is the specific absorption index of the State Standard sample of rutin at 412 nm; W is the mass loss during drying,%.EFFECT: described method is effective for quantifying the amount of flavonoids in the leaves of ordinary lilac.1 cl, 3 dwg, 4 tbl, 2 ex

Description

The invention relates to the chemical-pharmaceutical industry and can be used in quality control centers for medicines and analytical laboratories for quantitative determination of the amount of flavonoids in the leaves of common lilac (Syringa vulgaris L.).

The current drug quality control system requires constant improvement of approaches to the standardization of biologically active compounds (BAS) using modern methods of analysis and up-to-date data on their physicochemical, spectral and pharmacological properties, allowing to objectively and selectively determine the content of target substances [4].

Common lilac (Syringa vulgaris L.) is one of the plant species not included in the State Pharmacopoeia of the Russian Federation of the XIV edition, but as a medicinal plant material (MPR), as a source of the state standard sample (SSS) - syringin, bark is registered (VFS 42- 2106-92) [1,2]. Preparations based on lilac bark have a wide range of pharmacological activity: immunomodulatory, adaptogenic, anxiolytic and antidepressant effects [3].

Of particular interest, along with the pharmacopoeial raw material - bark, are the leaves of this plant. Common lilac leaves (Syringa vulgaris L.) are a promising source of ALS with diuretic and anti-inflammatory effects. However, the problem of standardizing this type of raw material remains unresolved.

The prototype for the development of this technique is a method for the quantitative determination of flavonoids in black poplar leaves. It is based on differential spectrophotometry using a standard sample of rutin. The disadvantage of this technique is the impossibility of its application to the raw material of common lilac leaves [8].

Thus, the aim of the invention is to develop a method for the quantitative determination of the amount of flavonoids in the leaves of common lilac, which has high specificity, accuracy and reproducibility.

The technical result is to create a method for the quantitative determination of the amount of flavonoids in the leaves of common lilac in terms of rutin.

The technical result is achieved by the fact that a water-alcohol extraction from plant raw materials is preliminarily obtained by extraction of 1 g of an exact weighed portion of a plant material crushed to a particle size of 1 mm with 70% ethyl alcohol, in terms of a substance of a flavonoid nature, by the method of differential spectrophotometry in relation to raw material-extractant "- 1:50, with the degree of grinding of raw materials 1 mm. Determination of flavonoids is carried out at a wavelength of 412 nm in terms of rutin and absolutely dry raw materials are calculated by the formula:

where:

x - the content of the sum of flavonoids in terms of rutin,%;

D is the optical density of the test solution;

D _o - optical density of the solution of the State standard sample rutin;

m is the mass of raw materials, g;

m _o - mass of the State standard sample rutin, g;

W is the loss in mass on drying,%;

in the absence of a standard sample of rutin for the calculation, it is advisable to use the theoretical value of its specific absorption index, equal to 240:

where:

x - the content of the sum of flavonoids in terms of rutin,%;

D is the optical density of the test solution;

m is the mass of raw materials, g;

Государственного стандартного образца рутина при длине волны 412 нм;240 - specific absorption rate

State standard sample rutin at a wavelength of 412 nm;

W - loss in mass on drying,%.

When studying the spectral characteristics, it was revealed that it is rutin that determines the nature of the absorption curve of aqueous-alcoholic extract from the leaves of common lilac. It was determined that in the UV spectrum of the aqueous-alcoholic extract of common lilac there is a bathochromic shift of the long-wavelength band of flavonoids (Figure 1), as in the case of rutin (Figure 2), where curve 1 in Figure 1 and Figure 2 shows the initial solution of the aqueous alcoholic extract from the leaves of common lilac or the initial solution of rutin, respectively, and curve 2 is a solution of aqueous-alcoholic extract from the leaves of common lilac in the presence of aluminum chloride or a solution of rutin in the presence of aluminum chloride, respectively.

The study of the UV spectra of figure 2 (where curve 1 is a rutin solution, and curve 2 is a rutin solution with the addition of aluminum chloride) showed that the GSO rutin solution in the presence of aluminum chloride has an absorption maximum at a wavelength of 412 nm. In the UV spectrum of the aqueous-alcoholic extract from the leaves of common lilac in the differential version in Figure 3, an absorption maximum is also found at a wavelength of 412 nm, which corresponds to the absorption maximum of an alcoholic solution of rutin.

This fact allows for the spectrophotometric determination of the amount of flavonoids in the leaves of common lilac at an analytical wavelength of 412 nm.

We also studied the effect of the extractant on the extraction process. Table 1 shows the dependence of the yield of flavonoids in common lilac leaves on the concentration of the extractant. As a result of the experiment, we chose 70% ethyl alcohol as the optimal extractant, since the yield of active substances from raw materials is maximum when it is used.

Next, we studied the issue of the duration of extraction in a boiling water bath, table 2 shows the dependence of the yield of flavonoids in common lilac leaves on the time of extraction in a boiling water bath, while the extraction time was selected at 45 minutes, at which the maximum yield of substances was observed.

Table 3 shows the dependence of the yield of flavonoids in common lilac leaves on the "raw material-extractant" ratio. From table 3 it can be seen that the maximum yield of active substances is observed at the ratio of "raw material-extractant" 1:50, for this reason, this ratio was chosen by us as the optimal one.

The final stage was the study of the influence of the degree of grinding of raw materials on the yield of phenylpropanoids from flowers of common lilac. From table 4 it follows that the maximum yield of substances was observed when extracting particles passing through a sieve with a diameter of 1 mm.

Taking into account that an increase in the number of operations at the stage of sample preparation leads to an increase in the error, the choice was made in favor of a one-stage extraction process with confirmation of the required accuracy of quantitative determination.

Thus, it was determined that the optimal extraction parameters are: a single extraction with 70% ethyl alcohol in a boiling water bath for 45 minutes in the ratio of "raw material-extractant" - 1:50, the degree of particle size is 1 mm.

Taking into account the fact that rutin is specific for common lilac leaves, and the absorption maxima of rutin solution and aqueous-alcoholic extract of common lilac leaves are in the region of 412 nm, it is expedient to determine the content of the sum of flavonoids in terms of rutin at a wavelength of 412 nm.

The method is implemented as follows.

An analytical sample of the air-dry raw material of common lilac is crushed to a particle size passing through a sieve with holes 1 mm in diameter. About 1 g of an exact sample of the crushed raw material is placed in a flask with a thin section with a capacity of 100 ml, 50 ml of 70% ethyl alcohol are added. Close the flask with a stopper and weigh on a tare balance with an accuracy of ± 0.01. The flask is connected to a reflux condenser and heated in a boiling water bath (moderate boil) for 45 minutes. Then the flask is cooled for 30 minutes, closed with the same stopper, weighed again, and the missing extractant is replenished to the original weight. The extract is filtered through a red stripe filter (herb extract).

The test solution for the analysis of the sum of flavonoids is prepared as follows: 1 ml of the obtained extract is placed in a 25 ml volumetric flask, 1 ml of a 3% alcoholic solution of aluminum chloride is added and the volume of the solution is brought to the mark with 96% ethyl alcohol (test solution).

The reference solution is prepared as follows: 1 ml of the obtained extract is placed in a 25 ml volumetric flask, the volume of the solution is brought to the mark with 96% ethyl alcohol (reference solution).

To calculate the content of the sum of flavonoids, a solution of a standard sample of rutin is prepared, a 3% alcohol solution of aluminum chloride is added to it, the optical density of the colored complex is measured at an analytical wavelength of 412 nm, and the determined value of the optical density is used in the calculation formula.

Preparation of rutin standard sample solution

About 0.025 g (accurately weighed) of rutin is placed in a volumetric flask with a capacity of 50 ml, dissolved in 30 ml of 70% ethyl alcohol when heated in a water bath. After cooling the contents of the flask to room temperature, bring the volume of the solution to the mark with 70% ethyl alcohol (rutin solution A). Then 1 ml of rutin solution A is placed in a 25 ml volumetric flask, 1 ml of 3% alcoholic solution of aluminum chloride is added and the volume of the solution is adjusted to the mark with 96% ethyl alcohol (test solution B of rutin). The reference solution is prepared as follows: 1 ml of the resulting solution is placed in a 25 ml volumetric flask, the volume of the solution is adjusted to the mark with ethyl alcohol 96%.

Measurement of optical density is carried out at a wavelength of 412 nm 40 minutes after the preparation of all solutions.

The content of the sum of flavonoids (x,%) in terms of rutin and absolutely dry raw materials is calculated by the formula:

where:

D is the optical density of the test solution;

D _o - optical density of the solution of the State standard sample rutin;

m is the mass of raw materials, g;

m _o - mass of the State standard sample rutin, g;

W is the percentage loss on drying.

In the absence of a standard sample of rutin for the calculation, it is advisable to use the theoretical value of its specific absorption index, equal to 240:

where:

D is the optical density of the test solution;

m is the mass of raw materials, g;

Государственного стандартного образца рутина при 412 нм;240 - specific absorption rate

State standard sample rutin at 412 nm;

W is the percentage loss on drying.

The proposed method is illustrated by the following examples.

Example 1.

An analytical sample of common lilac raw materials (harvested in the Botanical Garden of Samara University, 2016) is crushed to a particle size passing through a sieve with holes 1 mm in diameter. 0.9907 g of crushed raw materials are placed in a flask with a thin section with a capacity of 100 ml, add 50 ml of 70% ethyl alcohol. Close the flask with a stopper and weigh on a tare balance with an accuracy of ± 0.01. The flask is connected to a reflux condenser and heated in a boiling water bath (moderate boil) for 45 minutes. Then the flask is cooled for 30 minutes, closed with the same stopper, weighed again, and the missing extractant is replenished to the original weight. The extract is filtered through a filter paper (red stripe).

The test solution for the analysis of the sum of flavonoids is prepared as follows: 1 ml of the obtained extract is placed in a 25 ml volumetric flask, 1 ml of a 3% alcoholic solution of aluminum chloride is added and the volume of the solution is adjusted to the mark with 96% ethyl alcohol (test solution).

The reference solution is prepared as follows: 1 ml of the obtained extract is placed in a 25 ml volumetric flask, the volume of the solution is adjusted to the mark with ethyl alcohol 96% (reference solution).

To calculate the content of the sum of flavonoids, a solution of a standard sample of rutin is prepared, a 3% alcoholic solution of aluminum chloride is added to it, the optical density of the colored complex is measured at a wavelength of 412 nm, and the determined value of the optical density is used in the calculation formula.

Preparation of rutin standard sample solution

0.025 g of rutin is placed in a volumetric flask with a capacity of 50 ml, dissolved in 30 ml of 70% ethyl alcohol when heated in a water bath. After cooling the contents of the flask to room temperature, bring the volume of the solution to the mark with 70% ethyl alcohol (rutin solution A). Then 1 ml of rutin solution A is placed in a 25 ml volumetric flask, 1 ml of a 3% alcohol solution of aluminum chloride is added, then the volume of the solution is brought to the mark with 96% ethyl alcohol. The reference solution is prepared as follows: 1 ml of the resulting solution is placed in a 25 ml volumetric flask, the volume of the solution is adjusted to the mark with ethyl alcohol 96%.

Measurement of optical density is carried out at a wavelength of 412 nm 40 minutes after the preparation of all solutions.

The content of the sum of flavonoids (x,%) in terms of rutin and absolutely dry raw materials is calculated by the formula:

where:

0.6653 is the optical density of the test solution;

0.4202 - optical density of a solution of a standard sample of rutin;

0.9907 - weight of raw materials, g;

0.0202 - mass of a standard sample of rutin, g;

10 - loss in weight on drying in percent.

X = 3.59%.

The content of the sum of flavonoids in terms of rutin is 3.59%.

Example 2.

If it is necessary to determine the amount of flavonoids in the leaves of common lilac, in the absence of a standard sample of rutin, it is necessary to carry out all the steps from example 1 before preparing a solution of a standard sample of rutin.

After measuring the optical density of the extract from the leaves of common lilac at a wavelength of 412 nm, the content of the sum of flavonoids (x,%) in terms of rutin and absolutely dry raw materials is calculated by the formula using the theoretical value of the specific absorption index of rutin equal to 240:

where:

0.6653 is the optical density of the test solution;

0.9907 - weight of raw materials, g;

стандартного образца рутина при 412 нм;240 - specific absorption rate

a standard sample of rutin at 412 nm;

10 - loss in weight on drying in percent.

X = 3.89%.

The content of the sum of flavonoids in terms of rutin is 3.89%, which is comparable to the value obtained in example 1.

All results were statistically processed. The error of a single quantification was ± 6.12%.

Thus, the proposed method for the quantitative determination of the amount of flavonoids in terms of rutin in the leaves of common lilac using differential spectrophotometry was developed for the first time for this type of raw material and has the following advantages:

1. The developed method is more specific and selective, and also allows the extraction of raw materials once with 70% ethyl alcohol, which allows you to exhaustively extract the target substances (flavonoids).

2. The sum of flavonoids is recalculated for a flavonoid substance specific for common lilac leaves - rutin, which determines the nature of the absorption curve in the UV spectrum of the test solution (wavelength 412 nm)

3. The error of a single definition of the proposed method is ± 6.12%, which indicates the objectivity of the developed method.

This method can be used in drug quality control centers, pharmaceutical enterprises and control and analytical laboratories when conducting a quantitative analysis of the leaves of common lilac (Syringa vulgaris L.)

SOURCES OF INFORMATION

1. VFS 42-2106-92 "Common lilac bark".

2. State Pharmacopoeia of the Russian Federation. XIV edition. M., 2018 Access mode: http://femb.ru/femb/pharmacopea.php. (date of access: 27.11.2019)

3. Klimova I.Yu. Analytical and technical studies for the development of new drugs based on the common lilac bark. / I.Yu. Klimova - Samara, 2005 .-- 159 s

4. Kurkin V.A. On the issue of standardization of medicinal raw materials containing flavonoids and phenylpropanoids / V.A. Kurkin, O. V. Maevskaya, V.B. Braslavsky et al. // Application of chromatography in food, microbiological and medical industries: Materials of the All-Union conference. Gelendzhik, 1990 .-- S. 85-86.

5. Kurkin V.A. Pharmacognosy: A textbook for pharmacy students. universities - Ed. 4th, rev. and add. / V.A. Kurkin. - Samara: OOO "Etching", FSBEI HE SamGMU of the Ministry of Health of Russia, 2019. - 1278 p.

6. Kurkin V.A., Zapesochnaya G.G., Krivenchuk P.E. Flavonoids and mannitol from leaves of Syringa vulgaris II Chemistry of natural compounds - 1980. - 3. - P. 418-419.

7. RF patent No. 2701726 dated 1.10.2019 "Method for the quantitative determination of the amount of flavonoids in the leaves of black poplar."

Claims (16)

A method for the quantitative determination of the amount of flavonoids in the leaves of common lilac by obtaining an aqueous-alcoholic extract from plant raw materials by extraction of 1 g of an exact weighed portion of plant material crushed to a particle size of 1 mm with 70% ethyl alcohol, followed by sample preparation and determination of optical density by differential spectrophotometry using standard a sample of rutin, and in its absence - using a theoretical specific absorption index, characterized in that the extraction of crushed leaves of common lilac is carried out for 45 minutes, with a raw material: extractant ratio of 1:50; the quantitative determination of the sum of flavonoids in the leaves of common lilac is carried out at a wavelength of 412 nm in terms of rutin and the content of the sum of flavonoids in terms of rutin and absolutely dry raw materials is calculated by the formula:

where x is the content of the sum of flavonoids in terms of rutin,%; D is the optical density of the test solution; D _o - optical density of the solution of the State standard sample rutin; m is the mass of raw materials, g; m _o - mass of the State standard sample rutin, g; W - loss in mass on drying,%, in the absence of a standard sample of rutin, it is advisable to use the theoretical value of its specific absorption index, equal to 240:

where x is the content of the sum of flavonoids in terms of rutin,%; D is the optical density of the test solution; m is the mass of raw materials, g; m _o - mass of the State standard sample rutin, g; 240 - specific absorption rate

State standard sample rutin at 412 nm; W - loss in mass on drying,%.

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