RU2747483C1 - Method for quantitative determination of amount of phenylpropanoids in lilac flowers - Google Patents

Abstract

FIELD: chemical industry; pharmaceutical industry.

SUBSTANCE: invention relates to the chemical industry and the pharmaceutical industry, and in particular to a method for the quantitative determination of the amount of phenylpopanoids in flowers of common lilac (Syringa vulgaris L.). Disclosed is a method for the quantitative determination of phenylpropanoids, which consists in the preliminary obtaining of an aqueous-alcoholic extract from plant raw materials by extraction of 1 g of an exact weighed portion of a plant material crushed to a particle size of 1 mm with 60% ethyl alcohol for 45 minutes, in terms of a substance of a phenylpropanoic nature, by the method of direct spectrophotometry in the ratio of "raw material-extractant" - 1: 100, the determination of phenylpropanoids is carried out at a wavelength of 330 nm in terms of chlorogenic acid, the content of the sum of phenylpropanoids in terms of chlorogenic acid, and absolutely dry raw materials are calculated by the formula:

where x is the content of the sum of phenylpropanoids in terms of chlorogenic acid,%; D is the optical density of the test solution; D_o is the optical density of the solution of the state standard sample of chlorogenic acid; m is the mass of raw materials, g; m_o is the mass of the state standard sample of chlorogenic acid, g; W is the loss in mass on drying,%; in the absence of a standard sample of chlorogenic acid, it is advisable to use the theoretical value of its specific absorption index, equal to 497:

where x is the content of the sum of phenylpropanoids in terms of chlorogenic acid,%; D is the optical density of the test solution; m is the mass of raw materials, g; 497 - specific absorption rate

of the state standard sample of rutin at 330 nm; W - loss in mass on drying,%.

EFFECT: above method for quantifying the amount of phenylpropanoids in common lilac flowers is specific and accurate.

1 cl, 2 dwg, 4 tbl, 2 ex

Description

The invention relates to the pharmaceutical industry and can be used in drug quality control centers and analytical laboratories for quantitative determination of the amount of phenylpropanoids in flowers 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 bark (VFS 42-2106-92) is registered as medicinal plant material (MPR) [1, 2]. Preparations based on lilac bark have a wide spectrum of pharmacological activity: immunomodulatory, adaptogenic, anxiolytic and antidepressant effects [3].

Of particular interest, along with the pharmacopoeial raw material - bark, are the flowers of this plant. The flowers of common lilac (Syringa vulgaris L.) are a promising source of ALS with immunostimulating, anxiolytic and antidepressant effects. However, the problem of standardizing this type of raw material remains unresolved.

At the moment, there are no known methods for quantitative determination of the amount of phenylpropanoids in flowers of common lilac by direct spectrophotometry [2].

Thus, the aim of the invention is to develop a method for the quantitative determination of the amount of phenylpropanoids in flowers 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 phenylpropanoids in the flowers of common lilac in terms of chlorogenic acid.

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 60% ethyl alcohol, in terms of a substance of phenylpropanoid nature, by the method of direct spectrophotometry in relation to "raw material-extractant "- 1: 100, the determination of phenylpropanoids is carried out at a wavelength of 330 nm in terms of chlorogenic acid and absolutely dry raw material is calculated by the formula:

Where:

x - the content of the sum of phenylpropanoids in terms of chlorogenic acid,%;

D is the optical density of the test solution;

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

m is the mass of raw materials, g;

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

W is the loss in mass on drying,%;

in the absence of a standard sample of chlorogenic acid for the calculation, it is advisable to use the theoretical value of its specific absorption index, equal to 497:

Where:

x - the content of the sum of phenylpropanoids in terms of chlorogenic acid,%;

D is the optical density of the test solution;

m is the mass of raw materials, g;

) Государственного стандартного образца хлорогеновой кислоты при длине волны 330 нм;497 - specific absorption rate (

) State standard sample of chlorogenic acid at a wavelength of 330 nm;

W - loss in mass on drying,%.

When studying the spectral characteristics, it was revealed that it is chlorogenic acid that determines the nature of the absorption curve of aqueous-alcoholic extraction from flowers of common lilac. It was determined that in the UV spectrum of the aqueous-alcoholic extraction of common lilac, there is a maximum absorption of phenylpropanoids (Fig. 1 in Appendix 1), as in the case of chlorogenic acid (Fig. 2 in Appendix 2), where curve 1 in Fig. 1 and FIG. 2 shows an initial solution of aqueous-alcoholic extraction from ordinary lilac flowers or an initial solution of chlorogenic acid, respectively.

Examination of the UV spectra of FIG. 2 (where curve 1 is a chlorogenic acid solution) showed that the GSO solution of chlorogenic acid has an absorption maximum at a wavelength of 330 nm. In the UV spectrum of aqueous-alcoholic extraction from flowers of common lilac in the direct version in Fig. 1 (Appendix 1), an absorption maximum is also found at a wavelength of 330 nm, which corresponds to the absorption maximum of an alcoholic solution of chlorogenic acid.

This fact allows for the spectrophotometric determination of the amount of phenylpropanoids in the flowers of common lilac at an analytical wavelength of 330 nm.

We also studied the effect of the extractant on the extraction process. Table 1 (Appendix 3) shows the dependence of the yield of phenylpropanoids of common lilac flowers on the concentration of the extractant. As a result of the experiment, we chose 60% 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 (Appendix 4) shows the dependence of the yield of phenylpropanoids of ordinary lilac flowers on the extraction time in a boiling water bath, while the extraction time was chosen at 45 minutes, at which the maximum yield of substances was observed ...

Table 3 (Appendix 5) shows the dependence of the yield of phenylpropanoids of common lilac flowers 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: 100, for this reason, this ratio was chosen by us as optimal.

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 (Appendix 6) 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 60% ethyl alcohol in a boiling water bath for 45 minutes in the ratio "raw material-extractant" - 1: 100, the degree of grinding of raw materials 1 mm.

Taking into account the fact that chlorogenic acid is specific for ordinary lilac flowers, and the absorption maxima of chlorogenic acid solution and aqueous-alcoholic extract of ordinary lilac flowers are in the region of 330 nm, it is expedient to determine the content of the sum of phenylpropanoids in terms of chlorogenic acid at a wavelength 330 nm.

The method is implemented as follows.

An analytical sample of the air-dry raw material of common lilac is ground to a particle size passing through a sieve with holes 1 mm in diameter. About 1 g of an accurately weighed sample of the crushed raw material is placed in a flask with a thin section with a capacity of 250 ml, 100 ml of 60% ethyl alcohol is 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 amount of phenylpropanoids is prepared as follows: 1 ml of the obtained extract is placed in a 50 ml volumetric flask and the volume of the solution is brought to the mark with 96% ethyl alcohol (test solution).

To calculate the content of the sum of phenylpropanoids, a solution of a standard sample of chlorogenic acid is prepared, the optical density of the solution is measured at an analytical wavelength of 330 nm, and the determined value of the optical density is used in the calculation formula.

Preparation of a chlorogenic acid standard sample solution.

About 0.020 g (accurately weighed) of chlorogenic acid is placed in a volumetric flask with a capacity of 100 ml, dissolved in 70 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 (chlorogenic acid solution A). Then 1 ml of chlorogenic acid solution A is placed in a 25 ml volumetric flask, the volume of the solution is adjusted to the mark with 96% ethyl alcohol (test solution B of chlorogenic acid).

Measurement of optical density is carried out at a wavelength of 330 nm 40 minutes after the preparation of all solutions.

The content of the sum of phenylpropanoids (x,%) in terms of chlorogenic acid and absolutely dry raw material 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 of chlorogenic acid;

m is the mass of raw materials, g;

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

W is the percentage loss on drying.

In the absence of a standard sample of chlorogenic acid for the calculation, it is advisable to use the theoretical value of its specific absorption index, equal to 497:

Where:

D is the optical density of the test solution;

m is the mass of raw materials, g;

) Государственного стандартного образца хлорогеновой кислоты при 330 нм;497 - specific absorption rate (

) State standard sample of chlorogenic acid at 330 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. 0.9950 g of crushed raw materials are placed in a flask with a thin section with a capacity of 250 ml, add 100 ml of 60% 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 amount of phenylpropanoids is prepared as follows: 1 ml of the obtained extract is placed in a volumetric flask with a capacity of 50 ml and the volume of the solution is brought to the mark with ethyl alcohol 96% (test solution).

To calculate the content of the sum of phenylpropanoids, a solution of a standard sample of chlorogenic acid is prepared and the optical density of the solution is measured at a wavelength of 330 nm and the determined value of the optical density is used in the calculation formula.

Preparation of a chlorogenic acid standard sample solution.

0.020 g of chlorogenic acid is placed in a 100 ml volumetric flask, dissolved in 70 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 (chlorogenic acid solution A). Then 1 ml of chlorogenic acid solution A is placed in a 25 ml volumetric flask, then the volume of the solution is brought to the mark with ethyl alcohol 96%.

Measurement of optical density is carried out at a wavelength of 330 nm 40 minutes after the preparation of all solutions.

The content of the sum of phenylpropanoids (x,%) in terms of chlorogenic acid and absolutely dry raw material is calculated by the formula:

Where:

0.5511 is the optical density of the test solution;

0.3829 - optical density of a solution of a standard sample of chlorogenic acid;

0.9950 - weight of raw materials, g;

0.0202 - mass of a standard sample of chlorogenic acid, g;

9.8 is the percentage loss on drying.

X = 6.48%.

The content of the sum of phenylpropanoids in terms of chlorogenic acid is 6.48%.

Example 2

If it is necessary to determine the sum of phenylpropanoids in the flowers of common lilac in the absence of a standard sample of chlorogenic acid, it is necessary to carry out all the steps from example 1 before preparing a solution of a standard sample of chlorogenic acid.

After measuring the optical density of the extract from the flowers of common lilac at a wavelength of 330 nm, the content of the sum of phenylpropanoids (x,%) in terms of chlorogenic acid and absolutely dry raw materials is calculated by the formula using the theoretical value of the specific absorption index of rutin equal to 497:

Where:

0.5511 is the optical density of the test solution;

0.9950 - weight of raw materials, g;

) стандартного образца хлорогеновой кислоты при 330 нм;497 - specific absorption rate (

) a standard sample of chlorogenic acid at 330 nm;

9.8 is the percentage loss on drying.

X = 6.18%.

The content of the sum of phenylpopanoids in terms of chlorogenic acid is 6.18%, which is comparable to the value obtained in example 1.

All results were statistically processed. The error of a single quantification was ± 1.85%.

Thus, the proposed method for the quantitative determination of the amount of phenylpropanoids in terms of chlorogenic acid in the flowers of common lilac using direct spectrophotometry was developed for the first time for this type of raw material and has the following advantages:

1. The developed method is specific and selective, and also allows the extraction of raw materials once with 60% ethyl alcohol, which allows the exhaustive extraction of the target substances (phenylpropanoids).

2. The sum of phenylpropanoids is recalculated for phenylpropanoid substance, specific for flowers of common lilac, chlorogenic acid, which determines the nature of the absorption curve in the UV spectrum of the test solution (wavelength 330 nm).

3. The error of a single definition of the proposed method is ± 1.85%, which indicates the objectivity of the developed method.

4. The developed method makes it possible to exclude sample preparation, which consists in processing the extract with toxic solvents.

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

INFORMATION SOURCES:

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 research on the development of new drugs based on the common lilac bark. / I.Yu. Klimova - Samara, 2005.-159 p.

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 the food, microbiological and medical industries: Materials of the All-Union Conference. Gelendzhik, 1990 .-- S. 85-

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

6. Kurkin V.A., Zapesochnaya G.G., Grinenko N.A. Zolotarev B.M. Phenolic compounds of the bark of Syringa vulgaris // Chemistry of natural compounds. - 1989. - No. 4. -FROM. 581-582.

APPENDIX 3

APPENDIX 4

APPENDIX 5

APPENDIX 6

Claims (17)

A method for the quantitative determination of the amount of phenylpropanoids in the flowers of common lilac, which consists in the preliminary obtaining of an aqueous-alcoholic extract from the flowers of common lilac by a single extraction for 45 minutes with 60% ethyl alcohol of air-dry raw materials with an accurate weight of 1 g, crushed to the size of particles passing through a sieve with holes 1 mm in diameter, with a raw material-extractant ratio of 1: 100; the quantitative determination of the amount of phenylpropanoids in the flowers of common lilac is carried out at a wavelength of 330 nm in terms of chlorogenic acid and the content of the amount of phenylpropanoids in terms of chlorogenic acid, and absolutely dry raw materials are calculated by the formula:

Where: x - the content of the sum of phenylpropanoids in terms of chlorogenic acid,%; D is the optical density of the test solution; D _o - optical density of the solution of the State standard sample of chlorogenic acid; m is the mass of raw materials, g; m ₀ - mass of the State standard sample of chlorogenic acid, g; W is the loss in mass on drying,%; in the absence of a standard sample of chlorogenic acid, it is advisable to use the theoretical value of its specific absorption index, equal to 497:

Where: x - the content of the sum of phenylpropanoids in terms of chlorogenic acid,%; D is the optical density of the test solution; m is the mass of raw materials, g; 497 - specific absorption rate (

) State standard sample of chlorogenic acid at 330 nm; W - loss in mass on drying,%.

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