A method for quantifying the amount of flavonoids in flowers Acer tataricum L.

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INTRODUCTION

The genus Maple (Acer L.) belongs to the Maple family (Aceraceae), which includes about 129 species distributed mainly in the temperate latitudes of the Northern Hemisphere (East Asia, North America, Europe). Many types of maple have been used in folk medicine for centuries to treat various diseases [1]. Extracts of various types of maple (Acer) have been used in traditional medicine for a long time due to their wide range of pharmacological activity, including antioxidant, anti-inflammatory, hypoglycemic, hepatoprotective and potential anticarcinogenic effects [1]. These effects may be due to the presence of a variety of biologically active compounds in their composition [1].

The Tatar maple (Acer tataricum L.), also known as the black maple, is a deciduous tree-like plant or large shrub reaching a height of 8 meters. Its distribution range covers the regions of Central, Eastern, and Southeastern Europe, Asia Minor, and the Caucasus [2-4].

Tatar maple has a chemical composition that includes a variety of biologically active substances. The main group consists of phenolic compounds that determine the pharmacological properties of the plant, in particular its antimicrobial activity. The leaves of the Tatar maple contain phenolic compounds (including methyl gallate, tannins), cyclitols (for example, quebrachite), saponins [5].

Aim

Development of a method for quantitative determination of the amount of flavonoids in the flowers of the Tatar maple.

MATERIALS AND METHODS

To develop the technique, flowers of the Tatar maple were used, collected in the Dubki forest area (Sokolya Gora district, Samara). The drying of plant raw materials was carried out in natural conditions at room temperature, without exposure to direct sunlight. Direct and differential spectrophotometry in accordance with the OFS was used as a research method.1.2.1.1.0003.15 "Spectrophotometry in the ultraviolet and visible regions" of the RF State Budget XV edition [6]. The spectral characteristics of water-alcohol extracts were evaluated on a Specord 40 spectrophotometer (AnalytikJena AG, Germany) in the wavelength range of 190-500 nm in cuvettes with a layer thickness of 10 mm.

RESULTS

A comparative study of the UV spectra of water-alcohol extracts from Tatar maple flowers and rutin solution (Figure 1-4) showed that in both cases there is a bathochromic shift in the long-wavelength region of the UV spectra in the presence of AlCl3 at 412 ±2 nm, which is typical for rutin [7-10]. Accordingly, the developed technique is based on the reaction of complexation of flavonoids with aluminum chloride (AlCl3) using rutin and an analytical wavelength of 412 nm as a standard sample (CO).

Figure 1. UV spectra of an aqueous-alcohol extract of Acer tataricum L. flowers (1) and an aqueous-alcohol extract of Acer tataricum L. leaves with added aluminum chloride (2)

Figure 2. UV spectrum of an aqueous-alcohol extract of Acer tataricum L. flowers (differential spectrum)

Figure 3. UV spectra of a rutin solution (1) and a solution containing aluminum chloride (2)

Figure 4. UV spectrum of rutin solution (differential spectrum)

In the UV spectrum of water-alcohol extraction from Tatar maple flowers, a differential absorption maximum is found at a wavelength of 412 nm (Figure 4), which is close to the maximum of the alcohol solution of rutin (Figure 2) [7-10]. Extraction conditions were optimized for the quantitative determination of flavonoid compounds: 70% ethanol was used as an extractant, the ratio "raw material: extractant" was 1:50, the degree of grinding was 2 mm, and the extraction time was 45 minutes when heated in a water bath with a reverse refrigerator (Table 1).

 

№	Extractant	Ratio "raw material- extractant"	Extraction time, min	Degree of grinding, mm	Content of the amount of flavonoids in terms of rutin and absolutely dry raw materials (in %)
Extractant
1	ethyl alcohol 40%	1:50	45	2	1,62 ± 0,04%
2	ethyl alcohol 50%				1,65 ± 0,05%
3	ethyl alcohol 60%				1,76 ± 0,05%
4	ethyl alcohol 70%				2,01± 0,05%
5	ethyl alcohol 80%				1,82± 0,04%
6	ethyl alcohol 90%				1,80± 0,05%
7	ethyl alcohol 96%				1,75± 0,05%
Extraction time
8	ethyl alcohol 70%	1:50	30	2	1,85± 0,04%
9			45		2,29± 0,05%
10			60		1,89± 0,04%
11			90		1,75± 0,04%
Degree of grinding
12	ethyl alcohol 70%	1:50	45	1	1,85± 0,04%
13				2	1,92± 0,05%
14				3	1,86± 0,04%
The ratio "raw material-extractant"
15	ethyl alcohol 70%	1:20	45	2	1,82± 0,05%
16		1:30			1,87± 0,05%
17		1:50			1,90± 0,05%

 

Table 1. Influence of various factors on the completeness of extraction of flavonoids from the flowers of Acer tataricum L.

The method of quantitative determination of the amount of flavonoids in the flowers of the Tatar maple.

The analytical sample of the raw material is crushed to the size of particles passing through a sieve with holes with a diameter of 2 mm. About 1 g of crushed raw materials (exact weight) is placed in a flask with a slot with a capacity of 100 ml, 50 ml of 70% ethyl alcohol is added. The flask is closed with a stopper and weighed on a calibrated scale with an accuracy of ± 0.01. The flask is connected to a return refrigerator and heated in a boiling water bath (moderate boiling) for 45 minutes. Then it is cooled for 30 minutes, closed with the same stopper, weighed again and filled with the missing extractant to the original mass. Extraction is filtered through a paper filter (red stripe). The test solution is prepared as follows: 1 ml of the resulting extract is placed in a measuring flask with a capacity of 50 ml, 2 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 A). The optical density of the test solution is measured on a spectrophotometer at a wavelength of 412 nm 40 minutes after preparation. As a reference solution, a solution is used, obtained as follows: 1 ml of extraction (1:25) is placed in a measuring flask with a capacity of 25 ml and the volume of the solution is adjusted with 96% ethyl alcohol to the mark.

Preparation of a solution of a standard sample of routine.

About 0.0048 g (exact weight) of rutin is placed in a measuring flask with a capacity of 25 ml, dissolved in 15 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 with 70% ethyl alcohol to the mark (solution A rutin). 1 ml of solution A of rutin is placed in a 25 ml measuring flask, 2 ml of a 3% alcohol 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 solution A of rutin is placed in a 25 ml measuring flask and the volume of the solution is adjusted to the mark with ethyl alcohol 96% (rutin comparison solution).

The percentage of the amount of flavonoids in terms of rutin and absolutely dry raw materials (X) is calculated using the formula:

X = (A * mo ∙50 ∙25 ∙1∙ 100∙ 100)/( Ao* m *1* 25 *25 (100-W)), where A is the optical density of the test solution; Ao is the optical density of the CO rutin solution; m is the mass of the raw material, g; mo is the mass of CO rutin, g; W is the mass loss during drying, %. In the absence of CO rutin, it is advisable to use the calculated value of the specific absorption index at 412 nm – 240 [11].

x=(A ×50× 25 ×100)/(m ×240 (100-W)), where A is the optical density of the test solution; m is the mass of the raw material, g; 240 is the specific absorption index (E(1%)¦(1 cm)) Temperature at 412 nm; W is the loss in weight during drying, %. The criterion for evaluating an analytical technique is a validation assessment [6, 12-15]. The validation of the methodology was carried out in accordance with the RF State Budget of the XV edition [6]. The validation evaluation of the developed methodology was carried out according to the following indicators: specificity, linearity, correctness. The specificity of the technique was determined by the correspondence of the absorption maxima of the flavonoid complex of Tatar maple leaves and a solution of rutin with aluminum chloride (differential version). The linearity of the technique was determined for a series of rutin solutions (with concentrations ranging from 0.008 to 0.038 mg/ml) with aluminum chloride at a wavelength of 412 nm. Based on the data obtained, a graph of the dependence of the optical density values of rutin solutions with aluminum chloride on the concentration of rutin was constructed and then the linear regression equation was calculated (Figure 5; Table 2). When studying the linear dependence of the type y = bx + a, the correlation coefficient was 1, therefore, this technique can be used to analyze the amount of flavonoids in the leaves of the Tatar maple in terms of rutin in the specified concentration range (Figure 5; Table 2).

 

№ п/п	Concentration of the solution of the standard sample rutin, mg/ml	Optical density value, E.O.P. (average value of three consecutive measurements)
1	0,008	0,1641
2	0,015	0,3316
3	0,023	0,4856
4	0,038	0,8118

 

Table 2. Initial data for assessing the linearity of the methodology

Figure 5. Dependence of the optical density values of a rutin solution with aluminum chloride on the concentration of rutin (differential version)

The metrological characteristics of the methods for quantifying the amount of flavonoids in the water-alcohol extraction of Tatar maple flowers are shown in Table 3. The error in the single determination of the amount of flavonoids in Tatar maple flowers with a confidence probability of 95% is ± 1.67% (Table 3).

 

n	f	,%	S2	S		P, %	t (P, f)	±∆X	±∆	E, %	, %
11	10	1,90	0,0010	0,0319	0,014	95	2,23	±0,07	±0,03	±1,67	±3,74

 

Table 3. Results of the precision assessment of the method for quantifying the sum of flavonoids in the flowers of  Acer tataricum L.(repeatability level)

Thus, based on the results of the validation evaluation of the experimental results, it can be concluded that this technique is suitable for quantifying the amount of flavonoids in terms of rutin. Using this technique, four samples of Tatar maple flowers harvested in the summer were analyzed. It was determined that the amount of flavonoids in the analyzed samples collected in the Samara region varies from 1.89%±0.05% to 2.01%±0.05%, depending on the month of harvest of plant raw materials (Table 4). In our opinion, to substantiate the numerical indicator – the amount of flavonoids in the flowers of the Tatar maple, it is advisable to conduct further research using the example of raw material samples from other regions of the Russian Federation. 

№	Haracteristics of the raw material sample	He content of the amount of flavonoids in absolutely dry raw materials (in %) in terms of rutin
1.	The Dubki forest area in the Sokolikh Gory area of Samara (June 2025) Date of harvest: 06.06.2022	1,89±0,05%
2.	The Dubki forest area in the Sokolikh Gory area of Samara (June 2025) Date of collection: 13.06.2025	2,01±0,05%
3.	Dubki forest area in the Sokolikh Mountains region of Samara (June 2025) Date of harvest: 21.06.2025	1,95±0,05%

 

Table 4. Content of the sum of flavonoids in samples of Acer tataricum L. flowers (in %) in terms of rutin

CONCLUIONS

Thus, as a result of the conducted research, a methodology has been developed for the quantitative analysis of the amount of flavonoids in the flowers of the Tatar maple using CO-routine at an analytical wavelength of 412 nm. It was determined that the amount of flavonoids in terms of rutin in the flowers of the Tatar maple, collected in the Samara region, varies from 1.89% ± 0.05% to 2.01% ± 0.05%. The error of a single determination with a 95% confidence probability is ± 1.67%. A validation assessment of the developed methodology was carried out based on the indicators of specificity and linearity in accordance with the RF State Budget of the XV edition. Based on the results of the validation evaluation of the experimental results, we can talk about the suitability of using this technique to quantify the amount of flavonoids in terms of rutin in the flowers of the Tatar maple. The obtained research results can be used in the development of regulatory documentation for a promising type of medicinal plant raw materials "Tatar Maple flowers" for implementation in the State Pharmacopoeia of the Russian Federation.

About the authors

Komila Khozinova

Самарский государственный медицинский университет

Author for correspondence.
Email: kamilahozinova@gmail.com
ORCID iD: 0009-0007-8581-8563

Аспирант

Russian Federation

Vladimir Aleksandrovich Kurkin

Samara State Medical University

Email: v.a.kurkin@samsmu.ru
ORCID iD: 0000-0002-7513-9352

Head of the Department of Pharmacognosy with Botany and the Basics of Phytotherapy, Doctor of Pharmaceutical Sciences, Professor

Russian Federation, Chapaevskaya ul., 89, Samara, 443099

References

Supplementary files