RU2266544C2 - Unified assay of flavonoids in eyebright grass and extractive preparations - Google Patents

Abstract

FIELD: pharmacology.

SUBSTANCE: invention relates to unified assay of total flavonoids in three kinds of eyebright grass: Euphrasia brevipila Burnat@Gremli, Euphrasia parviflora Schag, and Euphrasia X reuteri Wettst, as well as in extractive preparations of Euphrasia brevipila Burnat@Gremli. Claimed method includes utilization of 2 % aluminum chloride alcohol solution as chelating agent and 8 % sodium acetate alcohol solution as ionizing agent; detection of colored complex by differential spectrophotometry at λ = 382±2 nm; and calculation of total flavonoids using specific absorption coefficient

for cinarozide as reference standard and individual dilution series for each extractive preparation.

EFFECT: method for standardization of drug raw materials and eyebright extractive preparations according to active ingredient (flavonoid) content, useful in development of normative and technical documentation.

6 cl, 8 tbl, 6 ex, 1 dwg

Description

The invention relates to the field of pharmacy and relates to the quantitative determination of the amount of flavonoids in the grass of various types of eyebrights and extraction preparations of eyebright short-haired.

Plants of the genus Ochanka (Fr.) are annual, semi-parasitic herbs of the family. Scrophulariaceae having difficult to distinguish morphological and anatomical signs. They are used in traditional medicine and homeopathy for the treatment of inflammatory and sclerotic diseases of the eyes (cataract, glaucoma, conjunctivitis, blepharitis), the gastrointestinal tract (hepatitis, colitis, enterocolitis), the upper respiratory tract (bronchitis, tonsillitis, pneumonia) [Plant resources of the USSR . Flowering plants, their use, of the family Caprifoliaceae - Plantaginaceae. - M .: Nauka, 1990, p.139].

In the course of phytochemical studies it was established that the main groups of active substances of grass on. short-haired, about. small-flowered and about. Reuters are flavonoids, iridoids and phenol carboxylic acids. The complex of flavonoid substances of these plants is represented by flavone derivatives - apigenin, luteolin, cinaroside [Sukhinina T.V. Pharmacognostic study of plants of the genus of pupil / Abstract. Cand. dis. Perm, 2002. 20 p.].

Pharmacological studies of extraction preparations showed that the presence of antimicrobial, hypotensive and anti-inflammatory activity, combined with low toxicity [T. Sukhinina Pharmacognostic study of plants of the genus of pupil / Abstract. Cand. dis. Perm, 2002. 20 p.]. Many years of experience in using traditional medicine and modern data from pharmacological studies open up prospects for introducing euphrasia herb and extraction preparations based on it into medical practice.

However, the development and industrial production of euphrasia herb preparations is difficult due to the possible falsification and use of poor-quality raw materials, the lack of intermediate technological control and quality assessment of the final products. These difficulties are due to the lack of a reliable method for the quantitative determination of active substances.

Analysis of patent and scientific literature showed: a prototype of the method for determining the active substances in the grass of various types of eyewear is absent. This, in particular, is evidenced by the absence of a section for determining the active substances in the normative and technical documentation for grass about. Rostovius and about. direct in Germany [Deutschen Arcneimittel Codex, Stuttgart, 1997, A-192].

Known methods for the quantitative determination of flavonoids in plant objects are not standardized and are developed or only for the raw materials of one plant species [Yartseva IB, Kurkin VA Quantitative determination of the amount of flavonoids in the grass of dandelion officinalis // Pharmacy, 1996, No. 4, P.24-26; Samylina I.A., Evdokimova O.V., Kashnikova M.V. The use of aluminum chloride to determine the amount of flavonoids in the flowers of hawthorn // Pharmacia, 1994, No. 6, P.42-45; Smirnova L.P., First L.N. Quantitative determination of the amount of flavonoids in the flowers of the sandy immortelle // Chemical and Pharmaceutical Journal, 1998, No. 6, S.35-36], or only for preparations derived from plant materials [Chemesova II, Chubarova SL, Sakanyan E.I. et al. Spectrophotometric method for quantifying the content of polyphenols in a dry extract from the aerial part of Mellilotus officinalis (L.) Pall. and in its dosage form (tablets) // Plant Resources, 2000, Issue 1, T.36, S.72-74; Kabishev K.E., Sakanyan E.I. Quantitative determination of the amount of flavonoid compounds in intranasal dosage forms of the drug “Oxophilum” with a polyextract from the aerial part of Oxytropis oxyphilla (Pall.) DC.// Plant Resources, 2002, Issue 4, T.38, S.120-127]. Using these methods to determine the amount of flavonoids in the grass and extraction preparations is not possible because of the difference:

- in the chemical composition of plants;

- in the consistency and structure of plant material;

- in spectral characteristics;

- in the sequence of analytical operations;

- in the used state standard samples (GSO).

The aim of the present invention is to develop a unified method for quantitative determination, allowing to determine the amount of flavonoids (active substances):

- in the raw materials of various types of glasses;

- in aqueous extracts-infusions (1:10), dry and liquid (1: 2) extracts;

- in alcoholic extracts - tinctures (1: 5), dry and liquid (1: 2) extracts, substances containing a complex of flavonoids (fraction of flavonoids).

To achieve this goal, the following tasks were solved:

- an affordable complexing reagent has been selected that allows spectrally detecting the presence of flavonoids in plants and herbal preparations by the formation of a complex compound that is stable over time;

- the method of spectral detection of flavonoids with a complexing reagent;

- An analytical wavelength has been selected to selectively determine flavonoid compounds;

- a state standard sample was selected for recalculating the sum of flavonoids and its specific absorption index (E ^1% _1cm ) was determined under the conditions of a quantitative determination method;

- determined the optimal conditions for the extraction of flavonoids from raw materials (extractant; ratio of raw materials and extractant; extraction time);

- optimal conditions have been developed for the quantitative determination of the sum of flavonoids in the extraction preparations of a short-haired specimen (working wavelength, weight, dilution);

- a metrological characteristic of the method for the quantitative determination of flavonoids in raw materials and extraction preparations was carried out.

The quantitative determination method is based on the use of the complexation reaction of eye flavonoids with aluminum chloride (AlCl ₃ ) in the presence of sodium acetate (CH ₃ COONa), followed by quantitative determination of this complex by differential spectrophotometry. Differential spectrophotometry involves the use of a test solution without reagents as a control, which eliminates the influence of colored and related substances, as well as substances that do not form complexes with reagents. Aluminum chloride is a cheap and affordable reagent and is used in combination with an ionizing additive - sodium acetate. The simultaneous use of complexing and ionizing reagents causes a more significant bathochromic shift of the absorption flavonoid absorption bands compared to using only the complexing reagent (Δλ = 6 nm), which allows the flavonoid complex to be determined in the long-wavelength part of the spectrum free of absorption of hydroxycinnamic acids.

To determine the method of spectral detection and analytical wavelength, we studied the UV spectra of alcoholic extracts of the aboveground part with the roots (hereinafter grass) of three types of eyewear - о. short-haired, about. small-flowered and about. Reuters and the state standard sample (GSO) of cinaroside (FS 42-3150-95). It was established that the UV spectra of the studied types of eyewear are the same (Table 1), as an example, the spectra of the eyewear of a short-haired one are shown (see drawing).

As follows from the obtained data, the alcohol extraction spectrum has one maximum (328 nm), which slightly shifts when aluminum chloride and sodium acetate are added to the test solution and is probably due to the presence of hydroxycinnamic and acylquinic acids in the studied extract, the absorption spectrum of which is in the region 310-330 nm [Bandyukova V.A. Phenolic acids of plants, their esters and glycosides // Chemistry of nature. compounds, 1983, No. 2, C.271-273]. The hypochromic effect observed in this case does not allow the direct spectrophotometry of the colored complex and the given wavelength to be used as the working one.

The differential absorption curve (see drawing) is an integrated overlay spectrum of a complex of flavonoid substances contained in the specimen, has only one maximum of 382 nm and overlaps with the long-wavelength band of GSO cinaroside in the presence of aluminum chloride and sodium acetate. Since the interval between the maxima of the differential absorption spectrum and the long-wavelength absorption band of the standard sample does not exceed half the half-width of the absorption band of the standard sample, the measurement error will not be significant [EA Lovtseva Improvement of quality control methods for medicines of purine derivatives of N-glycoside structure / Abstract. Cand. dis., Pyatigorsk, 1993. 20 p.]. This makes it possible to use λ = 382 nm as an analytical one, and taking into account measurements on different devices, the value of wavelengths can differ by ± 2 nm (λ = 382 ± 2 nm) [State Pharmacopoeia of the USSR, XIth ed., M; 1987, T.1, C.36].

To study the possibility of using the developed method for the quantitative determination of flavonoids in the analysis of extraction preparations about. short-haired were studied their differential UV spectra with aluminum chloride and sodium acetate (table 2). As follows from the data obtained, the maxima of the differential curves coincide, which allows the use of λ = 382 ± 2 nm as the working wavelength in the analysis of the studied preparations.

In developing optimal conditions for the extraction of flavonoids from raw materials, water and ethanol of various concentrations were used. The most complete extraction of the analytes was achieved by extraction with 80% ethanol with a ratio of raw material and extractant of 1:80. The time for complete extraction of flavonoids is 20 minutes. A further increase in the extraction time is impractical, since there is a significant decrease in the amount of flavonoids in the studied extracts. These extraction conditions are the same for the raw materials of three types of pupils - about. short-haired, about. small-flowered and about. Reuters. As an example, the results of a study of short-haired euphrasia grass (Table 3).

To recalculate the amount of flavonoids in the grass, the pupils on cinaroside we calculated (Table 4) the specific absorption rate (E ^1% _1cm ) of complexes of GSO cinaroside solutions with aluminum chloride and sodium acetate at an analytical wavelength of 382 nm, which is 357.4 ± 3.4. In this regard, in the calculation formula, we included the theoretical value of E ^1% _1cm = 357, which allows not using cinaroside in the GSO method.

The unified method for the quantitative determination of the sum of flavonoids is confirmed by the following examples.

Example 1. The determination of the amount of flavonoids in the grass about. short-haired, about. small-flowered and about. Reuters. An analytical sample of air-dry raw materials is crushed to the size of particles passing through a sieve according to GOST 214-83 with holes with a diameter of 2 mm. About 0.6 g (accurately weighed) of the raw material is placed in a 100 ml conical flask with a thin section, 50 ml of 80% ethanol are added. The flask is attached to a reflux condenser and heated in a boiling water bath for 20 minutes, counting from the moment the extractant is boiled. The flask is cooled to room temperature, the extraction is filtered through a cotton swab into a flat-bottomed volumetric flask with a capacity of 50 ml, and through the same filter the solution volume is brought up to 80% with ethanol. The resulting extract was filtered into a flat-bottomed flask through a paper filter (solution A), the first 10 ml of the filtrate was discarded. 1 ml of solution A was placed in a 50 ml volumetric flask, 1 ml of a 2% alcohol solution of aluminum chloride and 1 ml of an 8% alcohol solution of sodium acetate were added, the solution volume was adjusted to the mark with 80% ethanol and stirred (solution B). After 30 minutes, the optical density of solution B was measured on a spectrophotometer at a wavelength of 382 nm in a cuvette with an absorbing layer thickness of 10 mm. As a comparison solution, use 1 ml of solution A, brought to 80% ethanol to the mark in a 50 ml volumetric flask. The content of the sum of flavonoids in the dry aerial part with the eyebright roots in terms of cinaroside is calculated by the formula:

where D is the optical density of the test solution; m is a sample of raw materials, g; V is the volume of solution A taken for dilution, ml; 50 - the volume of solution A, ml; 50 - the volume of solution B, ml; W is the loss in mass upon drying of the raw material,%; 357 is the specific absorption rate of the complex of cinaroside with reagents at a wavelength of 382 nm.

Statistical processing of the results of quantitative determination was performed according to the generally accepted methodology of GF-XI [State Pharmacopoeia of the USSR, XI ed., M., 1987, 4.1, 336 pp.]. The results (Table 5) showed that a single determination error with a reliable probability of 95% was in the range of 4.5-5.5%.

Check for the absence of a systematic error, by adding GSO cinaroside to a sample of raw materials, was performed on the grass about. short-haired. It was established that the systematic error of the developed method is absent (Table 6).

Example 2. Determination of the amount of flavonoids in the infusions (1:10) of the euphorbia short-haired. 1 ml of the infusion is placed in a 50 ml volumetric flask, 1 ml of a 2% alcohol solution of aluminum chloride and 1 ml of an 8% alcohol solution of sodium acetate are added, the solution volume is adjusted to the mark with 80% ethanol and stirred (solution A). After 30 minutes, the optical density of solution A was measured on a spectrophotometer at a wavelength of 382 nm in a cuvette with an absorbing layer thickness of 10 mm. As a comparison solution, use 1 ml of infusion, brought up to 80% ethanol to the mark in a 50 ml volumetric flask. The content of the sum of flavonoids in the infusions of eyebright short-haired in terms of cinaroside is calculated by the formula:

where D is the optical density of the test solution; a - a sample of infusion, ml; 50 - the volume of solution A, ml; 357 is the specific absorption rate of the complex of cinaroside with reagents at a wavelength of 382 nm.

Example 3. Determination of the sum of flavonoids in a liquid alcoholic extract (1: 2) of a short-haired eye. 1 ml of liquid alcoholic extract is placed in a flat-bottomed volumetric flask with a capacity of 25 ml and the volume of the solution is brought up to the mark with 80% ethanol. The resulting dilution was filtered into a flat-bottomed flask through a paper filter (solution A), the first 10 ml of the filtrate was discarded. 1 ml of solution A was placed in a 50 ml volumetric flask, 1 ml of a 2% alcohol solution of aluminum chloride and 1 ml of an 8% alcohol solution of sodium acetate were added, the solution volume was adjusted to the mark with 80% ethanol and stirred (solution B). After 30 minutes, the optical density of solution B was measured on a spectrophotometer at a wavelength of 382 nm in a cuvette with an absorbing layer thickness of 10 mm. As a comparison solution, use 1 ml of solution A, brought to 80% ethanol to the mark in a 50 ml volumetric flask. The content of the sum of flavonoids in the liquid alcohol extract (1: 2) short-haired eyeballs in terms of cynaroside is calculated by the formula:

where D is the optical density of the test solution; a - a sample of raw materials, g; V is the volume of solution A taken for dilution, ml; 25 - the volume of solution A, ml; 50 - the volume of solution B, ml; 357 is the specific absorption rate of the complex of cinaroside with reagents at a wavelength of 382 nm.

Example 4. Determination of the sum of flavonoids in a dry alcohol and dry aqueous extract of a short-haired pupil. About 0.1 g (accurately weighed) of a dry alcohol or water extract is placed in a flat-bottomed volumetric flask with a capacity of 25 ml, dissolved and the solution volume is brought up to 80% with ethanol to the mark. The resulting dilution was filtered into a flat-bottomed flask through a paper filter (solution A), the first 10 ml of the filtrate was discarded. 1 ml of solution A was placed in a 50 ml volumetric flask, 1 ml of a 2% alcohol solution of aluminum chloride and 1 ml of an 8% alcohol solution of sodium acetate were added, the solution volume was adjusted to the mark with 80% ethanol and stirred (solution B). After 30 minutes, the optical density of solution B was measured on a spectrophotometer at a wavelength of 382 nm in a cuvette with an absorbing layer thickness of 10 mm. As a comparison solution, use 1 ml of solution A, brought to 80% ethanol to the mark in a 50 ml volumetric flask. The content of the sum of flavonoids in the dry alcohol and water extract of the short-haired eyebright in terms of cinaroside is calculated by the formula:

where D is the optical density of the test solution; a - a sample of raw materials, g; V is the volume of solution A taken for dilution, ml; 25 - the volume of solution A, ml; 50 - the volume of solution B, ml; W is the loss in mass upon drying of the dry extract,%; 357 is the specific absorption rate of the complex of cinaroside with reagents at a wavelength of 382 nm.

Example 5. Determination of the sum of flavonoids in a substance containing a complex of flavonoids of an eyebright short-hair (fraction of flavonoids). About 0.1 g (accurately weighed) of a substance containing the sum of flavonoids is placed in a flat-bottomed volumetric flask with a capacity of 25 ml, dissolved and the volume of the solution is brought to 80% with ethanol to the mark. The resulting dilution was filtered into a flat-bottomed flask through a paper filter (solution A), the first 10 ml of the filtrate was discarded. 1 ml of solution A was placed in a 50 ml volumetric flask, 1 ml of a 2% alcohol solution of aluminum chloride and 1 ml of an 8% alcohol solution of sodium acetate were added, the solution volume was adjusted to the mark with 80% ethanol and stirred (solution B). After 30 minutes, the optical density of solution B was measured on a spectrophotometer at a wavelength of 382 nm in a cuvette with an absorbing layer thickness of 10 mm. As a comparison solution, use 1 ml of solution A, brought to 80% ethanol to the mark in a 50 ml volumetric flask. The content of the amount of flavonoids in the substance in terms of cinaroside is calculated by the formula:

where D is the optical density of the test solution; a - a sample of the substance, g; V is the volume of solution A taken for dilution, ml; 25 - the volume of solution A, ml; 50 - the volume of solution B, ml; W is the loss in mass upon drying of the substance,%; 357 is the specific absorption rate of the complex of cinaroside with reagents at a wavelength of 382 nm.

Example 6. Determination of the sum of flavonoids in a liquid aqueous extract (1: 2) of a short-haired eye. 1 ml of a liquid aqueous extract was placed in a 25 ml flat-bottomed volumetric flask and the volume of the solution was adjusted to the mark with 80% ethanol. The resulting dilution is filtered into a flat-bottomed flask through a paper filter (solution A), the first 10 ml of the filtrate is discarded, 1 ml of solution A is placed a 50 ml volumetric flask, 1 ml of a 2% alcohol solution of aluminum chloride and 1 ml of 8% alcohol are added sodium acetate solution, bring the volume of the solution to the mark with 80% ethanol and mix (solution B). After 30 minutes, the optical density of solution B was measured on a spectrophotometer at a wavelength of 382 nm in a cuvette with an absorbing layer thickness of 10 mm. As a comparison solution, use 1 ml of solution A, brought to 80% ethanol to the mark in a 50 ml volumetric flask. The content of the sum of flavonoids in the liquid aqueous extract of the pupils of the short-haired in terms of cinaroside is calculated by the formula:

where D is the optical density of the test solution; a - a portion of a liquid aqueous extract, ml; V is the volume of solution A taken for dilution, ml; 25 - the volume of solution A, ml; 50 - the volume of solution B, ml; 357 is the specific absorption rate of the complex of cinaroside with reagents at a wavelength of 382 nm.

Statistical processing of the results of the quantitative determination of the sum of flavonoids in extraction preparations is given in Table 7. As follows from the data obtained, the error of a single determination with a reliable probability of 95% is in the range from 0.49 to 2.65%.

Thus, the method for quantitative determination of the amount of flavonoids is unified, since it allows the determination of active substances in the raw materials of various types of eyewear and extraction preparations of eyelid short-hair according to a single method, the difference in the analysis is only in the size of the weighed portions and the degree of dilution to the photometric solution table 8).

The developed method can be used in the pharmaceutical and medical industries to standardize the raw materials of three types of eyewear and extraction preparations of short-haired asthma according to the content of active substances (flavonoids), and carrying out technological control of production.

Literature

Plant resources of the USSR. Flowering plants, their use, of the family Caprifoliaceae - Plantaginaceae. - M .: Nauka, 1990, p.139.

Sukhinina T.V. Pharmacognostic study of plants of the genus of pupil / Abstract. Cand. dis., Perm, 2002.20 s.

Deutschen Arcneimittel Codex, Stuttgart, 1997, A-192.

Yartseva I.B., Kurkin V.A. Quantitative determination of the amount of flavonoids in the grass of dandelion officinalis // Pharmacy, 1996, No. 4, pp. 24-26.

Samylina I.A., Evdokimova O.V. Kashnikova M.V. The use of aluminum chloride to determine the amount of flavonoids in the flowers of hawthorn // Pharmacy, 1994, No. 6, S.42-45.

Smirnova L.P., First L.N. Quantitative determination of the amount of flavonoids in the flowers of a sandy immortelle // Chemical and Pharmaceutical Journal, 1998, No. 6, S.35-36.

Chemesova I.I., Chubarova S.L., Sakanyan E.I. et al. Spectrophotometric method for quantifying the content of polyphenols in a dry extract from the aerial part of Mellilotus officinalis (L.) Pall. and in its dosage form (tablets) // Plant Resources, 2000, Issue 1, T.36, S.72-74.

Kabishev K.E., Sakanyan E.I. Quantitative determination of the amount of flavonoid compounds in intranasal dosage forms of the drug “Oxophilum” with a polyextract from the aerial part of Oxytropis oxyphilla (Pall.) DC.// Plant Resources, 2002, Issue 4, T.38, S.120-127.

Bandyukova V.A. Phenolic acids of plants, their esters and glycosides // Chemistry of nature. compounds, 1983, No. 2, C.271-273.

Lovtseva E.A. Improvement of quality control methods for medicines of purine derivatives of N-glycoside structure / Abstract. Cand. dis., Pyatigorsk, 1993.20 s.

The State Pharmacopoeia of the USSR, XI ed., M., 1987, T.I., 336 p.

Table 1 Spectral characteristics (λ _max ) of extracts from grass of various types of pupils in the UV region The conditions of the invention Types of Eyebright O. short-haired O. small-flowered O. Reuters 1. Alcohol extraction 328 nm 328 nm 328 nm 2. The differential spectrum of alcohol extraction with AlCl ₃ 376 nm 376 nm 376 nm 3. The differential spectrum of alcohol extraction with AlCl ₃ and CH ₃ COONa 382 nm 382 nm 382 nm

table 2 Spectral characteristics of the differential UV spectra of extraction preparations of a short-haired eyebright with aluminum chloride and sodium acetate Extract preparations λ _max , NM 1. Infusion (1:10) 382 2. Dry water extract 382 3. Liquid water extract (1: 2) 382 4. Tincture (1: 5) 382 5. Dry alcohol extract 382 6. Liquid alcoholic extract (1: 2) 382 7. A substance containing a complex of flavonoids (fraction of flavonoids) 382

Table 3 The influence of extraction conditions on the content of the sum of flavonoids in the grass Extraction conditions The content of the amount of flavonoids,% Extractant: Water 2.6 Ethanol,%: 40 3.2 60 3.3 80 3,5 90 3.4 The ratio of raw materials and extractant (80% ethanol): 1:10 2.6 1:20 3,5 1:40 3.8 1:60 4,5 1:80 5.2 1: 100 5.1 Extraction time, min
(80% ethanol; ratio of raw material to extractant 1:80): 10 4.7 twenty 5.2 40 5.1 60 4.9

Table 4 Calculation of the specific absorption coefficient of GSO cinaroside at λ = 382 nm The concentration of the solution,% Optical density The specific absorption coefficient, E ^1% _1cm The average value of specific coefficient. absorption., E ^1% _1cm 0.000132 0,049 371.21




357 ± 3.4 0,000264 0,093 352.27 0,000393 0.140 356.23 0,000528 0.180 340.91 0,000659 0.232 351.52 0,000792 0,300 378.78 0,000924 0.349 378.79 0,001056 0.367 348.48 0.001188 0.421 354.98 0.00132 0.451 341.75

Table 5 Metrological characteristics of the method for quantitative determination of the sum of flavonoids in grass of three types Research species Statistical Parameters f X S _x P% t Δx E% O. short-haired nine 4.98 0.1195 95 2.26 0.27 ± 5.5 O. small-flowered nine 4.69 0.0929 95 2.26 0.21 ± 4,5 O. Reuters nine 3.12 0,0708 95 2.26 0.16 ± 5.1

Table 6 The results of the quantitative determination of the amount of flavonoids in the grass of the euphorbia short-haired using the GSO cinaroside additive method The content of the total flavonoid 1.0 g of raw material, mg Added GSO cinaroside, mg Sum of flavonoids Relative error,% Found mg Calculated, mg 49.8 3.7 (in raw materials) 52.3 53.5 -2.2 49.8 3.7 (in raw materials) 52.9 53.5 -1.1 50,2 3.7 (in raw materials) 52,2 53.9 +3.2 51.5 3.7 (to extract) 56.1 55.2 -1.6 61.3 3.7 (to extract) 66.7 65.0 -2.6 61.3 3.7 (to extract) 62.6 0.9 +3.7

Table 7 Metrological characteristics of the method for the quantitative determination of the amount of flavonoids in extraction preparations Extract preparations Statistical Parameters f x S _x P% t Δx E% 1. Infusion (1:10) 4 0.15 0,001549 95 2,57 0,00398 ± 2.65 2. Liquid water extract (1: 2) 4 0.51 0.003162 95 2,57 0.008126 ± 1.59 3. Tincture (1: 5) 4 0.65 0,003878 95 2,57 0,00997 ± 1.53 4. Liquid alcohol extract (1: 2) 4 2.85 0.005711 95 2,57 0.01468 ± 0.52 5. Dry water extract 4 12.80 0,04472 95 2,57 0.11494 ± 0.90 6. Dry alcohol extract 4 19.82 0,03748 95 2,57 0,09634 ± 0.49 7. A substance containing a complex of flavonoids (sum of flavonoids) 4 26.00 0.1789 95 2,57 0.4598 ± 1.77

Claims (6)

1. A standardized method for the quantitative determination of flavonoids in grass and extractive preparations of eyebright, characterized in that a complexing agent — a 2% alcohol solution of aluminum chloride and an ionizing agent — an 8% alcohol solution of sodium acetate are used together in the analytical reaction; the optical density of the colored complex is determined by differential spectrophotometry at λ = (382 ± 2) nm; to calculate the amount of flavonoids, a specific absorption rate of E ^1% _1cm = 357 state standard sample of cinaroside is used. 2. The unified quantitative determination method according to claim 1, characterized in that for determining the amount of flavonoids in the grass, the pupils are short-haired, pupils of small-color and pupils of Reuters are taken 0.6 g of crushed raw materials and extracted with 80% ethyl alcohol, in the ratio of raw materials: extractant 1:80 for 20 minutes; dilute the initial extraction to the ratio of the sample: spectrophotometric solution of 0.6: 2500. 3. The unified method of quantitative determination according to claim 1, characterized in that to determine the amount of flavonoids in the infusions, the pupils of a short-haired specimen take a 1 ml sample and dilute it to a ratio of the sample: spectrophotometric solution of 1:50. 4. The unified method of quantitative determination according to claim 1, characterized in that for determining the amount of flavonoids in a liquid alcohol extract, the pupils of a short-haired specimen take a weighed portion of 1 ml and dilute it to a ratio of a weighed portion: spectrophotometric solution of 1: 2500. 5. The unified method of quantitative determination according to claim 1, characterized in that to determine the sum of flavonoids in dry alcohol and dry water extracts in a substance containing a complex of flavonoids, they take a sample of 0.1 g and dilute it to the ratio of sample: spectrophotometric solution 0, 1: 1250. 6. The unified quantitative determination method according to claim 1, characterized in that to determine the amount of flavonoids in a liquid aqueous extract, a 1 ml sample is taken and diluted to a sample: spectrophotometric solution ratio of 1: 1250.