Tannins (tanides) are plant high-molecular phenolic compounds that can precipitate proteins and have an astringent taste.
The term "tannins" has developed historically, thanks to the ability of these compounds to turn raw animal skin into durable skin, resistant to moisture and microorganisms. The use of this term was officially proposed in 1796 by Seguin to designate substances in the extracts of certain plants that can carry out the tanning process.
Tanning is a complex chemical interaction of tannins with collagen molecules, the main protein of connective tissue. Tanning properties are possessed by polynuclear phenols containing more than one hydroxyl in the molecule. With a flat arrangement of tanide on a protein molecule, stable hydrogen bonds arise between them:
Fragment of a protein molecule Fragment of a tanide molecule
The strength of the interaction of tanide with protein depends on the number of hydrogen bonds and is limited by the size of the molecule of the polyphenolic compound. The molecular weight of tannins can be up to 20,000. At the same time, there are 1-2 phenolic hydroxy groups per 100 molecular weight units in tannins. Therefore, the number of hydrogen bonds formed is numerous and the tanning process is irreversible. Hydrophobic radicals oriented to the external environment make the skin inaccessible to moisture and microorganisms.
Not all tannins are capable of true tanning. This property distinguishes compounds having a molecular weight of 1,000 or more. Polyphenolic compounds with a mass of less than 1,000 are not capable of tanning leather and have only an astringent effect.
Tannins are very widely used in industry. Suffice it to say that the world production of tannins exceeds 1,500,000 tons per year, and the share of vegetable tannins is up to 50-60% of the total.
Distribution in the plant world and the role of tannins in plants. Tannins are widely found in representatives of angiosperms and gymnosperms, algae, fungi, lichens, in club mosses and ferns. They are found in many higher plants, especially dicots. Their greatest number was found in a number of representatives of the families Fabaceae, Myrtaceae, Rosaceae, Anacardiaceae, Fagaceae, Polygonaceae.
Tannins in the plant are located in cell vacuoles and are adsorbed on cell walls during cell aging. They accumulate in large quantities in underground organs, bark, but can be found in leaves and fruits.
Tannins perform mainly protective functions in plants. With mechanical damage to tissues, an increased formation of tannins begins, accompanied by their oxidative condensation in the surface layers, thereby protecting the plant from further damage and the negative influence of pathogens. Due to the large amount of phenolic hydroxyls, tannins have pronounced bacteriostatic and fungicidal properties, thereby protecting plant organisms from various diseases.
Classification of tannins. In 1894, G. Procter, studying the end products of the pyrolysis of tannins, discovered 2 groups of compounds - pyrogallic (pyrogallol is formed) and pyrocatechin (pyrocatechin is formed during decomposition):
K. Freudenberg in 1933 specified the classification of G. Procter. He, like Procter, classified tannins according to the end products of their decomposition, but not under pyrolysis conditions, but under acid hydrolysis. Depending on the ability to hydrolyze, K. Freudenberg proposed to distinguish two groups of tannins: hydrolysable and condensed. Currently, the classification of K. Freudenberg is more often used.
To the group hydrolysable tannins include compounds built according to the type of esters and decomposing during acid hydrolysis into constituent components. The central link is most often glucose, less often other sugars or alicyclic compounds (for example, quinic acid). The alcohol hydroxyls of the central residue can be ether bonded to gallic acid, forming a group gallotannins, or ellagic acid, forming a group ellagitannins.
Gallotannins- esters of gallic acid, the most common in the group of hydrolysable tannins. There are mono-, di-, tri-, tetra-, penta- and polygalloy ethers. The representative of monogalloyl ethers is b-D-glucogallin:
An example of polygalloyl ethers is Chinese tannin, the structure of which was first established in 1963 by Haworth:
ellagitannins are esters of sugar and ellagic acid or its derivatives. Ellagic acid is formed by the oxidation of two molecules of gallic acid to hexaoxydiphenic acid, which immediately forms a lactone - ellagic acid:
As in the previous case, the sugar component of ellagitannins is most often glucose.
Non-sugar esters of gallic acids are esters of gallic acid and a non-sugar component, such as quinic acid, hydroxycinnamic, etc. An example of this group of substances is 3,4,5-trigalloylquinic acid.
condensed tannins differ from hydrolysable ones in that during acid hydrolysis they are not split into constituent components, but, on the contrary, under the action of mineral acids, dense red-brown polymerization products are formed - flobaphenes.
Condensed tannins are formed mainly by catechins and leucocyanidins, and, much less frequently, by other reduced forms of flavonoids. Condensed tannins do not belong to the "Glycosides" group: there is no sugar component in condensed tannins.
The formation of condensed tannins can occur in two ways. K. Freidenberg (30s of the XX century) established that the formation of condensed tannins is a non-enzymatic process of autocondensation of catechins or leukocyanidins (or their cross-condensation) as a result of exposure to atmospheric oxygen, heat and an acidic environment. Autocondensation is accompanied by rupture of the pyran ring of catechins and the C-2 carbon atom of one molecule is connected by a carbon-carbon bond to the C-6 or C-8 carbon atom of another molecule. In this case, a sufficiently extended chain can be formed:
According to another scientist, D. Hathway, condensed tannins can be formed as a result of enzymatic oxidative condensation of molecules according to the “head to tail” type (ring A to ring B) or “tail to tail” (ring B to ring B):
In plants containing condensed tannins, there are necessarily their precursors - free catechins or leukocyanidins. Often there are mixed condensed polymers consisting of catechins and leucocyanidins.
As a rule, tannins of both condensed and hydrolysable groups are simultaneously present in plants.
Physical and chemical properties of tannins. Tannins are characterized by high molecular weight - up to 20,000. Natural tannins, with a few exceptions, are known to date only in an amorphous state. The reason for this is that these substances are mixtures of compounds that are similar in chemical structure but differ in molecular weight.
Tannins are yellow or brown compounds that form colloidal solutions in water. Soluble in ethanol, acetone, butanol and insoluble in solvents with pronounced hydrophobicity - chloroform, benzene, etc.
Gallotannins are poorly soluble in cold water and relatively well in hot water.
Tannins have optical activity and are easily oxidized in air.
Due to the presence of phenolic hydroxyls, they are precipitated by salts of heavy metals and form colored compounds with Fe +3.
Isolation of tannins from vegetable raw materials. Since tannins are a mixture of various polyphenols, their isolation and analysis presents a certain difficulty.
Often, to obtain the amount of tannins, raw materials are extracted with hot water (tannins are poorly soluble in cold water) and the cooled extract is treated with an organic solvent (chloroform, benzene, etc.) to remove lipophilic substances. Then the tannins are precipitated with salts of heavy metals, followed by the destruction of the complex with sulfuric acid or sulfides.
To obtain a fraction of tannins similar in chemical structure, it is possible to use the extraction of raw materials with diethyl ether, methyl or ethyl alcohol with preliminary removal of lipophilic components using solvents with pronounced hydrophobicity - petroleum ether, benzene, chloroform.
The isolation of some components of tannins by precipitation from aqueous or water-alcohol solutions with lead salts is widespread. The resulting precipitates are then treated with dilute sulfuric acid.
When isolating the individual components of tannins, chromatographic methods are used: adsorption chromatography on cellulose, polyamide; ion exchange on various cation exchangers; distribution on silica gel; gel filtration on molecular sieves.
Identification of individual components of tannins is carried out using chromatography on paper or in a thin layer of sorbent, using spectral analysis, qualitative reactions and the study of cleavage products.
Qualitative analysis of tannins. Qualitative reactions to tannins can be divided into two groups: precipitation reactions and color reactions. To carry out qualitative reactions, raw materials are most often extracted with hot water.
Precipitation reactions. 1. When tannins interact with a 1% gelatin solution prepared in a 10% sodium chloride solution, a precipitate forms or the solution becomes cloudy. When excess gelatin is added, the turbidity disappears.
2. Tanides give abundant precipitation with alkaloids (caffeine, pachycarpine), as well as some nitrogenous bases (urotropine, novocaine, dibazol).
3. When interacting with a 10% solution of lead acetate, tannins of the hydrolysable group form a flocculent precipitate.
4. Tannins of the condensed group form a flocculent precipitate in reaction with bromine water.
color reactions. The tannins of the hydrolyzable group with a solution of iron ammonium alum form black-blue colored compounds, and the condensed group - black-green.
If the plant simultaneously contains tannins and hydrolysable and condensed groups, then first hydrolysable tannins are precipitated with a 10% solution of lead acetate, the precipitate is filtered off, and then the filtrate is reacted with a solution of iron ammonium alum. The appearance of a dark green color indicates the presence of substances of the condensed group.
Quantitative determination of tannins. While there are about 100 different methods for the quantitative determination of tannins, an accurate quantitative analysis of this group of biologically active substances is difficult.
Among the widely used methods for the quantitative determination of tannins, the following can be distinguished.
1. Gravimetric - based on the quantitative precipitation of tannins by gelatin, salts of heavy metals, etc.
2. Titrimetric - based on oxidative reactions, primarily with potassium permanganate.
3. Photoelectrocolorimetric - based on the ability of tannins to form stable colored reaction products with iron oxide salts, phosphotungstic acid, etc.
The State Pharmacopoeia of X and XI editions recommends a titrimetric method for the quantitative determination of tannins.
Contents
OFS.1.5.3.0008.15 Determination of the content of tannins in medicinal herbal raw materials and medicinal herbal preparations
Instead of Art. GF XI
Determination of the content of tannins in medicinal herbal raw materials and medicinal herbal preparations is carried out by titrimetric and/or spectrophotometric methods. The titrimetric method consists in determining the amount of tannins in terms of tannin, and the spectrophotometric method allows you to determine the amount of tannins in terms of pyrogallol.
Method 1. Determination of the amount of tannins in terms of tannin
About 2 g (accurately weighed) of crushed medicinal herbal raw materials or herbal medicinal products, sifted through a sieve with holes of 3 mm, are placed in a 500 ml conical flask, poured into 250 ml of water heated to boiling and boiled under reflux on an electric stove with a closed spiral for 30 min with occasional stirring. The resulting extract is cooled to room temperature and filtered through cotton wool into a volumetric flask with a capacity of 250 ml so that the particles of the raw material / preparation do not fall into the flask, the volume of the solution is adjusted to the mark with water and mixed. 25.0 ml of the resulting aqueous extract is placed in a 1000 ml conical flask, 500 ml of water, 25 ml of indigo sulfonic acid solution are added and titrated with constant stirring of potassium permanganate with a solution of 0.02 M until golden yellow color.
In parallel, a control experiment is carried out: 525 ml of water, 25 ml of a solution of indigo sulfonic acid are placed in a conical flask with a capacity of 1000 ml and titrated with constant stirring of potassium permanganate with a solution of 0.02 M until golden yellow color.
1 ml of potassium permanganate solution of 0.02 M corresponds to 0.004157 g of tannins in terms of tannin.
(V – V 1 ) 0.004157 250 100 100
X = ————————————————— ,
a 25 (100 – W)
V is the volume of potassium permanganate solution of 0.02 M used for titration of aqueous extract, ml;
V 1 is the volume of potassium permanganate solution of 0.02 M used for titration in the control experiment, ml;
0.004157 - the amount of tannins corresponding to 1 ml of potassium permanganate solution 0.02 M (in terms of tannin), g;
a- a sample of raw materials or herbal medicinal product, g;
W– humidity of medicinal plant material or medicinal plant product, %;
250 – total volume of water extraction, ml;
25 – volume of water extract taken for titration, ml.
Note.Preparation of a solution of indigo sulfonic acid. 1 g of indigo carmine is dissolved in 25 ml of concentrated sulfuric acid, then an additional 25 ml of concentrated sulfuric acid is added and diluted with water to 1000 ml, carefully pouring the resulting solution into water, in a volumetric flask with a capacity of 1000 ml, stirred.
Method 2. Determination of the amount of tanninsin terms of pyrogallol
About 0.5 - 1.0 g (accurately weighed or otherwise specified in the pharmacopoeial monograph or regulatory documentation) of crushed medicinal plant material or herbal medicinal product, sifted through a sieve with holes of 0.18 mm, is placed in a conical flask with a capacity of 250 ml , add 150 ml of water and boil on a water bath under reflux for 30 minutes. The resulting aqueous extract in the flask is cooled to room temperature, filtered through cotton wool into a volumetric flask with a capacity of 250 ml so that the particles of the raw material do not fall into the flask, the volume of the solution is adjusted to the mark with water and mixed. The resulting solution is filtered through a paper filter with a diameter of about 125 mm, discarding the first 50 ml of the filtrate.
The determination is carried out in a place protected from light.
Determination of the amount of tannins. Place 5.0 ml of the filtrate in a 25 ml volumetric flask, dilute the volume of the solution with water to the mark and mix. 2.0 ml of the resulting solution is placed in a volumetric flask with a capacity of 25 ml, 1 ml of phosphomolybdenum-tungsten reagent, 10 ml of water are added and the volume of the solution is adjusted to the mark of sodium carbonate with a solution of 10.6% (test solution). After 30 minutes, measure the optical density of the test solution (A 1) on a spectrophotometer at a wavelength of 760 nm in a cuvette with a layer thickness of 10 mm, using water as a reference solution.
Determination of the amount of tannins not adsorbed by skin powder. 0.1 g of skin powder is added to 10.0 ml of the filtrate, the resulting mixture is stirred for 60 minutes and filtered through a paper filter. 5.0 ml of the resulting filtrate is placed in a volumetric flask with a capacity of 25 ml, the volume of the solution is adjusted to the mark with water and mixed. 2.0 ml of the resulting solution is placed in a volumetric flask with a capacity of 25 ml, 1 ml of phosphomolybdenum-tungsten reagent, 10 ml of water are added, the volume of the solution is adjusted to the mark of sodium carbonate with a solution of 10.6% and mixed (test solution). After 30 min, measure the optical density of the test solution (A 2) on a spectrophotometer at a wavelength of 760 nm in a cuvette with a layer thickness of 10 mm, using water as a reference solution.
In parallel, measure the optical density of the standard solution.
2.0 ml of pyrogallol SS solution is placed in a 25 ml volumetric flask, 1 ml of phosphomolybdenum-tungsten reagent, 10 ml of water are added, the volume of the solution is adjusted to the mark of sodium carbonate with a solution of 10.6% and mixed (standard solution). After 30 min, measure the optical density of the standard solution (A 3) on a spectrophotometer at a wavelength of 760 nm in a cuvette with a layer thickness of 10 mm, using water as a reference solution.
A 1- optical density of the test solution when determining the amount of tannins;
A 2 - the optical density of the test solution when determining the amount of tannins not adsorbed by the skin powder, in terms of pyrogallol;
A 3— optical density of the standard solution;
a- weight of medicinal plant material or medicinal plant preparation, g;
a 0 is a sample of pyrogallol SS, g;
W– humidity of medicinal plant material or medicinal plant product, %.
Note. Preparation of pyrogallol RS solution. 0.05 g (accurately weighed) pyrogallol SS is placed in a volumetric flask with a capacity of 100 ml, dissolved in water, the volume of the solution is adjusted to the mark with water, and mixed. 5.0 ml of the resulting solution is placed in a volumetric flask with a capacity of 100 ml, the volume of the solution is adjusted to the mark with water and mixed. The solution is used freshly prepared.
Introduction
In plants, one of the most common groups of biologically active substances (BAS) are tannins (tannins), which have a wide range of pharmacological activity.Tanninshave a hemostatic, astringent, anti-inflammatory, antimicrobial effect, and also show high P-vitamin activity, anti-sclerotic and antihypoxic effect. Condensed tannins are antioxidants and have an antitumor effect. Tanninsused as an antidote for poisoning with glycosides, alkaloids, salts of heavy metals. In medicine, tannins are used in the treatment of diseases such as stomatitis, gingivitis, pharyngitis, tonsillitis, colitis, enterocolitis, dysentery, they are also used for burns, uterine, gastric and hemorrhoidal bleeding..
Definition of contenttannins is an important component in establishing the quality of plant materials containing tannins. There are various methods for the determination of tannins, but the most commonly used are titrimetric and spectrophotometric methods.
Objective- validation evaluation of methods for the quantitative determination of tannins in terms of convergence, correctness, linearity.
Materials and methods of research
The raw material used as the object of study was air-dry grass.common cuff (Alchemilla vulgaris L.) fam. Rosaceae (Rosaceae).
For the validation evaluation of methods for the quantitative determination of tannins in air-dry grasscuff vulgaris, two methods were chosen: permanganometric titration and spectrophotometric determination based on the reaction with the Folin-Ciocalteu reagent. The choice of methods is justified by the frequency of their use in practice.
air dry grasscuff vulgaris harvested in September 2015 in the Primorsky district of the Arkhangelsk region, which was the raw material for the study and quantitative determination of tannins (tannins).
The method of permanganometric determination is a pharmacopoeial one, whichbased on the oxidation reaction of tannins with a solution of potassium permanganate.About 2 g (accurately weighed) of crushed raw material, sifted through a sieve with a hole size of 3 mm, was placed in a 500 ml conical flask, 250 ml of water heated to boiling was added and boiled under reflux on an electric stove with a closed spiral for 30 minutes with occasional stirring. The resulting extract was cooled to room temperature and a 250 ml conical flask was filtered through cotton so that the raw material particles did not enter the flask. 25 ml of the obtained extract was taken with a pipette and transferredinto another conical flask with a capacity of 750 ml, add 500 ml of water, 25 ml of indigo sulfonic acid solution and titrate with constant stirring with a potassium solutionpermanganate (0.02 mol/l) until golden yellow.
In parallel, a control experiment was carried out.
1 ml of potassium permanganate solution (0.02 mol/l) corresponds to 0.004157 g of tannins in terms of tannin.
The content of tannins (X), in percent, in terms of absolute dry raw materials, was calculated by the formula (1):
Where (1)
V is the volume of potassium permanganate solution (0.02 mol/l) used for titration of the extract, ml;
is the volume of potassium permanganate solution (0.02 mol/l) used for titration in the control experiment, ml;
0.004157 - the amount of tannins corresponding to 1 ml of potassium permanganate solution (0.02 mol / l) (in terms of tannin), g;
250 – total volume of extraction, ml;
25 – volume of extract taken for titration, ml.
m– mass of raw materials, g;
W- loss in mass during drying of raw materials, g;
For the quantitative determination of tannins by spectrophotometry, about 1 g (accurately weighed) of the studied plant material, crushed to a particle size passing through sieves with a hole size of 1 mm, was placed in a conical flask with a thin section with a capacity of 50 ml, 25 ml of a mixture of acetone-water was added in a ratio of 7:3 (70% acetone solution). The flask was closed and placed in a laboratory mixer (LAB PU-2, Russia) for 60 minutes. The obtained extract was filtered into a volumetric flask with a capacity of 50 ml and the volume was brought to the mark with 70% acetone solution (solution A).
In a volumetric flask with a capacity of 10 ml, 1 ml of solution A was placed, the volume of the solution in the flask was adjusted with purified water to the mark (solution B).
0.5 ml of solution B was placed in a volumetric flask with a capacity of 10 ml, 2 ml of purified water, 0.25 ml of the Folin-Ciocalteu reagent, 1.25 ml of a 20% sodium carbonate solution were added, and the volume of the solution was brought to the mark with water. The flask was left for 40 minutes in a place protected from light. The optical density of the solution was determined at a wavelength of 750 nm. A mixture of reagents without extract was used as a reference solution.
The content of tannins in the extracts from vegetable raw materials was calculated from the values of the calibration graph for the construction of which, a 0.1 mg/ml solution of the tannin CO standard sample was used. For this purpose, 0.05 g (exact mass) of tannin CO was placed in a 100 ml volumetric flask, dissolved in 30 ml of water, and the volume in the flask was adjusted to the mark with the same solvent (solution A).
1 ml of the resulting solution was transferred into a 10 ml volumetric flask. The volume of the solution in the flask was made up to the mark with water (solution B).
A series of solutions containing 1; 2; 3; four; Tannin CO (5 μg/mL) was prepared by placing weighed portions of solution B into 10-mL volumetric flasks, the Folin–Ciocalteu reagent and 20% aqueous sodium carbonate solution were added, and the volume of the solutions in the flask was made up to the mark with water.
The solutions were mixed, the flasks were sealed and kept at room temperature in a place protected from light for 40 min.
The optical density of the resulting solutions was determined spectrophotometrically in quartz cuvettes with a layer thickness of 1 cm at a wavelength of 725 nm relative to the reference solution.
The reference solution was a mixture of reagents without the addition of CO tannin (solution B).
Based on the results of the studies, a graph of the dependence of optical density on tannin concentration was built (Fig. 1).
Taking into account the obtained values, the amount of tannins was calculated in terms of tannin according to the formula:
, where
results
The results of the quantitative determination of tannins by titration are presented in table. one.
Table 1. Results of the quantitative determination of tannins by permanganatometry
| Weight of plant raw materials, g | The volume of potassium permanganate (0.02 mol/l) used for titration of the obtained extract from vegetable raw materials, ml | The amount of tannins, % (X i) | |
|
2,10250 |
15,34892 |
15,72%
|
|
|
2,03255 |
15,21262 |
||
|
2,18345 |
15,84713 |
||
|
2,24350 |
16,24333 |
||
|
2,12465 |
15,85257 |
||
|
2,07055 |
15,80574 |
The average content of tannins in raw materials was 15.7%. The calculated value of the relative standard deviation (0.024%), which does not exceed 2%, which characterizes the satisfactory convergence of the results obtained.
The addition method was used to determine the correctness of the method. For this purpose, 1 ml of 0.05%, 0.1% and 0.15% tannin CO was added to the titration flask and titrated three times for each case. The results of the research are presented in table. 2.
Table 2. Determination of the correctness of the method of permanganometric titration of tannins
| The amount of added CO tannin, g | Mass of raw materials, g | Calculated amount of tannins, g | Found amount of tannins, g | Opening rate, % | Metrological characteristics |
|
0,0005 |
2,2435 |
0,0357 |
0,0353 |
98,87 |
99,91%
|
|
2,1247 |
0,0339 |
0,0340 |
100,29 |
||
|
2,0706 |
0,0330 |
0,0337 |
102,12 |
||
|
0,001 |
2,2435 |
0,0362 |
0,0357 |
98,61 |
|
|
2,1247 |
0,0344 |
0,0340 |
98,84 |
||
|
2,0706 |
0,0335 |
0,0336 |
100,51 |
||
|
0,0015 |
2,2435 |
0,0367 |
0,0366 |
99,73 |
|
|
2,1247 |
0,0349 |
0,0353 |
101,14 |
||
|
2,0706 |
0,0340 |
0,0337 |
99,12 |
The results obtained indicate that the calculated Student's coefficient is less than the tabular value andThe technique does not contain a systematic error, which allows us to conclude that it is correct.
To study the linearity, the dependence of the found values of the quantitative content of tannins on the weight of the studied plant material was determined. For this purpose, quantitative determination of tannins was carried out in six samples of air-dry raw materials of the common cuff, differing in weight (Table 3).
table 3
|
|
Volume of potassium permanganate used for titration, ml |
|
|
2,0706 |
0,3159 |
|
|
3,0013 |
10,8 |
0,4490 |
|
4,0595 |
13,0 |
0,5404 |
|
5,1180 |
15,3 |
0,6360 |
|
6,1385 |
18,2 |
0,7566 |
Based on the data obtained in the course of the studies, a graph of the dependence of a certain content of tannins on the mass of a sample of the studied plant material was plotted (Fig. 2) and the correlation coefficient was calculated.
Rice. Fig. 2. Graph of the dependence of the found amount of tannins on the weight of the sample of air-dry raw materials of the common cuff
The calculated correlation coefficient did not exceed 0.95, which indicates the linearity of the results of determining the content of the studied substances from the weight of the sample of the analyzed plant material in the indicated concentration range.
The results of the quantitative determination of tannins in air-dry raw materials of the herb of the common cuff by spectrophotometry are presented in table. four.
Table 4. Results of quantitative determination of tannins by spectrophotometry
|
Sample weight, g |
Solution Optical Density |
The amount of tannins found, % (X i) |
Metrological characteristics |
|
|
1,02755 |
0,5957 |
7,30920 |
7,87340 |
7,84%
|
|
0,99745 |
0,6130 |
7,52147 |
8,34656 |
|
|
1,0068 |
0,5678 |
6,96687 |
7,65932 |
|
|
0,99580 |
0,5742 |
7,04539 |
7,83120 |
|
|
1,0060 |
0,5750 |
7,05521 |
7,76261 |
|
|
1,00670 |
0,5617 |
6,89202 |
7,57779 |
The average content of tannins in vegetable raw materials is 7.8% with a relative standard deviation (0.034%) not exceeding 2%, which characterizes the satisfactory convergence of the results.
The addition method was used to determine the correctness of the method. For this purpose, 1 ml of 0.05%, 0.1% and 0.15% tannin CO solution was added to the flask with primary acetone extraction, and then the tannins were quantified three times for each concentration. The results of the research are presented in table. 5.
The owners of the patent RU 2439568:
The invention relates to the field of pharmacology and can be used to determine tannins in plant materials. The method for determining tannins in plant raw materials consists in extracting a sample of raw materials with water during boiling, cooling, filtering, measuring the optical density of an aliquot sample at a wavelength of 277 nm and calculating the content of the sum of all tannins according to a certain formula, then adding to the aliquot sample of the filtrate A 1% solution of collagen in 1% acetic acid is shaken, filtered, the optical density of the filtrate is measured at a wavelength of 277 nm and the content of precipitated tannins is calculated using a certain formula. The method makes it possible to increase the accuracy of determining the content of tannins in vegetable raw materials and to selectively determine precipitated and non-precipitated tannins in vegetable raw materials.
The invention relates to the pharmaceutical industry, the field of pharmacognosy and pharmaceutical chemistry, and can be used to control the quality of plant materials containing tannins.
A known method for the determination of tannins in medicinal plant materials (MPM) by coulometry in terms of tannin (S. G. Abdullina and others. Coulometric determination of tannins in medicinal plant materials. // Pharmacy. No. 4. - 2010. - P. 13 -fifteen).
The disadvantage of this method is the use of additional equipment (coulometer), a specific titrant (potassium hypoiodide), which in terms of oxidizing properties approaches potassium permanganate and does not make it possible to differentiate between high and low molecular weight tannins.
There is also known a method for determining the content of tannin and gallic acid derivatives in tea by conductometry (Patent No. 2127878. Method for separate determination of tannin and catechins (in terms of gallic acid) in tea. M.: 1999).
The disadvantage of this method is the use of toxic organic solvents (isobutyl alcohol), as well as the use of a color reaction with Fe (III), the product of which is a colored compound that is unstable in color over time.
There is also known a method for the quantitative determination of tannins in terms of tannin in the leaves of skumpia and sumac by the method of complexometry after precipitation of tannins with zinc salts (GOST 4564-79. Leaf of skumpia. Specifications; GOST 4565-79. Leaf of sumac. Specifications).
The disadvantage of this method is the duration of the analysis and the difficulty of determining the equivalence point.
There is also known a method for the quantitative determination of tannins by the spectrophotometric method after reaction with the Folin-Ciocalteu reagent in terms of gallic acid (Guidelines for methods of quality control and safety of biologically active food supplements. Guide. R 4.1.1672-03. - M. - 2004 - p.94-95).
The disadvantage of this method is the impossibility of separate determination of low and high molecular weight tannins.
Closest to the proposed method is that tannins are determined by spectrophotometry in terms of gallic acid (Guidelines for methods of quality control and safety of biologically active food supplements. Guide. R 4.1.1672-03. - M. - 2004 g. - P. 120).
The disadvantage of this method is the repeated dilution of the test sample, as a result of which the concentration of tannins in the solution is poorly determined. Also in this method, the reference solution is a buffer solution, which makes the analysis difficult. In addition, this method does not make it possible to separately determine the content of low molecular weight and high molecular weight tannins.
The objective of the invention is to improve the accuracy of the determination of tannins and the possibility of separate determination of precipitated and non-precipitable tannins in vegetable raw materials.
The problem is solved by the fact that a sample of the raw material is extracted with water during boiling, cooled, filtered, the optical density of an aliquot sample is measured at a wavelength of 277 nm and the content of the sum of all tannins is calculated by the formula
50 - flask volume, ml,
W - raw material moisture content, %,
a 1% solution of collagen in 1% acetic acid is added to an aliquot of the filtrate, shaken, filtered, the optical density of the filtrate is measured at a wavelength of 277 nm and the content of precipitated tannins is calculated by the formula
D 1 - optical density of solution 1,
D 2 - optical density of solution 2,
m nav - the weight of the sample of raw materials, g,
V a - volume of aliquot sample, ml,
250 - total extraction volume, ml,
50 - flask volume, ml,
508 - specific absorption index of gallic acid (optical density of 1% solution of gallic acid 1 mg / ml),
W - raw material moisture content, %.
Practically, the method is carried out as follows. About 2.0 (accurately weighed) crushed raw materials, sifted through a sieve with a hole diameter of 3 mm, are placed in a 500 ml flask, pour 250 ml of water heated to boiling point and boil for 30 minutes under reflux with occasional stirring. Cool to room temperature, dilute with water to 250 ml, filter through cotton wool so that the raw material particles do not get into the aqueous extract. The first 50 ml of the filtrate are discarded.
1-4 ml of aqueous extract is placed in a 50 ml volumetric flask, adjusted to the mark with water (solution 1). Measure the optical density of solution 1 at a wavelength of 277 nm. Water is used as a comparison.
30 ml of aqueous extract is placed in a measuring container with a capacity of 50 ml, 2-10 ml of precipitation reagent is added, shaken for 30-60 minutes, settled, filtered. 1-4 ml of the obtained filtrate is transferred into a flask with a capacity of 50 ml, adjusted to the mark with water (solution 2). Measure the optical density of solution 2 at a wavelength of 277 nm. Water is used as a comparison.
The invention is illustrated by the following examples.
Example 1. For analysis taken vegetable raw materials - oak bark.
About 2.0 (accurately weighed) crushed raw oak bark, sifted through a sieve with a hole diameter of 3 mm, is placed in a 500 ml flask, poured with 250 ml of water heated to boiling and boiled for 30 minutes under reflux with occasional stirring. Cool to room temperature, dilute with water to 250 ml, filter through cotton wool so that the raw material particles do not get into the aqueous extract. The first 50 ml of the filtrate are discarded.
2 ml of an aqueous extract from the oak bark is placed in a volumetric flask with a capacity of 50 ml, adjusted with water to the mark (solution 1). Measure the optical density of solution 1 at a wavelength of 277 nm. Water is used as a comparison. D 1 for oak bark is 0.595.
30 ml of aqueous extract is placed in a 50 ml volumetric container, 2 ml of precipitation reagent is added, shaken for 30 minutes, settled, filtered. 2 ml of the obtained filtrate is transferred into a flask with a capacity of 50 ml, adjusted to the mark with water (solution 2). Measure the optical density of solution 2 at a wavelength of 277 nm. Water is used as a comparison. D 2 for oak bark is 0.276.
Example 2. For analysis, the plant material of the serpentine rhizome was taken.
About 2.0 (accurately weighed) of the crushed raw material of the serpentine rhizome, sifted through a sieve with a hole diameter of 3 mm, is placed in a flask with a capacity of 500 ml, pour 250 ml of water heated to boiling and boil for 30 minutes under reflux with occasional stirring. Cool to room temperature, dilute with water to 250 ml, filter through cotton wool so that the raw material particles do not get into the aqueous extract. The first 50 ml of the filtrate are discarded.
1 ml of an aqueous extract from the rhizome of the coil is placed in a volumetric flask with a capacity of 50 ml, adjusted with water to the mark (solution 1). Measure the optical density of solution 1 at a wavelength of 277 nm. Water is used as a comparison.
30 ml of aqueous extract is placed in a measuring container with a capacity of 50 ml, 7 ml of precipitation reagent is added, shaken for 60 minutes, settled, filtered. 1 ml of the obtained filtrate is transferred into a flask with a capacity of 50 ml, adjusted to the mark with water (solution 2). Measure the optical density of solution 2 at a wavelength of 277 nm. Water is used as a comparison.
The proposed method improves the accuracy of determining the content of tannins in vegetable raw materials and selectively determine the precipitated and non-precipitated tannins in vegetable raw materials.
A method for determining tannins in vegetable raw materials in terms of gallic acid, which consists in extracting a sample of raw materials with water during boiling, cooling, filtering, measuring the optical density of an aliquot sample at a wavelength of 277 nm and calculating the content of the sum of all tannins according to the formula: 
where x a - the content of the sum of tannins in terms of gallic acid,%;
50 - flask volume, ml;
508 - specific absorption index of gallic acid (optical density of 1% solution of gallic acid 1 mg/ml);
W - raw material moisture content, %,
a 1% solution of collagen in 1% acetic acid is added to an aliquot of the filtrate, shaken, filtered, the optical density of the filtrate is measured at a wavelength of 277 nm, and the content of precipitated tannins is calculated using the formula: 
where X is the content of precipitated tannins in terms of gallic acid,%;
D 1 - optical density of solution 1;
D 2 - optical density of solution 2;
m nav - mass of sample of raw materials, g;
V a - volume of aliquot sample, ml;
250 - total volume of extraction, ml;
50 - flask volume, ml;
508 - specific absorption index of gallic acid (optical density of a 1% solution of gallic acid 1 mg/ml);
W - raw material moisture content, %.
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