RU2700787C1 - Method of determining total content of phenol compounds in plant objects - Google Patents

Abstract

FIELD: biochemistry of plants.

SUBSTANCE: invention relates to physiology and biochemistry of plants and can be used in determining content of phenol compounds (PC) in plant objects. Method involves milling vegetal raw materials, extraction in alcohol while stirring, subsequent centrifugation, adding Folin-Ciocalteu reagent and sodium carbonate to the extract. Raw material with weight of 0.05 g is ground with 1.5 cm³ 96 % ethanol, extraction of phenol compounds is carried out for 45 minutes at 45 °C with periodic stirring (every 15 minutes) and subsequent centrifugation for 2 minutes at rotation speed of 16,000 rpm, from obtained extract samples are taken, with volume of 0.075 cm³, adding to them at 0.075 cm³ Folin-Ciocalteu reagent diluted 5-fold is mixed, after 3 min 0.15 cm³ 20 % solution of sodium carbonate and 1.2 cm³ distilled water, closed with a cover, stirred and left at room temperature, and after 1 hour, the optical density of the formed tungsten blue is measured at wavelength 725 nm, the length of the optical path is 1 cm. Total content of PC is expressed in mg-equivalent gallic acid per g raw material weight.

EFFECT: use of the invention enables to develop a safe, cost-effective method of determining total content of PC in plant objects using standard equipment for laboratory analysis, without using methanol, while reducing extraction time.

1 cl, 3 tbl, 2 ex

Description

Application area

The invention relates to the field of plant physiology and biochemistry and can be used in basic research in physiology, biochemistry, plant breeding and biotechnology, and also find application in the pharmaceutical industry in assessing the quality of plant materials.

State of the art

Phenolic compounds (PS) are unique substances of secondary metabolism with antiradical properties and antioxidant activity, the formation of which is characteristic of plants (Menshikova EB, Lankin V.Z., Kandalintseva N.V. “Phenolic antioxidants in biology and medicine “Structure, Properties, Mechanisms of Action.” Berlin, Academic Publishing, 2012, 488).

A characteristic feature of PS is the ability to interact with reactive oxygen species (ROS), thereby contributing to their inactivation in all living organisms (plants, animals, humans). Excessive amounts of ROS are formed in cells under the influence of various stressors, including heavy metals, toxic waste from chemical industries, etc., as well as with increased levels of ozone and ultraviolet radiation in the atmosphere (Tarakhovsky Yu.S., Kim Yu.A., Abdrasilov B. S., Muzafarov E.N. “Flavonoids: biochemistry, biophysics, medicine” M., Pushchino, 2013, 380). ROS interact with proteins, lipids and DNA, damaging them and even causing cell death (Tyukavkina N.A. "Bioflavonoids", M., Russian doctor, 2002, 56). In this aspect, the presence of PS in plant objects, including their number, is an important indicator of the resistance of plants to stressful effects, as well as the criterion of the appropriateness of using plant materials as a product with antioxidant activity in the human diet (Kurkin VA "Actual issues of improving standardization medicinal plant materials and medicinal plant preparations containing phenolic compounds "// Modern high technology. 2016. No. 8-2. S. 247-250).

A known method for determining the total content of PS by the colorimetric method using the Folin-Chocalteu reagent containing phosphofungsten and phosphomolybdenum acids, which upon reduction with phenolic compounds (PS) in an alkaline medium, forms a blue complex (tungsten blue), the color intensity of which is proportional to the amount of PS (Singleton, VL, Orthofer, R., Lamuela-Raventos, RM "Analysis of total phenols and other oxidation substrates and antioxidants by means of Folin-Ciocalteu reagent", in Methods in Enzymology, Academic Press Inc., San-Diego, 1999, Vol. 299, P. 152-178).

There is also a method for determining the total PS content in water-methanol extracts from 24 exotic fruits by the method of Folina-Ciocalteu (J. Contreras-Calderon, L. Calderon-Jaimes, E. Guerra-Hernandez, B. Garcia-Villanova "Antioxidant capacity, phenolic content and vitamin C in pulp, peel and seed from 24 exotic fruits from Colombia "Food Research International. 2011. Vol. 44. P. 2047-2053).

In addition, a method is known for determining the total PS content in methanol extracts from quince and tea leaves by the method of Folina-Chocalteu (R. M. Costa, AS Magalhes, JA Pereira, R. B. Andrade, P. Valentro "Evaluation of free radical-scavenging and antihemolytic activities of quince (Cydonia oblonga) leaf: A comparative study with green tea (Camellia sinensis) Food and Chemical Toxicology. 2009. Vol. 47. P. 860-865).

The disadvantages of these methods are the high consumption of reagents, the duration of the procedure for determining the total content of PS, as well as the use of a toxic solvent - methanol.

Also known is a method for determining the total PS content by the Folin-Ciocalteu method using microplates in methanol extracts from strawberries, kiwi and carrots ("The Folin-Ciocalteu assay revisited: improvement of its specificity for total phenolic content determination", Analytical Methods. 2013. Vol. 5. P. 5990-5999). In this method, 0.015 cm ^{3 of the} extract containing PS are added to the microplate cell, 0.240 cm ^{3 of} distilled water, 0.015 cm ^{3 of the} Folin-Ciocalteu reagent, 0.030 cm ^{3 of a} 5.3% aqueous solution of sodium carbonate are added. Using a special spectrophotometer, which allows measuring the optical density of solutions on microplates, the density of the formed color is measured at 765 nm.

The disadvantage of this method, despite the minimum consumption of the Folin-Ciocalteu reagent, is the need to use special equipment: microplates and a spectrophotometer, which allows measuring the optical density of solutions on microplates, as well as the use of a toxic solvent - methanol.

The closest way to the proposed is a method for determining the total content of FS in tea (GOST R ISO 14502-1-2010. Tea. Method for determining the total content of polyphenols) - prototype.

The method involves 2-fold extraction of PS from a ground tea sample (weighing 0.2 g) with a 70% aqueous methanol solution at a temperature of 70 ° C with periodic stirring, followed by centrifugation and washing of the precipitate. The extracts are combined, their volume is adjusted to 10.0 cm ³ with a cold 70% aqueous methanol solution and mixed. The extract thus prepared is diluted 100 times with distilled water, and 1.0 cm ^{3 of the} diluted extract are placed in separate graduated tubes. To each tube, 5.0 cm ^{3 of} Folin-Ciocalteu reagent diluted 10 times are added and mixed. After 3 minutes, 4.0 cm ^{3 of a} 7.5% aqueous solution of sodium carbonate was added to each tube, the tubes were closed with stoppers, mixed and left at room temperature. After 1 h, the total content of PS in the extracts is determined by the colorimetric method using the Folin-Ciocalteu reagent. The optical density of the solution is measured relative to water at a wavelength of 765 nm. The optical path length is 1 cm. The total content of PS is expressed in mg equivalents (mEq) of gallic acid per g of dry weight of tea.

The disadvantage of this method is the high consumption of reagents, the duration of the extraction of PS and the use of a toxic solvent of methanol, as well as the high temperature of extraction, which can lead to the oxidation of part of the PS and, as a consequence, an incorrect assessment of the determination results.

Object of the invention

The objective of our invention is to develop a safe, cost-effective way to determine the total content of PS in plant objects using standard equipment for laboratory research, without the use of methanol, while reducing the extraction time.

The solution of the problem

This problem is solved by creating a new method for determining the total PS content in plant objects (buckwheat, wheat, callus culture of tea plant tissue), which consists in grinding them, extracting them in alcohol with stirring, subsequent centrifugation, and adding Folin-Ciocalteu reagent and carbonic sodium to the extract, moreover a sample of plant material weighing 0.05 g is ground, triturated with 1.5 cm ^{3 of} 96% ethanol in test tubes, infused for 45 minutes at a temperature of 45 ° C with periodic stirring, followed by by centrifugation for 2 minutes at a speed of 16,000 rpm, samples with a volume of 0.075 cm ³ are taken from the obtained extract containing PS, 0.075 cm ^{3 of} Folin-Ciocalteu reagent diluted 5 times are added to them, mixed after 3 minutes add 0.15 cm ^{3 of a} 20% solution of sodium carbonate and 1.2 cm ^{3 of} distilled water, close the lid, mix and leave at room temperature, after 1 h measure the optical density of the resulting tungsten blue at a wavelength of 725 nm, optical path length - 1 cm.

The total PS content is expressed in mEq. gallic acid per g wet weight of raw materials

A significant difference of the proposed method from the prototype is that the extraction of the PS from the ground raw material is carried out in one stage at a lower temperature using a non-toxic solvent, and the ratio of reagents is changed when determining the total content of the FS.

The novelty of the invention.

The novelty of the present invention is the creation of a new method for determining the total content of PS in plant objects, which allows to obtain reliable results with significant savings in reagents and raw materials and without the use of a toxic solvent.

SUMMARY OF THE INVENTION

According to the authors, the present invention consists in the fact that replacing a toxic methanol solvent with ethanol increases the safety of work, and lowering the extraction temperature reduces the likelihood of PS oxidation. Changes in the ratios of raw materials and reagents, as well as a decrease in their quantities, can reduce the consumption of reagents and plant materials with high sensitivity of the method and the reliability of the obtained data on the content of PS in plant objects.

The invention is illustrated by the following examples.

Example 1 (prototype)

0.700 g of callus culture of tea plant tissue, freshly chopped wheat leaves (Amir variety) or buckwheat (Daria variety), respectively, are placed in extraction tubes. The tubes are placed in a water bath with a temperature of 70 ° C, add 5.0 cm ^{3 of a} 70% aqueous solution of methanol heated to 70 ° C, close the cork and mix. Extraction is carried out in a water bath for 10 minutes, stirring after 5 and 10 minutes. The tubes are removed from the water bath, cooled to room temperature and centrifuged for 10 minutes at a speed of 3500 rpm. The supernatant (extract) is transferred to a graduated tube. Re-extraction of the precipitate is carried out under the same conditions. The extracts are combined, their volume is adjusted to 10.0 cm ³ with a cold 70% aqueous methanol solution and mixed. 1.0 cm ^{3 of} thus prepared extracts from callus culture of tea tissue, wheat leaves and buckwheat are placed in separate graduated tubes. Instead of an extract containing PS, 1.0 cm ^{3 of} distilled water for blank samples are placed in separate graduated tubes. To each tube, 5.0 cm ^{3 of} Folin-Ciocalteu reagent diluted 10 times are added and mixed. After 3 minutes, 4.0 cm ^{3 of a} 7.5% aqueous solution of sodium carbonate was added to each tube, the tubes were closed with stoppers, mixed and left at room temperature. After 1 h, the optical density of the solution was measured with respect to water at a wavelength of 765 nm. The optical path length is 1 cm.

The total amount of PS contained in 1 g of raw material is calculated by the formula:

C = (D _op -D _k ) ⋅V / K⋅М⋅1000, where

C is the total amount of FS in mEq gallic acid / g wet weight of raw materials

D _op - the optical density of the sample at 765 nm

D _to - the optical density of the blank experiment at 765 nm

K is the slope of the calibration curve (0.0037)

V is the volume of extract (10 cm ³ )

m is the mass of raw materials (0.7 g), g

1000 - Convert mcg to mg

The analysis is carried out in three biological and two analytical replicates. The time spent on the experiment is 215 minutes. All results are statistically processed in Excel. Table 1 presents the mean values and their standard errors.

To build a calibration curve in a volumetric flask with a volume of 100 cm ³ , prepare the initial standard aqueous solution of gallic acid with a mass concentration of 1 mg / cm ³ . Transfer the initial standard solution of gallic acid in the volumes indicated in table 2 to volumetric flasks per 100 cm ³ and bring to the mark with distilled water. Then put 1.0 cm ³ standard solutions of gallic acid, according to the table. 2 into separate graduated tubes. Instead of gallic acid solutions, 1.0 cm ^{3 of} distilled water are placed in separate graduated tubes for blank experiments. To each tube, 5.0 cm ^{3 of} Folin-Ciocalteu reagent diluted 10 times are added and mixed. After 3 minutes, 4.0 cm ^{3 of a} 7.5% aqueous solution of sodium carbonate was added to each tube, the tubes were closed with stoppers, mixed and left at room temperature. After 1 h, the optical density of the solutions was measured with respect to water at a wavelength of 765 nm. The optical path length is 1 cm.

The analysis is carried out in duplicate. A calibration curve is constructed for the dependence of the optical density of the gallic acid solution on the mass concentration of gallic acid in the solution. Determine the coefficient of linear dependence K (the tangent of the slope of the calibration curve).

Example 2 (the proposed method)

Ground samples of raw materials (callus culture of tea tissue, fresh wheat leaves (Amir cultivar) or buckwheat (Daria cultivar) weighing 0.05 g are triturated with 1.5 cm ^{3 of} 96% ethanol in Eppendorf tubes, cover, insist for 45 min at a temperature of 45 ° C with periodic stirring (every 15 min) and centrifuged for 2 minutes at a speed of 16000 rpm, Samples of 0.075 cm ^{3 were} taken from the obtained extracts, 0.075 cm ^{3 of} reagent were added to each sample Folina-Ciocalteu, diluted 5 times, stirred, after 3 minutes add 0.15 cm ³ 2 A 0% sodium carbonate solution and 1.2 cm ^{3 of} distilled water are stirred and left at room temperature. After 1 h, the optical density of the solution is measured at a wavelength of 725 nm against a blank experiment. The optical path length is 1 cm.

The total content of PS is expressed in mEq. gallic acid per g wet weight of raw materials.

The total amount of PS contained in 1 g of raw material is calculated by the formula:

C = D⋅V / K⋅М⋅1000, where

C is the total amount of FS in mEq gallic acid / g wet weight of raw materials

D is the optical density of the solution at 725 nm

K is the slope of the calibration curve (0.0040)

V is the volume of extract (1.5 cm ³ ), cm ³

m is the mass of raw materials (0.05 g), g

1000 - Convert mcg to mg

The analysis is carried out in three biological and two analytical replicates. The time spent on the experiment is 120 minutes. All results are statistically processed in Excel. Table 1 presents the mean values and their standard errors.

To build a calibration curve in a volumetric flask with a volume of 50 cm ³ , an initial standard solution of gallic acid in 96% ethanol with a mass concentration of 0.200 mg / cm ³ is prepared. Diluting the initial gallic acid solution with 96% ethanol in the proportions indicated in table 3, solutions of lower concentration are obtained. Then put on 0,075 cm ³ standard solutions of gallic acid, according to the table. 2 into separate graduated tubes. Instead of gallic acid solutions, 0.075 cm ^{3 of} 96% ethanol is placed in separate graduated tubes for blank experiments; 0.075 cm ^{3 of} Folin-Ciocalteu reagent diluted 5 times is added to each tube, mixed after 3 minutes. add 0.150 cm ^{3 of a} 20% sodium carbonate solution and 1.2 cm ^{3 of} distilled water, mix. The reaction proceeds at room temperature. After 1 h, the optical density of the solution was measured against a blank experiment at a wavelength of 725 nm. The optical path length is 1 cm.

The analysis is carried out in duplicate. A calibration curve is constructed for the dependence of the optical density of the gallic acid solution on the mass concentration of gallic acid in the solution. Determine the coefficient of linear dependence K (the tangent of the slope of the calibration curve).

List of graphic materials

Table 1. The total content of FS in the callus culture of tea plant tissue, wheat and buckwheat with different methods for their determination.

Table 2. The construction of a calibration curve for gallic acid for a known method (prototype).

Table 3. The construction of a calibration curve for gallic acid for the proposed method.

The results of the invention

As follows from the analysis of data (table 1), when determining the total content of PS in plant objects according to the prototype, it is necessary to increase the mass of the sample of raw materials (0.7 g) compared to tea (0.2 g). This is due to the fact that tea belongs to the so-called “phenol accumulator plants” and the total content of PS in it exceeds 60 mg / g dry weight (Zaprometov MN “Biochemistry of Catechins”, M., “Science”, 1964). The number of such plants in nature is very small.

The decrease in the number of FS in example 2 (by the proposed method) compared with example 1 (by the prototype) is due to different extraction conditions. When using water-alcohol extractants at high temperature, not only PS, but also sugars and some amino acids, which also interact with the Folin-Ciocalteu reagent, pass into the extract, which leads to an overestimation of the determination results.

The main goal of our development is to create a new sensitive method for determining the total content of PS in plant objects, which allows obtaining reliable data with a significant reduction in the consumption of raw materials and reagents, as well as increasing the safety of work while shortening the extraction time.

In contrast to the prototype in the proposed method, a new ratio of reagents: the volume of extract, sodium carbonate solution and the Folin-Ciocalteu reagent obtained during laboratory tests, provides a significant reduction in the consumption of reagents with high sensitivity of the method (from 0.010 mg / cm ³ extract). Raw material consumption is also reduced. Replacing the toxic methanol extractant with ethanol increases safety.

Thus, the goal of the present invention is achieved - the creation of a new sensitive method for determining the total content of PS in plant objects.

The proposed method for determining the total content of PS in plant objects allows to reduce the consumption of raw materials, the duration of the extraction of PS at a lower temperature, which prevents the transfer of non-phenolic substances interacting with the Folina-Chocalteu reagent to the extract, the consumption of the Folina-Chocalteu reagent by 20 times, and increase the safety by replacing the toxic solvent of methanol with ethanol, obtain reliable data on the content of PS in plant objects.

The difference of the proposed method from the prototype is that the extraction of PS from raw materials is carried out in one stage at a lower temperature using a non-toxic solvent, and the ratio of reagents is changed when determining the total content of phenolic compounds.

Claims (1)

The method for determining the total content of phenolic compounds in plant objects, which consists in grinding them, extraction in alcohol with stirring, subsequent centrifugation, adding Folin-Ciocalteu reagent and carbonic acid to the extract, characterized in that grinding of a sample of raw materials weighing 0.05 g is carried out with grinding 1.5 cm ³ of 96% ethanol extraction of phenolic compounds is carried out for 45 minutes at 45 ° C with occasional stirring (every 15 min) and subsequent centrifugation for e 2 minutes at a speed of 16000 rev / min, from the resulting extract was sampled, the volume of 0,075 cm ^3, was added thereto at 0.075 cm ³ reagent Folin-Ciocalteu diluted 5 times, stirred 3 min, to 0, 15 cm ^{3 of a} 20% solution of sodium carbonate and 1.2 cm ^{3 of} distilled water, cover, stir and leave at room temperature and after 1 h the optical density of the formed tungsten blue at a wavelength of 725 nm is measured, the optical path length is 1 cm, total phenolic compounds are expressed in mg equiv tapes gallic acid per g wet weight of raw materials.