RU2531940C1 - Method for spectrophotometric measurement of combination of chlorophyll, carotinoids and hydroxycinnamic acid in great nettle leaves - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: invention refers to medicine, namely to studying and analysing medical preparations, and can be used for standardising herbal raw materials. A method for identification and qualitative measurement of chlorophyll, carotinoids and hydroxycinnamic acids in a combination in great nettle leaves involves a 1-hour fractional extraction, 30 min each of the ground raw material having a particle size of 1.0 mm on a water bath at a temperature of 100°C with 70% ethanol in a ratio of the herbal raw material to the extractant of 1:100, the combination of the extracts and reduction to 100 ml with a solvent, dilution of the prepared solution in a ratio of 2:25 in 96% ethanol, measuring an optical density of the solution in relation to 96% ethanol at maximum absorption 328±1 nm, 442±1 nm and 667±1 nm, calculation of the total content of hydroxycinnamic acids equivalent to chlorogenic acid, carotinoids equivalent to violaxanthin and chlorophyll in the percentage equivalent to an absolute dry mass of the raw material by formulas.

EFFECT: method provides availability, simplicity, efficiency and low error of measurement.

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Description

The invention relates to medicine, namely to the study and analysis of medicines, and can be used to standardize medicinal plant materials, for example, dioica nettle, as well as medicines based on it based on the content of the main groups of biologically active substances (BAS), and also used in the pharmaceutical, cosmetic and food industries for the identification and quantification of medicinal plant raw materials, dioecious nettle and drugs based on it ove chlorophylls, carotenoids and hydroxycinnamic acids in the presence of a joint.

Known methods for the separate determination of chlorophylls, carotenoids and hydroxycinnamic acids in various types of medicinal plant materials by paper and thin-layer chromatography, photoelectrocolorimetry, mass spectroscopy and spectrophotometry.

A known method for determining the composition of the lipophilic fraction of tinctures of homeopathic matrix nettle and stinging nettle obtained from freshly collected and dried raw materials according to the method of the General Pharmacopoeia of the State Pharmacopoeia using 86 and 62% (by weight) ethanol as extractant, respectively (Lapinskaya E.S., Kopytko YF The study of the composition of the lipophilic fraction of tinctures of homeopathic matrix nettle dioica and stinging nettle. Chem. - Pharm. Journal. - 2008. - Volume 42. - No. 12. - SS.26-29). The method consists in the following: 10 ml of tincture and 20 ml of n-hexane are placed in a 50 ml separatory funnel, and shaken for 10 minutes. The hexane extract was poured into a 50 ml round bottom flask. The extraction is repeated with the same amount of solvent. The extracts are combined and distilled to dryness on a vacuum evaporation rotary apparatus at a temperature of about 50 ° C. The dry residue is dissolved in 2 ml of ethyl acetate. For analysis, 0.2 μl of the sample is introduced into the evaporator of the gas chromatography mass spectrometer. Identification of the separated components was performed using the NIST 98 mass spectra library and Saturn V / S software comparison algorithms (Varian). A quantitative assessment of the content of biologically active substances was carried out by the method of internal normalization by the peak area (total ion current) of the identified compounds using an automatic data processing system.

A known method for the determination of pigments (chlorophyll and carotenoids) in fresh plant material (Strusovskaya OG Determination of the pigment composition of Cochlearia officinalis, growing on the islands of the Solovetsky archipelago. Materials of the V international conference "Pharmacy and public health. Ekaterinburg, 2012. - pp. 184 -187. 3. Zemlyanukhin AA, Zemlyanukhin LA Large workshop on the physiology and biochemistry of plants: Textbook. - Voronezh: Voronezh State University, 1996. - 188 p.), Including grinding of raw materials and processing it with an ammonia solution with concentration of 1 mol / l and 10-fold volume acetone followed by filtration and spectrometric measurement at wavelengths of 440.5 nm, 644, 649, 662 and 665 nm, which contain the main absorption maxima of carotenoids, chlorophyll a, f and b, and the calculation of BAS data for Holm-Wetstein (for 100% acetone) or according to Verton (for 80% acetone).

There is a method for the joint determination of hydroxycinnamic acids and flavonoids in the leaves of nettle (Matyuschenko N.V. Influence of drying conditions on the content of flavonoids and hydroxycinnamic acids in the leaves of nettle. - Issue 67. - 2012. - S.78-79), including grinding the raw material and treating it with 70% ethanol with alcohol, followed by direct spectrometric determination of hydroxycinnamic acids in terms of caffeic acid at ine wavelength 328 nm. Flavonoids are determined separately by differential spectrophotometry by reaction with aluminum chloride.

In the literature (Lupinskaya S.M., Orekhova S.V., Vasilieva O.G. Study of biologically active substances of linden, nettle and oregano and whey extracts based on them. Chemistry of plant raw materials. - 2010.- No. 3. pp. 143-145; Yudina N.V., Ivanov A.A., Loskutova Yu.V. et al. Effect of dispersion parameters of dioica nettle on changes in the degree of grinding, yields and properties of extractives. Chemistry of plant raw materials. - 2012. - No. 1. - SS.137-142.) Methods of qualitative joint determination of chlorophylls and carotenoids in various plant objects by chromatography are presented on paper or in a thin layer using different chromatography conditions. However, studies on the determination of carotenoids, hydroxycinnamic acids, and chlorophylls with the joint presence by TLC were not detected.

A known method for the determination of chlorophyll in buckwheat plants (RU 2244916, G01N 21/25, C09B 61/00, 2005), including grinding the raw material and treating it with alcohol, followed by spectrometric measurement at two wavelengths of 665 and 649 nm, which contain the main absorption maxima chlorophyll "a" and "b" and the calculation of chlorophyll according to the formulas. Use 96% alcohol. Extraction is carried out in hermetically sealed containers for 24-48 hours.

The disadvantages of these methods are the bulkiness and duration of the allocation of biologically active substances from medicinal plant materials, the use of various extractants and extraction modes, the use of expensive standard samples.

In the scientific, pharmaceutical, medical, and patent literature, no methods have been identified for the simultaneous spectrophotometric determination of hydroxycinnamic acids, carotenoids, and chlorophyll in dioecious nettle leaves without the prior separation of these groups of biologically active substances during a single extraction procedure of medicinal plant materials.

The objective of the invention is the development of a method for the simultaneous determination of the authenticity and quantitative content of chlorophylls, carotenoids and hydroxycinnamic acids in the leaves of the nettle by spectrophotometry with the joint presence for standardization of medicinal plant materials and medicines, food and cosmetic products.

The technical result consists in accessibility, ease of implementation, cost-effectiveness and a small error of determination.

The technical result consists in the fact that the method of spectrophotometric quantitative determination of dioecious nettle leaves in the combined presence of chlorophyll, carotenoids and hydroxy brown acids involves fractional extraction for 1 hour for 30 minutes each of the crushed raw materials with a grinding degree of 1.0 mm in a 70% ethanol water bath in the ratio of raw materials: extractant 1: 100, combining the extracts and bringing the used solvent to 100 ml, followed by dilution of the resulting solution in a ratio of 2:25 to 96% ethanol, measured the optical density of the solution with respect to 95% ethanol at the absorption maxima of 328 ± 1 nm, 442 ± 1 nm and 667 ± 1 nm, the calculation of the content of the sum of hydroxycinnamic acids in terms of chlorogenic acid, carotenoids in terms of violoxanthine and chlorophyll as a percentage, based on absolutely dry mass of raw materials according to the formulas.

Statistical processing of the experimental results showed that the average determination error does not exceed 6.17%.

Figure 1 presents the absorption spectrum of the extract from the leaves of nettle dioecious; figure 2 presents the effect of the composition of the extractant on the extraction of the studied groups of biologically active substances; figure 3 presents a table with metrological characteristics of the method of quantitative determination of biologically active substances (%) (n = 9; f = 8; P = 95%).

The method is illustrated by the following example.

As the object of study, we used finished crushed raw material of nettle leaves of a dioecious domestic producer. Electronic absorption spectra were measured on an SF-2000 (RF) spectrophotometer in quartz cuvettes with an absorbing layer thickness of 10 mm in the wavelength range of 200-800 nm.

When studying the spectral characteristics of extracts from dicotyledonous nettle leaves obtained using various extractants (water, water-alcohol mixtures of various concentrations, acetone), the main absorbances in the 323 ± 5 nm region typical for hydroxycinnamic acids were observed; 442 ± 2 nm, characteristic of carotenoids (violoxanthin) and 667 ± 3 nm, characteristic of chlorophyll (figure 1).

Since absorption maxima of the studied biologically active substances are not superimposed (Fig. 1), it is possible to carry out the simultaneous determination of hydroxycinnamic acids, carotenoids and chlorophyll in nettle leaves by direct spectrophotometric method without prior separation.

When developing the methodology, the optimal parameters for the extraction of hydroxycinnamic acids, carotenoids and chlorophyll from raw materials were studied and established: the degree of grinding of the raw material was 1.0 mm; extractant - 70% ethanol (ethyl alcohol); ratio of raw materials: extractant 1: 100; extraction time - 1 h (fractionally 30 min). The choice of extractant was carried out on the basis of the data on the influence of the polarity of the solvent on the extraction of biologically active substances (figure 2). It has been established that with an increase in polarity to 6.0 units, the extractability of all studied groups of biologically active substances increases. A further increase in the polarity of the extractant is impractical due to the reduction of their content in the extraction (figure 2).

Example.

About 0.5 g (accurately weighed) of the raw material, crushed to the size of particles passing through a sieve with holes with a diameter of 1 mm, was placed in a flat-bottomed flask with a thin section with a capacity of 100 ml and extracted with 50 ml of 70% ethyl alcohol for 30 minutes. After cooling, the extract was decanted and filtered through a paper filter into a 100 ml volumetric flask. The residue in the flask was poured into 50 ml of 70% ethyl alcohol and extracted again for 30 minutes. The combined extracts in a volumetric flask were brought up to the mark with 70% ethyl alcohol (solution A). 2 ml of solution A was transferred into a volumetric flask with a capacity of 25 ml and the volume was adjusted with 95% ethyl alcohol to the mark (solution B). The optical density of the resulting solution was measured on an SF-2000 spectrophotometer (RF) in a cuvette with a layer thickness of 10 mm at a wavelength of 328 ± 1 nm, 442 ± 1 nm, and 667 ± 1 nm. A comparison solution was 95% ethyl alcohol. The content of the amount of hydroxycinnamic acids in terms of chlorogenic acid, carotenoids in terms of viloxanthin and chlorophyll in percent (X) in terms of the absolutely dry mass of raw materials was calculated by the formulas (1-3)

$$X=\frac{A\cdot 25\cdot \mathrm{one\; hundred}\cdot \mathrm{one\; hundred}\cdot \mathrm{one\; hundred}}{m\cdot 507\cdot 2\cdot \mathrm{one\; hundred}\cdot (\mathrm{one\; hundred}-W)}=\frac{A\cdot 250000}{m\cdot 507\cdot 2\cdot (\mathrm{one\; hundred}-W)}(\mathrm{one})$$

$$X=\frac{A\cdot 25\cdot \mathrm{one\; hundred}\cdot \mathrm{one\; hundred}\cdot \mathrm{one\; hundred}}{m\cdot 2500\cdot 2\cdot \mathrm{one\; hundred}\cdot (\mathrm{one\; hundred}-W)}=\frac{A\cdot \mathrm{one\; hundred}}{m\cdot 2\cdot (\mathrm{one\; hundred}-W)}(2)$$

$$X=\frac{A\cdot 25\cdot \mathrm{one\; hundred}\cdot \mathrm{one\; hundred}\cdot \mathrm{one\; hundred}}{m\cdot 944.5\cdot 2\cdot \mathrm{one\; hundred}\cdot (\mathrm{one\; hundred}-W)}=\frac{A\cdot 250000}{m\cdot 944.5\cdot 2\cdot (\mathrm{one\; hundred}-W)}(3)$$

where A is the optical density of solution B at the corresponding absorption maximum; m is the mass of raw materials, g; W is the loss in mass upon drying of the raw material,%; 507 — specific absorption rate of chlorogenic acid at 327 ± 1 nm; 2500 - specific absorption rate of violoxanthin at 442 ± 2 nm; 944.5 is the specific absorption rate of chlorophyll at 663 ± 5 nm.

The results of the quantitative determination of the studied groups of biologically active substances in the leaves of the nettle dioecious and metrological characteristics of the technique are presented in the table (figure 3).

Statistical processing of the experimental results showed that the average determination error does not exceed 6.17% (Table 3), which indicates the possibility of its use and inclusion in modern regulatory documentation for standardizing and assessing the quality of dioica nettle leaves.

The method allows to simultaneously determine the authenticity and quantitative content of biologically active substances in the leaves of the nettle by direct spectrophotometry, and to standardize medicinal plant materials and drugs by the content of chlorophylls, carotenoids and hydroxycinnamic acids in the joint presence. The developed method is distinguished by accessibility, ease of implementation, cost-effectiveness and low error of determination.

Claims (1)

The method of spectrophotometric qualitative and quantitative determination of chlorophyll, carotenoids and hydroxycinnamic acids with the combined presence of dioecious nettle leaves, comprising fractional extraction for 30 minutes each of crushed raw materials with a particle size of 1.0 mm in a water bath at a temperature of 100 ° C 70% ethanol in the ratio of raw materials: extractant 1: 100, combining the extracts and bringing the used solvent to 100 ml, followed by dilution of the resulting solution in a ratio of 2:25 to 96% ethanol, optical measurement the density of the solution relative to 96% ethanol at the absorption maxima of 328 ± 1 nm, 442 ± 1 nm and 667 ± 1 nm, the calculation of the content of the sum of hydroxycinnamic acids in terms of chlorogenic acid, carotenoids in terms of violoxanthin and chlorophyll in percent, calculated on absolutely dry mass of raw materials according to the formulas:
$$X=\frac{A\cdot 25\cdot \mathrm{one\; hundred}\cdot \mathrm{one\; hundred}\cdot \mathrm{one\; hundred}}{m\cdot 507\cdot 2\cdot \mathrm{one\; hundred}\cdot (\mathrm{one\; hundred}-W)}=\frac{A\cdot 125000}{m\cdot 507\cdot (\mathrm{one\; hundred}-W)}(\mathrm{one})$$

$$X=\frac{A\cdot 25\cdot \mathrm{one\; hundred}\cdot \mathrm{one\; hundred}\cdot \mathrm{one\; hundred}}{m\cdot 2500\cdot 2\cdot \mathrm{one\; hundred}\cdot (\mathrm{one\; hundred}-W)}=\frac{A\cdot \mathrm{fifty}}{m\cdot (\mathrm{one\; hundred}-W)}(2)$$

$$X=\frac{A\cdot 25\cdot \mathrm{one\; hundred}\cdot \mathrm{one\; hundred}\cdot \mathrm{one\; hundred}}{m\cdot 944.5\cdot 2\cdot \mathrm{one\; hundred}\cdot (\mathrm{one\; hundred}-W)}=\frac{A\cdot 125000}{m\cdot 944.5\cdot (\mathrm{one\; hundred}-W)}(3)$$

where A is the optical density of the combined solution at the corresponding absorption maximum; m is the mass of raw materials, g; W is the loss in mass upon drying of the raw material,%; 507 — specific absorption rate of chlorogenic acid at 327 ± 1 nm; 2500 - specific absorption rate of violoxanthin at 442 ± 2 nm; 944.5 is the specific absorption rate of chlorophyll at 663 ± 5 nm.

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