SU1541515A1 - Method of quantitative determination of alkaloids in seeds and vegetative mass of lupine - Google Patents

Abstract

The invention relates to the biochemistry of plants and can be used in the selection and seed production of lupine, as well as in animal husbandry, in determining the quality of feed containing lupine. The aim of the invention is to reduce the analysis time and reduce its labor intensity while maintaining high accuracy of determination. The basis of the proposed method is the introduction of a new method for the extraction of alkaloids from plant material, which involves the use of a 10% aqueous solution of trichloroacetic acid (TCA). Replacing the multi-stage extraction with the use of a complex of mineral and organic substances with a single-stage procedure for treating the sample with TCA solution combined with the rapid staining of the extract with iodate-iodide reagent reduced the analysis time (compared to the known method) by about 10 times. The new method is suitable for determining the content of alkaloids both in the seeds and in the vegetative mass; it can be used for the analysis of high-alkaloid and non-alkaloid types of lupine. The results of the determination completely coincide with the data of the most accurate weighting method. The application of the invention in practice will provide significant savings in time, labor and reagents in the work on the selection and seed production of lupine, to obtain feed based on it. 3 tab.

Description

The invention relates to the biochemistry of plants and can be used in the selection and seed production of lupine, as well as in determining the quality of feed containing lupine.

The aim of the invention is to reduce the analysis time and reduce its labor intensity while maintaining the accuracy of the determination.

Achieving this goal is provided by introducing a fundamentally new method of extracting alkaloids using a 10% TCA solution followed by determining the alkaloids in the extract by dyeing them with an iodine iodine reagent in an acidic medium. using TCA and iodate-iodide reagent used in the color reaction, which ensures rapid color development, reduces the analysis time to 1 hour with Compared with 9-10 h in a known way. At the same time, according to the statistical analysis data, the data obtained by the proposed method almost completely coincide (have a high convergence rate) with the results of parallel analyzes performed using the standard method (and 31.

Table 01 presents comparative operations for analyzing the content of alkaloids by known and proposed methods.

Table 2 presents the assessment of the metrological characteristics of the proposed method (A) and the weight method (c) in determining the content of alkaloids in the seeds and the vegetative mass of lupine (t Q $$ 2.09).

Table 3 presents an estimate of the convergence index.

Example. A weighed 0, -5-10.001 g (for alkaloid types of lupine) or 2.5-5tO, 001 g (for low-alkaloid species) of an air-dry sample of plant material, ground in a mill, placed in a conical flask (250 ml), poured 50 ml of 10% trichloroacetic acid solution. The contents are well stirred by rocking and incubated for 45 minutes to enlarge the protein sediment and transfer the alkaloids into solution. During the infusion time, the mixture is stirred two or three times at intervals of 10-15 minutes, after settling, shake vigorously again and transfer the contents to a medium density filter paper. 1-2 ml of filtrate for alkaloid and 5-10 ml of filtrate for low alkaloid species are taken for determination. The selected volume of filtrate is placed in a 100 ml volumetric flask, 10 ml 0.2 N is added. NgNW + and distilled water to 80 ml, then stirred.

Add 5 ml of iodate-iodide reagent, finish with water to 100 ml and mix by 3-fold by inverting the flask. After 5 minutes, the optical density is recorded with respect to the control sample, which contains all COMPONRENTS, except for the filtrate. Measurements are carried out in a flow cell with an optical path length of 10 mm at a wavelength of 500 nm.

The content of alkaloids (%) is calculated by the formula

--but

-Om:

1,000

AND ABOUT

n

100

vz lOO-b)

Yoo V,

where a is the content of alkaloids in

100 ml of solution, determined by the calibration curve mg;

V., - total volume of the extract, ml; V is the volume of filtrate taken on

definition, ml;

And - a hinge of plant material, g;

B is the humidity of the sample,%. Table 4 shows the content (%) in the seeds and vegetative mass of various species of lupine, determined by the proposed (A) and known (B) methods.

These data indicate that the proposed method can be used to determine the content of alkaloids both in the seeds and in the vegetative mass of high alkaloid and non-alkaloid types of lupine. At the same time, the results of the analysis almost completely coincide with the data of the weighting method (according to Mach and Lederlei,), although the proposed method allows obtaining them in 10 times faster

The application of the invention provides a significant saving of time, labor and reagents in the work on the selection of lupine and obtaining feed based on it.

Claims (1)

Invention Formula The method of quantitative determination of the content of alkaloids in the seeds and the vegetative mass of lupine, including their extraction from plant matter, carrying out a color reaction, colorimetrication of the colored solution and comparison of the color intensity of the experimental and standard samples characterized in that, in order to reduce the analysis time and reduce its labor intensity , alkaloids are extracted with 10% solution of TCA, Iodate-iodide reagent is used as a color reagent, and its treatment is carried out in the presence of sulfuric acid, Table 1 Continuation of table 1 Operations and their duration according to the method Operations and their duration according to the method ten Known Proposed Hitch 1 min. Infusion with 10% NaOH solution 30 min Extraction with chloroform in a Soxlet apparatus for 2 h 30 min. Flushing the extraction flask with ethyl ether for 2 min. 15 Evaporation of the acid extract and measuring the volume of I h. Alkaloids are precipitated into the colored precipitate by adding 10% phosphoric molybdic acid for 4 h. Filtering the precipitate through a glass filter for 30 min. Washing with water and reducing the precipitate with an alkaline solution of tin chloride for 30 min. Transferring the colored complex to dimensional  „„ 100 ml bottle, volume and 0.2 n. what gh ju bringing water Hitch 1 min. Infusion with I0% trichloroacetic acid 45 min Filter through paper filter 5 min 20 Transferring part of the filtrate to a measured 25 flask per 10 ml, adding water, staining with iodine-iodine reagent. Shaking the extract in 10 ml portions of 0.02 N,% SC, 5 times 30 min. 1 min. Adding water to the mark and settling for 5 min. up to the mark and settling 15 minutes. Colorimetry 1 min 35 Total time 9 h 53 min Known Proposed Colorimetry 1 min Total time 58 min 15 20 up to the mark and settling 15 minutes. Colorimetry 1 min. Total time 9 h 53 min Table 2 Table 3 T a b i c a A