RU2076318C1 - Method of determining phytohormones in vegetable material - Google Patents

Abstract

FIELD: analytical methods for vegetable materials. SUBSTANCE: method includes homogenization of vegetable material, extraction of phytohormones from homogenizate, purification of extract by high-performance liquid chromatography with reversed-phase sorbent and moving phase, and controlling phase separation process by measuring UV absorption, which makes it possible to determine concentration of individual phytohormones in fractions and thereby to calculate their content in vegetable material. As reversed-phase sorbent, Zorba ODS is used; as moving phase 0.35% solution of methanol in 0.1% trichloroacetic acid solution with pH 6.0. Separation process is controlled at wavelength 214 nm. EFFECT: improved analytical procedure. 3 cl, 1 tbl

Description

 The invention relates to analytical chemistry, in particular to the quantitative determination of phytohormones in plant material, and can be used in chemical, biochemical, physiological studies.

 Known methods for the quantitative determination of plant growth regulators, which include homogenization of plant material, extraction with water-alcohol mixtures and isolation of individual compounds from the extract at the final stage using high performance liquid chromatography (HPLC). The number of individual phytohormones is determined by the peak area or spectrophotometrically [1-3] or by isotopic dilution of the labeled compound added after the extraction step [4] or before the extraction step [5] by the endogenous phytohormone contained in the plant.

 However, the known methods are limited to the determination of auxins [4] or auxins and cytokinins [5]. This does not allow to determine the whole hormonal status of the plant, including mainly auxins, cytokines and gibberellins.

 A known method for the quantitative determination of auxins and cytokinins in plant material is to homogenize the material, add to the homogenate labeled with tritium auxins and cytokinins, extract a preliminary sample with an organic solvent, chromatographic purification using Amberlite XAD-4 and Lichroprene C-18 and isolate individual compounds using HPLC (aut. St. USSR N 1704065, G 01 N 30/06, 1989).

 The disadvantage of this method is that an important class of plant growth hormones of gibberellins is not determined in the plant material. For studies on plant physiology, it is important to simultaneously determine in plant extracts all the main classes of plant hormones, namely: auxins, cytokinins and gibberellins. Moreover, it is essential that this determination is carried out in a single technological process.

 In addition, for the quantitative determination of phytohormones in a known manner, a relatively large amount of plant material (about 10 g) is required, which does not allow determination with a limited amount of the studied plant material.

 The aim of the invention is the ability to use to determine small quantities of the studied plant material (about 1-3 g), which is especially important when working with transgenic plants or when separately determining phytohormones in different parts of the same plant. In addition, when implementing the invention, it becomes possible to simultaneously determine in one plant sample three classes of phytohormones: cytokinins, auxins and gibberellins. The sensitivity of the determination increases, the process of purification of the extract is accelerated.

 In a method for the quantitative determination of phytohormones in plant material, including its homogenization, adding labeled analogues of defined phytohormones to the homogenate, extracting phytohormones from the homogenate with lower alcohol, removing ballast substances from the extract, concentrating phytohormones in the purified extract by passing it through Amberlite XAD-4 and then through Lichroprene C-18 using UV registration, chromatographic isolation of individual phytohormones by passing purified and end nitrated extract through a reversed-phase sorbent, determining the molar activity of the isolated individual phytohormones and calculating their content in the starting material, performing the removal of ballast substances from the extract by repeatedly evaporating it and dissolving the precipitates formed in this process in decreasing volumes of lower alcohol with discarding components insoluble in alcohol precipitation and UV registration at 214 nm, can accelerate the purification of the extract, increase the purity of the purified of concentrated extract, increase sensitivity to UV-registration, thereby reducing the amount of plant material required for determining and use as labeled analogues labeled with tritium auxins, cytokinins and gibberellins can simultaneously determine the content of the plant material three classes phytohormones.

 It was found that repeated sequential evaporation of the alcohol extract and dissolution of the precipitates resulting from it in decreasing volumes of lower alcohol with the rejection of precipitate insoluble in alcohol allows increasing the degree of purification of the extract and accelerating the purification process. UV registration of the purified concentrated extract at 214 nm increases the sensitivity of the registration process, and therefore, to obtain the extract, it becomes possible to reduce the amount of plant material required to perform the determination. In addition, gibberellins are detected only at 214 nm and are not detected at 260 nm (prototype).

 Example.

 Objects of study: indolyl-3-acetic acid (IAA), zeatin (Zea), zeatin riboside (Zeo), gibberellin A1 (GAI).

1-3 g of fresh biomass leaves, tomato stems (7 lines) were fixed with liquid nitrogen, thoroughly triturated, quantitatively transferred to a flask using 80% methanol (ethanol) and tritium-labeled phytohormones (auxins, cytokinins, gibberellins) were added. The volume was adjusted to 100 ml with 80% methanol (ethanol) and left to be extracted overnight on a magnetic stirrer at a temperature of 5-10 ^o C.

 The next day, the extract was centrifuged at 6000 rpm for 20 minutes. The supernatant was poured into a flask for evaporation, evaporated to half the initial volume and centrifuged again at 6000 rpm for 10 min (10% label loss). The supernatant was evaporated to dryness. The precipitate was dissolved in 10 ml of 95% ethanol (not all of the precipitate was dissolved). The solution was transferred to a flask and again evaporated to dryness. The precipitate was redissolved in 5 ml of 95% ethanol (not all of the precipitate was also dissolved). The solution was transferred back to the flask and evaporated to dryness. The precipitate was redissolved in 500 μl of 95% ethanol. If not all of the precipitate was dissolved, then the evaporation and dissolution steps (in 500 μl of alcohol) were repeated until the entire precipitate was dissolved. Thus, it is possible at this stage to get rid of the water-soluble substances contained in the plant extract.

 The resulting solution was evaporated to dryness, dissolved in 300-500 μl of 10% ethanol and applied onto a 10x40 mm cartridge with Amberlite XAD-4 (Serva), the remaining water-soluble substances were washed off with water (10 ml), and 70% cytokinins, auxins and gibberellins ethanol (10 ml) (no label loss). The alcohol fraction was evaporated, 5 ml of 35% methanol in 0.1% trifluoroacetic acid (TFA) was added to the precipitate (pH 6.0, mobile phase). This solution was passed through a cartridge with the sorbent Lichropren S-18 (CSFR, 25-40 microns). The first fraction was collected, another 5 ml of the mobile phase was washed off, the fractions were combined, evaporated to dryness, and dissolved in 200 μl of the mobile phase (0.5% label loss).

где A_o молярная активность исходного ³H-меченого фитогормона (мКu/мкм);
A_к молярная активность выделенного фитогормона, (мКu/мкм);
a количество добавленного ³H-меченого фитогормона (мкм);
Х количество эндогенного фитогормона (мкм).The target products were isolated by high performance liquid chromatography (HPLC) on a 4.6 mm x 25 cm column, Zorbax ODS. Mobile phase 35% methanol in 0.1% TFA (pH 6.0), speed 1 ml / min, UV detector, 214 nm. Retention time: IAA 7.3 min; Zea 10 min; Zeo - 13.5 minutes; GAI 2.7 min. Peaks corresponding to IAA, Zea, Zeo, GAI were collected, and specific activity was measured. The content of endogenous auxins, cytokinins and gibberellins in the original plant was calculated by the formula:

where A _{o the} molar activity of the original ³ H-labeled phytohormone (mKu / μm);
A _{to the} molar activity of the selected phytohormone, (mKu / μm);
a the amount of added ³ H-labeled phytohormone (μm);
X amount of endogenous phytohormone (μm).

 If necessary, rechromatography of the target products was carried out by HPLC on a 4.6 mm x 25 cm Zorbax ODS column. Mobile phase 25% methanol in 0.1 M triethylaminobicarbonate. Retention times: for IAA 12 min; Zea 19.4 min; Zeo 28.2 min; GAI 24.8 minutes

 The time required to determine (for one analysis) in a new way - 8-10 hours, known (prototype) 20-22 hours.

 The results of the quantitative determination of IAA, Zea, Zeo and GAI are shown in the table.

 Thus, the new method allows us to expand the spectrum of determination of phytohormones due to gibberellins, increase the sensitivity of determination by almost an order of magnitude due to the use of a UV detector with registration at 214 nm at the HPLC stage, simplify and speed up the process by reducing the processing and extraction with n-butanol. The method allows you to work with a minimum amount of biomass, which also leads to greater sensitivity of the determined method. Fundamental is the fact that the new method makes it possible to determine the hormonal status in various parts of the plant, for example, in the roots, stems and leaves of the same plant. This is important when working in laboratory conditions with sterile plants, especially in the initial stages.

Claims (2)

1. A method for determining the content of phytohormones in plant material, including its homogenization, extraction of a mixture of phytohormones from the obtained homogenate, purification of the obtained extract, separation of the components of the purified extract by HPLC using a reversed-phase sorbent, a mobile phase and monitoring the process of separation into fractions by the presence of UV absorption, the determination of the content of individual phytohormones in the obtained fractions and the calculation of their content in plant material according to the obtained value, distinguishing The fact is that Zorbax ODS is used as a reversed-phase sorbent, having a pH 6.0 of a 35% solution of methanol in a 0.1% solution of trichloroacetic acid as a mobile phase, and the separation process is controlled at λ = 214 nm
2. The method according to p. 1, characterized in that labeled analogues of phytohormones are added to the homogenate, and their molar activity is determined in the obtained fractions containing individual phytohormones, using which the content of individual phytohormones in the starting material is calculated.  3. The method according to p. 2, characterized in that as labeled analogues of phytohormones use tritium-labeled auxins, cytokinins and gibberemins.

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