RU2622023C1 - Method for determining vegetation phase of medicinal plant raw material of knotweed, water pepper and lady's thumb by malate dehydrogenase by means of electrophoresis in polyacrylamide gel - Google Patents

Abstract

FIELD: pharmacology.

SUBSTANCE: method for definition the vegetation phase of the medicinal plant raw material of knotweed, water pepper and lady's thumb by malate dehydrogenase by means of electrophoresis in polyacrylamide gel includes the collection of the medicinal raw material, the determination of the molecular forms of malate dehydrogenase (MF MDH) by means of the electrophoretic method with the use of polyacrylamide gel, the calculation of the relative electrophoretic mobility (REM) of the detected MDH forms for the medicinal plant raw material of knotweed, water pepper and lady's thumb at the following phases of vegetation: budding, flowering, fruiting, the definition of the vegetation phase of the knotweed species by physiological markers - the molecular forms of the malate dehydrogenase enzyme. The plants flowering phase is determined as follows: for knotweed - if one MF MDH 1 with OEP 0.50±0.02 is detected, for water pepper - if only MDG 1 with OEP 0.60±0.02 is detected, for lady's thumb - if only MDG 1 with OEP 0.76±0.02 is detected.

EFFECT: improving the determination accuracy and reliability.

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Description

The invention relates to the field of pharmacy and can be used to determine the phase of flowering of medicinal raw materials, in particular the mountaineer avian, the mountaineer peppercorns and the mountaineer.

Currently, in accordance with the current regulatory documentation, the medicinal plant material of the mountaineer poultry (PF XI, Article 56), the mountain paprika (PF XI, Article 57) and the mountainea pomacea (PF XI, Article 58) should be collected in phase flowering.

However, methods for determining the flowering phase are not provided for in normative and technical documentation. The existing section “External signs” in the pharmacopoeial articles on the highlanders provides for the definition and proof of the type of plant itself, rather than the desired phase of vegetation (flowering).

Closest to the claimed invention is a method for studying the dynamics of the fractional composition of proteins and the activity of MF malate dehydrogenase in raw materials of common oregano, thyme, monarda duode by electrophoresis in a 7.5% polyacrylamide gel. The gel plates were stained with an amide black B. solution. The detection of MF enzymes was carried out by the phenazine metasulfate tetrazolium reaction. Quantitative analysis of electrophoregrams was carried out on a DenScan densitometer (Z. Mashchenko, dissertation author's abstract on the degree of candidate of pharmaceutical sciences, Perm, 2004, pp. 17-20).

A disadvantage of the known method is that it is not intended to determine the flowering phase of the mountaineer, the mountaineer pepper and the mountaineer.

The optimum phase for the harvesting of raw materials is precisely the flowering stage, since it was during this period that the raw materials of poultry, mountain and pepper and potato contains the largest number of active pharmacologically active compounds (flavonoids).

If medicinal plant raw materials are collected at an earlier stage of vegetation (budding earlier) or later (fruiting later), the raw materials will not contain the necessary amount of biologically active compounds that determine the pharmacological activity of the raw materials and preparations based on them.

Thus, an objective way to determine the flowering phase of the mountaineer, bird and mountaineer, and potato mountaineer will improve the quality of the collected medicinal plant materials and preparations based on it.

The objective of the invention is the creation of an objective method for determining the phase of flowering of the mountaineer avian, the mountain peppercorn and the mountaineer.

Malate dehydrogenase (MDH) was used as a marker for determining the vegetation phase. Malate dehydrogenase (L-malate: NAD-oxidoreductase, 1.1.1.37, MDH, malate dehydrogenase) is a key enzyme in the tricarboxylic acid cycle that catalyzes the reversible oxidation of malate (malic acid) to oxaloacetate (oxalic acetic acid). MDH is a multifunctional enzyme. It has a virtually universal distribution among living organisms, found in yeast cells, the vast majority of bacteria, as well as in the tissues of all animals and plants. In many organisms, it is a dimer.

MDH is present in the cells of various organisms in the form of multiple molecular forms, the number of which varies depending on the group of living creatures, on their lifestyle and on the complexity of metabolism.

For plants, the following characteristics of the isoenzyme spectrum of MDH are found: genetic polymorphism, variability, species and tissue specificity. The relations between the MDH isoenzymes in the tissues and cells of the body vary depending on the stage of its ontogenetic development and on the physiological state. When studying the isoenzyme spectrum of MDH from beet, the existence of an early enzyme polymorphism was established and the appearance of molecular forms at the first stages of ontogenesis of plants that are not characteristic of other stages of their development.

The number of MDH isoenzymes in the scab also decreases at later stages of maize development to four molecular forms. Age-related changes in the isoenzyme composition of the malate dehydrogenase system were found in the study of animal and plant enzymes, as well as the enzyme of microorganisms.

The data of electrophoretic analysis show that the isoenzyme spectra of MDH from organs of different plants (corn, castor oil plants, clover and soybeans) are unstable and can vary depending on age, ontogenesis stages and on the physiological state of the body. Changes in the isoenzyme spectrum of the MDH of these plants are associated either with the appearance and synthesis of additional molecular forms of the enzyme, or with their repression (inhibition) during the development of the organism.

A number of researchers confirmed the presence of species specificity of the isoenzyme spectrum of MDH of higher plants. These properties of the isoenzyme spectrum of MDH allow using it as a marker protein for breeding or as a biochemical trait for classifying species and creating new natural taxonomic groups.

Based on this, information on the structural organization and dynamics of the relative activity of MP MDH - one of the key enzymes of the Krebs cycle - can be used to identify LRS by the phases of vegetation.

The essence of the invention lies in the fact that as the objects of study at the molecular level, malate dehydrogenase of medicinal plants of poultry, pylori, and pompous, selected in raw materials using polyacrylamide gel electrophoresis is selected.

The proposed method is implemented as follows.

Samples of fresh plant materials were collected at different periods of vegetation: budding, flowering, fruiting.

To obtain an extract from 1.0 g (accurately weighed) of fresh medicinal plant material, 10.0 ml of 1/15 M phosphate buffer pH 7.2 was used.

Fresh plant material was disintegrated in a porcelain mortar with 5.0 ml of 1/15 M phosphate buffer, pH 7.2, cooled to 4 ° С for 5 minutes.

Next, the disintegrate was quantitatively transferred to the flask, 5.0 ml of 1/15 M phosphate buffer pH 7.2 and 10 drops of X-100 triton were added at a final concentration of 20 mg / ml. The flask was placed on a magnetic stir bar to solubilize the enzyme for 1 hour.

The homogenate from the flask was quantitatively transferred to a centrifuge tube, centrifuged at 5000 rpm for 10 min.

The resulting supernatant was placed in a tube with a stopper and stored frozen for no more than 6 days.

The molecular forms of malate dehydrogenase were determined in the supernatant.

To carry out the studies, the electrophoretic method of analysis was used. All reagents used in the experiment were of the brand “ХЧ” and “ЧДА”. Samples of substances were weighed using a VLT-150-P balance.

Stock solutions for electrophoresis were prepared according to the formulations proposed for electrophoresis in an alkaline system. To prepare the gel, acrylamide and methylene bisacrylamide from Sigma were used. Before use, recrystallization of these components was performed to remove acrylic acid. The reagents prepared and stored in the refrigerator were kept for some time at room temperature before work. For electrophoresis, a 7.5% polyacrylamide gel was prepared. The resulting solution of the separating gel was poured into the cuvettes of the device, then cold water was layered onto the surface with a syringe. The end of the polymerization was fixed by the formation of a clearly visible interface between the gel and layered water. After removing water, a solution of coarse gel was layered onto the surface of the finely porous gel, onto which a cooled upper electrode buffer was carefully applied. The photopolymerization of the large-pore gel was carried out using an ultraviolet lamp. The end of the polymerization of the large-pore concentrating gel was established by the formation of a clearly visible boundary between the gel and the layered buffer solution.

After polymerization of the gels, the chambers were poured with upper and lower buffer solutions. The electrode buffer used was 1 M Tris-EDTA borate buffer, pH 9.2.

Analyzed samples of the supernatant immediately before the start of electrophoresis were mixed with a 40% sucrose solution in a ratio of 2: 1 and 0.5 ml of the resulting mixture was applied to the start line.

The electrophoresis mode was selected empirically in preliminary experiments. The first 30 min electrophoresis was performed at a current strength of 5.0 mA / cm, then 10.0 mA / cm until the end of electrophoresis. The time of completion of the electrophoresis procedure was judged by the position of the disc of the dye bromphenol blue.

At the end of electrophoresis, the buffer solutions were poured, the gels from the cuvette were removed by introducing a distilled water between the gel surface and the cell wall with a syringe.

The molecular forms of malate dehydrogenase were detected on gel plates by the phenazine methasulfate-tetrazolium reaction in Petri dishes.

In the claimed invention, an optimized incubation medium was used: aqueous NAD solutions (1 mg / ml) - 40 ml, nitrosine tetrazolium (1 mg / ml) - 30 ml, 1 M sodium malate solution (pH = 7.0) - 10 ml, phenazine metasulfate (1 mg / ml) - 4 ml, 0.2 M Tris-HCl buffer solution (pH = 7.1) - up to 100 ml.

Gel plates were incubated 12 hours at 37 ° C. Molecular forms of MDH were detected in the form of dark blue stripes.

The relative electrophoretic mobility of the molecular forms of malate dehydrogenase was calculated as the ratio of the path covered by electrophoresis to the length of the gel plate using the TLC Manager computer program. The device used in the work: a chamber for vertical electrophoresis VE-10 (manufacturer "Helikon").

The obtained experimental data were statistically processed using methods of testing statistical hypotheses.

The molecular forms of malate dehydrogenase (MDH) were determined, the relative electrophoretic mobility (OEP) of the detected forms of MDH was calculated for the medicinal plant material of poultry, paprika and paprika on the following vegetation phases: budding, flowering, fruiting.

The results were presented in table form:

OEP (relative electrophoretic mobility) of malate dehydrogenase enzyme is a qualitative characteristic that is specific and characteristic for a particular type of raw material during electrophoresis under the specific conditions described above.

In the proposed method, the determination of the vegetation phase of highlanders is carried out by physiological markers - molecular forms of the enzyme malate dehydrogenase. The number of molecular forms and their OEP values are qualitative characteristics inherent in specific types of raw materials under certain electrophoresis conditions.

The research results made it possible to distinguish precisely the flowering phase, as optimal and regulated for the assembly of raw materials. There are 3 main molecular forms (MF) of malate dehydrogenase:

- Malate dehydrogenase - 1 (MDG-1);

- Malate dehydrogenase - 2 (MDG-2);

- Malate dehydrogenase - 3 (MDG-3).

Three MDF MDHs differ in molecular weight and in what processes are involved in the life of the plant. As can be seen from the table, in the raw materials of the mountaineers can be found as one (any) MF MDG, and all three MF MDGs at once. The amount of MF MDH depends on the phase of plant vegetation and its physiological state.

MDG-1 detection is possible, starting from the early stages of plant development, to flowering at the budding stage. And it indicates that there is an active growth of the plant, the accumulation of the mass of the plant itself and the biosynthesis of biologically active substances.

MDG-2 detection is possible at different stages of vegetation, to a greater extent indicates the stabilization of metabolic processes in the plant.

MDG-3 detection is possible mainly after the flowering period of plants before its death, since during this period oxidative processes prevail in the plant body and the plant withers.

The molecular forms of MDH as a result of electrophoresis are found in the form of dark blue stripes on gel plates (electrophoregrams).

OEP (relative electrophoretic mobility) is a relative value. Shows where exactly the detected MDH fraction is located. The EIA is calculated as the ratio of the distance traveled by the MD MDF from the beginning of the gel plate to the end point of electrophoresis. The EIA value will always be less than 1.0.

Distinctive features by which it is possible to diagnose the phase of the vegetation of a plant is a combination of features, namely:

- The value of the EIA for a specific MF MDG;

- The number of determined MDF MDH in the plant in a certain phase of vegetation.

For the mountaineer

- At the budding stage, one MDF-1 MF is detected with an OEP value of 0.83 ± 0.02;

- At the flowering stage, one MF MDG-1 with an OEP value of 0.50 ± 0.02;

- At the fruiting stage, two MDF MDGs are detected: MDG 1 with an OEP of 0.65 ± 0.02 and MDG-2 with an OEP of 0.45 ± 0.01;

For the mountaineer pepper

- At the budding stage, two MDF MDHs are detected: MDG 1 with an OEP of 0.63 ± 0.02 and MDG 2 with an OEP of 0.42 ± 0.02;

- At the flowering stage, only MDG 1 with an OEP of 0.60 ± 0.02 is detected;

- At the fruiting stage, all three MDF MDHs are detected: MDG 1 with an OEP of 0.64 ± 0.02, MDG 2 with an OEP of 0.44 ± 0.02 and MDG 3 with an OEP of 0.12 ± 0.01.

For the Highlander

- At the budding stage, one MDF MDH is detected: MDG 1 with an OEP of 0.63 ± 0.02;

- At the flowering stage, only MDG 1 with an OEP of 0.76 ± 0.02 is detected;

- At the fruiting stage, all three MDF MDHs are detected: MDG 1 with OEP 0.64 ± 0.02, MDG 2 with OEP 0.42 ± 0.02 and MDG 3 with OEP 0.10 ± 0.01.

Thus, we can draw a conclusion about the phase in which the plant, for example, highlander, is located after electrophoresis in a 7.5% polyacrylamide gel and detection of MDH, if one form of MDH 1 with an OEP of 0.60 ± 0 is detected on the electrophoregram 02, this means that the plant is in the flowering phase.

The invention creates an objective way to determine the flowering phase of the mountaineer, pylori and mountaineer, which will improve the quality of drugs made from raw materials of medicinal plants.

Claims (1)

A method for determining the vegetation phase of medicinal plant material of poultry, paprika, and pseudomonas by malate dehydrogenase using polyacrylamide gel electrophoresis, containing the collection of medicinal raw materials, determination of molecular forms of malate dehydrogenase (MP MDG) by electrophoretic method using polyacrylamide electrophoretic gel ) of the discovered forms of MDH for medicinal plant material of the mountaineer, pylori, and mountaineer on traveling vegetation phases: budding, flowering, fruiting, determination of the highlanders vegetation phase by physiological markers - molecular forms of the enzyme malate dehydrogenase, while the flowering phase of plants is determined as follows: for a mountaineer - if one MF MDH 1 with an OEP of 0.50 ± 0 is detected, 02, for the mountaineer of the pepper - if only MDG 1 with an OEP of 0.60 ± 0.02 is detected, for the mountaineer of the kidney - if only MDG 1 with an OEP of 0.76 ± 0.02 is detected.