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Raimova Guli Matmuradovna, Master of biology, scientific researcher A. S. Sadikov Institute of Bioorganic Chemistry, Academy of Sciences of the Republic of Uzbekistan, Tashkent city
E-mail: Guli-raimova@mail.ru Khoshimov Nozim Numonjonovich, Master of biology, scientific researcher A. S. Sadikov Institute of Bioorganic Chemistry, Academy of Sciences of the Republic of Uzbekistan, Tashkent city
E-mail: Nozimka@inbox.ru Nasirov Kabil Erkinovich, Doctor biological sciences, leading scientific researcher A. S. Sadikov Institute of Bioorganic Chemistry, Academy of Sciences of the Republic of Uzbekistan, Tashkent city
E-mail: K-nasirov@front.ru Ziyavitdinov J. F., Candidate of Chemical Sciences A. S. Sadikov Institute of Bioorganic Chemistry, Academy of Sciences of the Republic of Uzbekistan, Tashkent city
ISOLATION AND PHYSICAL AND CHEMICAL CHARACTERISTICS OF ANTICOAGULANTS FROM THE FIRE-BLIND CLEVER TRIFOLIEAE
Abstract: The obtained results indicate that in the concentrate isolated from the moldy clover, the anticoagulant components are contained as indirect so direct action that mutually reinforces its anticoagulant to the hemostasis system. These properties of the moldy clover concentrate are of some interest, which in the long run will allow using it as the basis of the rodenticide preparation against the rodents' fights.
Keywords: platelet, CMC, coumarin, HPLC, dicumarol.
Introduction. Anticoagulants of indirect action are coumarins (dicumarin, warfarin, syncumar neodicumarin, or others), which interfere with the formation of prothrombin of the liver and involved in blood clotting [1]. However, in itself, coumarin does not affect blood coagulation, but can be initially metabolized by various fungi in combination such as 4-hydroxycoumarin, and then (in the presence of natural formaldehyde) into dicumarol, which has anticoagulant properties. The fungal origin of dicumarol explains the presence of this anticoagulant only in a damaged clover silo. Thus, dicoumarol is a product of fermentation and mycotoxin [2].
The mechanism of indirect action of these anticoagulants is concluded in stopping normal formation of coagulation factors -coagulation, which is based on a complex system of various metabolic processes.
At present, dicumaroles have found a wide range of applications not only in medicine, but also the quality of rodenticides for the destruction of rodents. The purpose of this work is to isolate from the clover Trifolieae dicoumarins and the development of bait formulations of complex compounds based on anticoagulants and synergists to combat the synanthropic rodents and pests of agricultural crops.
Materials and methods
Spectrophotometric analysis was conducted in the range of characteristic absorption maxima (220-350 nm) for the quantitative determination of the content of coumarins [3; 4].
Fluorescence analysis was carried out on a USB 2000 fluorim-eter.In size the identification of coumarins at a maximum excitation (380 nm) and fluorescence (480 nm) was carried out using the Stokes shift and the fluorescence maximum. With the help of fluorescence spectra, a linear dependence of its intensity on the concentration was obtained and established [5; 6].
HPLC was run on an Agilent Technologies 1200 series chromatograph with a DAD detector and an automatic sampler. A chromatographic column of4.6 x 150 mm Eclipse XDBC-18, 5 ^m was used. Mobile phase: A - 0.1% phosphoric acid, B - acetonitrile. Gradient 5% - 3 min, 80% - 18-23 min, 5% -25 min at flow rate -1 ml/min. Absorption at 278 nm. The duration of the analysis is 25 minutes. As a standard sample, dicoumarol was used with a concentration of 1 mg/ml, previously isolated and dried from the dosage form of the dicumarin preparation (Sigma Aldrich. M 1390). The concentration of the test sample is 5 mg/ml [7].
Toxicological experiments were performed on non-native white male rats weighing 200-250 g. All experiments were carried out in accordance with the requirements of the World Society for the Protection of Animals and the European Convention for the Protection of Experimental Animals [12].
Thrombin and prothrobin time, activated plasma recalcification time (APRT) and activated partial thromboplastin time (APTT) -the test was performed on a coagulometer (CYANCoag, Belgium).
The results were statistically processed for Origin 6.1 (Origin Lab Corporation, USA).
Results and discussion
In preliminary studies it was found that when feeding laboratory rats and mice with bait containing an alcohol extract of moldy clover Trifolieae, during 2-3 days causes severe bleeding in animals, which served as the basis for obtaining a concentrate from moldy clover (CMC) and studying its anticoagulant and Toxicological characteristics, as well as the development of technology of rodenticidal preparations against rodent control.
The raw clover was moldy in a closed bag in a dark place for 10 days. After the formation of the mold, the moldy clover was crushed to a particle size of not more than 2 mm and subjected to extraction at a raw material/extractant ratio ofl: 100. The most complete extraction of coumarins (in free form and in the form of glycosides) was achieved by using 45-96% ethanol both in the cold and under heating. At the same time, the yield of extractive substances was 32-36% of the initial mass of moldy clover.
To purify the amount of coumarins from the concomitant substances, the thick extract obtained after distillation of the extractant was treated with chloroform and a mixture of coumarins was recovered. After concentrating the extract, a mixture of coumarins in a crystalline state was obtained.
The quantitative and qualitative composition of the coumarin mixture was studied using spectrophotometry, fluorescence analysis and high-performance liquid chromatography, as described in [8].
The spectrophotometric method is based on the ability of coumarins to absorb in the UV region of the spectrum and does not require a preliminary separation. We have found that in the UV spectra of the coumarin mixture, two characteristic maxima for dicoumarins (control) of high intensity bands in the 220-380 nm range are observed, the first of which is of an oscillatory nature,
and the second corresponds to the pt-conjugation of the benzene ring (Fig. 1.)
Figurel. UV spectrum of coumarins. 1 - Spectrum of dicumarol (control); 2 - The spectrum of a mixture of coumarins
Many coumarins exhibit characteristic (yellow, green, blue or violet) fluorescence under UV excitation in neutral alcohol solutions and in alkali solutions [9].
Fluorescence is enhanced in an alkaline medium due to the formation of a quinoid structure. By the size of the Stokes shift and the maximum offluorescence, it is proposed to carry out identification of phytochemical preparations [10]. For coumarins and dicoumarins,
fluorescence spectra were obtained and a linear dependence of its intensity on concentration was established, which made it possible to apply fluorescence analysis for the quantitative determination of coumarins [5; 6]. Fluorescent analysis of the extract (a mixture of coumarins) showed that with a saturated solution of bromine in an alkaline medium it was possible to identify them on the basis of equal excitation maxima (380 nm) and emission (480 nm) [11].
Wav*lffnolh (nm)
Figure 2. Indications of intensity from the concentration of the fluorescence spectrum
The obtained fluorescence spectra as a function of the concentration of the extract showed a linear dependence of their intensity on the concentration, which made it possible to estimate the quantitative content of coumarins in this extract (Fig. 2).
At present, the method of HPLC is widely used in the analysis of coumarin derivatives. The method in the isocratic mode of separation allows simultaneous qualitative and quantitative analysis of coumarins without preliminary purification of the extract.
In Analysis HPLC of a thick extract of moldy clover revealed a component with retention time identical with standard dicumarol of 15.22 minutes. (Fig. 3). The amount of dicoumarol in the thick extract was calculated by comparing the peak areas in the chromato-gram of the standard sample and in the test sample. As a result of the studies, it was found that in a dense extract the amount of dicoumarol was 0.64%, and in the crystalline fraction the sum of coumarins obtained with the help of chloroform was 30.5%.
Figure 3. Chromatogram of a thick extract of moldy clover
2,0
1,5.
t
■J 1,0.
0,5
0,0
CMC alcogol 45% CMC water CMC alcogol 96% Coumarin
200
250
300
350
400
Figure 4. Spectrophotometric readings of CMC and Coumarin. Electronic absorption spectrum of coumarin and CMC in 45-96% alcohol extraction
This material was enough to test anticoagulant actions and compare anticoagulant indirect effects with dicumarol, which caused bleeding.
A study of the toxicological properties of the extract extracted from clover in vivo, both with single and multiple oral administration to mice, revealed that depending on the dose of the extract (50-500 ^ l/g) in bait, the symptoms of intoxication were mani-
Table 1. - Toxicological features of CMC with a single consumption of bait by rats
fested for several days. Among the common manifestations of intoxication, a significant place was occupied by symptoms, massive damage to the blood supply of organs and tissues, and thrombo-hemorrhagic syndromes (hemorrhages of the mucous membranes of the skin, bleeding), like dicumarol poisoning, an anticoagulant of indirect action.
1
3
4
2
Dose of the CMC ml/gr Number of rats Number of surviving rats Deaths of rats Over time
50 6 6 1 5 days
100 6 2 4 3 days
250 6 1 5 3 days
500 6 0 6 3 days
In in vitro studies at a concentration of 50 mg/ml, CMC also prolonged the recalcification time, thrombin, prothrombin and AP'ni In these cases, lengthening the clotting time of blood plasma may be due to inhibition of one or more clotting factors. Violations of one or more of these processes can lead to disturbances in the functional activity of hemostasis and the development of hemor-rhagic syndrome.
The obtained data indicate that, in the concentrate isolated from the moldy clover, there are anticoagulant components as indirect, so direct actions that mutually reinforce its anticoagulant properties.
These properties of these concentrates are of some interest and require further detailed study of the physicochemical characteristics and mechanisms of their action, which will eventually allow them to be used as a rodenticide against rodent control.
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