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Section 2. Chemistry
https://doi.org/10.29013/AJT-20-3.4-23-31
Abdurazakov Asqar, Ph.D in Chemistry., Senior researcher, Institute of chemistry of plant substances Academy of sciences of the Republic of Uzbekistan, Republic of Uzbekistan, Tashkent
Е-mail: [email protected] Khvan Аllа,
Ph.D. in Chemistry., Senior researcher, Institute of chemistry of plant substances Academy of sciences of the Republic of Uzbekistan, Republic of Uzbekistan, Tashkent
Е-mail: [email protected] Zukhurova Gulnara, Ph.D. in Technical., Senior researcher, Institute of chemistry of plant substances Academy of sciences of the Republic of Uzbekistan, Republic of Uzbekistan, Tashkent
Е-mail: ре[email protected] Islamova Jannat, Ph.D. in Medical., Senior researcher, Institute of chemistry of plant substances Academy of sciences of the Republic of Uzbekistan, Republic of Uzbekistan, Tashkent
Е-mail: [email protected] Mirzakhidov Khayrulla, Ph.D., Associate Professor, National University of Uzbekistan, Republic of Uzbekistan, Tashkent Е-mail: [email protected]
COMPLEX FORMATION OF ALBENDAZOLE WITH PECTIN AND BIOLOGICAL ACTIVITY OF THE PRODUCT
Abstract. The features of the interaction of the anthelmintic drug adbendazole with citrus pectin have been studied. By the methods of potentiometric titration, viscometry, IR spectroscopy, and X-ray phase analysis it was shown that the interaction takes place in the narrow pH range using salt and hydrophobic bonds. It was established that the obtained polymer complex of albendazole "Alpec"
exhibits the more pronounced anthelmintic activity compared to the initial albendazole. It have been found that the LD50 of Alpec is 680 (601,8 ^ 768,4) mg.kg -1, which is almost 1,5 times higher than the LD50 of albendazole. The resulting polymer form of albendazole can be recommended for the application in medicine and veterinary practice.
Keywords: albendazole, pectin, alpec, medicine, polymer complex.
1. Introduction
The growing demand for anthelminthic medicines is caused by the extremely wide distribution of various helminths, especially in hot climates. According to WHO, at least 4 billion people are affected by parasitic diseases in the world, tens of millions of them die annually as a result of those diseases [1].
In some territories of the CIS countries, despite
the success achieved in recent years in combating
parasitic diseases, the epidemiological situation remains tense.
Therefore, one of the most important tasks of chemical science dealing with physiologically active compounds is the search and production of new highly effective antiparasitic drugs for medical practice.
Among the currently known anthelminthic drugs, the products belonging to the group of benz-imidazole, in particular, albendazole is notable for its wide range of effects. Albendazole is characterized by a fairly high biological activity. In addition, unlike the majority of practiced drugs, albendazole is now actively used to treat such a serious disease as echinococcosis.
At the same time, albendazole, like most modern medicines, is not devoid of certain shortcomings. First of all, it is insoluble in water, which in some cases severely limits its use in medical practice.
At the same time, in the intensively developing nowadays field of chemistry of medicinal compounds it is well known that the solubility and therapeutic efficacy of known drugs can be significantly increased in many cases by their chemical modification with various high-molecular compounds. The introduction of new polymer forms of medicinal products into pharmaceutical practice, in general, can allow to control the speed and place of their absorption,
give them prolonged effect, reduce toxicity, increase resistance and other valuable qualities. Polymers of both synthetic and natural origin can be used as carrier polymer. Polysaccharides, in particular, pectin, have special advantages in this respect, as it can degrade in the gastrointestinal tract and, being an en-terosorbent, can simultaneously remove slags, toxins and radionuclides from the body [2-5].
2. Research purpose and objective
Given the prospects for the use of polymeric drug form of anthelminthic drug in medical and veterinary practice, this paper aims to study the peculiarities of the process of complexation of albendazole with pectin, the study of biological properties of the obtained albendazole complex to obtain a polymeric form of albendazole with increased activity.
3. Experimental part
IR spectra were shot using KBr pallets, on the FT-IR spectrometer 2000, manufactured by Perkin-Elmer (USA).
Potentiometric titration was carried out on the device I-120.0 (H-120.0) ionomer. Viscometry was carried out using Ubbelohde viscometer.
X-ray phase analysis of powders of initial components of pectin, albendazole and Alpec polymer complex was conducted on powder diffractometer ShimadzuXRD-6100 (Japan). The samples were measured in Bragg-Brentano mode with scanning of 2-0 from 4 0 to 50 0.
Albendazole produced by the Institute of the Chemistry of plant substances of the Academy of Sciences of the Republic of Uzbekistan and citrus pectin (Genu pectin typedz Manufacturer CP Kelso, lilleskensved, Denmark.) Etherification degree 5862%, pH (1% solution) 2.9-3.4, gelling property of 145-155%, humidity not more than 12%.
The process of obtaining polymer com-plex of albendazole with pectin. The process of obtaining pectin polymer complex with albendazole. 8.34 g of albendazole was dissolved in 200 ml of distilled water, 20 ml of concentrated hydrochloric acid and 35 ml of ethyl alcohol under continuous stirring at 45-50 °C. 4.17 g pectin was dissolved separately in 237 ml of distilled water at continuous stirring. To the obtained solution of pectin at intensive stirring at 45-48 °C the prepared solution of albendazole was added 15-20 ml every 3-5 minutes and stirred for two hours [6]. Then the reaction mixture was filtered through a 0.5 mm sieve and dried on spray drying.
The range of antiparasitic action and efficiency of the investigated compounds were determined on the following experimental models of parasitosis: aspiculurosis (larval and semi-mature stage of As-piculuristetraptera oxyuride) ofwhite mice (the latter model is used as an experimental model of enterobiasis); giardiasis of white mice (vegetative forms of Lambliamuris and their cysts).
Efficiency of drugs was determined by the number or percentage of departing parasites - Intense Efficiency (IE). Due to the difficulty or impossibility to calculate departed parasites, the average number of helminths in exposed animals in experimental and control groups was compared and the IE (in %) was determined by the formula:
IE = 100 (C - O) / C, where C is the average number of helminths in the control group; O is the average number of helminths in the experimental group.
All animals were preliminary examined for parasite infestation by coproscopy before being taken into the experiment.
During the experimental infection of mice with aspiculurosis they were inj ected with peros by 100 A. tetraptera invasive eggs. Egg excretion from the faeces of spontaneously infected mice was carried out using flotation centrifuge method. After that, the amount of suspension containing the required amount of invasive material was calculated. Only viable eggs were counted [7]. When studying the activity against larvae
oxyuride chemopreparations were administered on the 5th day after aspiculurosis infection. For the study of the chemotherapeutic activity of drugs at the mature stage A. tetrapera the drugs were injected on the 10th day after infection. In both cases, the drugs were injected once a day, once. Accounting of the results of chemotherapy was carried out 3 days after the end of treatment by extracting and counting larvae (blind intestine) and adults (a section of the large intestine length of10 cm) oxyuridium, using binocular magnifier MBS-1 (MBC-1) simultaneously in the experimental and control groups [8].
In experiments to determine antiprotozoic activity, white non-breeding mice of both sexes weighing 13-15 g were used. To reproduce the experimental giardiasis model, the animals were infested with oral suspension containing Giardiamuris 5x103 cysts and trophosoites in 0.5 ml. The suspension was prepared from the content of small intestine of spontaneously infected mice (Roberts-Thomson, Mitchell method, 1978). The investigated compounds were injected into the stomach with a special atraumatic probe on the 5th day after infection within the next 5 days. All experiments were conducted in accordance with the requirements of the "European Convention for the Protection ofVertebrate Animals Used for Experimental Purposes and Other Scientific Purposes" (Strasbourg, 1986). After the end of treatment, mice were slaughtered under mild ether anesthesia by cervical dislocation and 10 cm ofsmall intestine were taken from each animal starting with gastroduodenal articulation. The efficiency ofthe investigated compounds was judged by calculating the average number of vegetative forms and giardial cysts per one animal. Intense-efficacy ofcompounds was determined by the formula: IE= 100 (C - O)/C, where C is the number of trophosoites and giamblion cysts in control, O - in experiment. Examination was made at magnification 10x40 [9].
4. Results and discussion
The study of the chemical interaction of albendazole (MS) with pectin (P) was conducted by various physical and chemical methods.
The results of potentiometric titration are shown in (Fig. 1). We can see that the addition of increasing amounts ofalbendazole to the aqueous solution of pectin leads to a decrease of the pH value of the medium.
Figure 1. Dependence of medium pH changes on the ratio of [LV]/[P] components
This indicates that the process of complexation of the anthelminthic drug with the polymer is characterized by proton release according to the scheme.
According to the data of potentiometric titration the value of О - electrostatic bonding has been calculated. This parameter is determined by the ratio of proton concentration changes when mixing solutions of MS and polymer. As can be seen from (Fig. 2), the degree of electrostatic bonding increases with increasing content of low molecular weight MS in the solution.
It was found that the maximum degree of electrostatic bonding of albendazole to pectin is 28.2%.
One of the factors influencing the conversion depth of Q is the medium pH environment, since the degree of electrostatic bonding of polyelectrolytes to counterions will obviously depend on the charge density on the polymer macromolecule, which is determined by the pH value of the solution.
Figure 2. Dependence of electrostatic bonding (0) on the component ratio [LV]/[P]
Figure 3 shows the dependence of the degree of electrostatic bonding of pectin to albendazole on pH values.
From (Fig. 3) we can see that the interaction takes place in a narrow pH range of 2.3-3.3. This indicates the cooperative nature of the process of interaction between pectin and albendazole.
In albendazole-pectin system it is possible to perform both ionic and hydrophobic interaction. All this should affect the conformation state of the complex, i.e. its hydrodynamic behavior in the solution. It can be seen from (Fig. 4), that addition of increasing
amounts ofMS to the ratio of 0.01 leads to a decrease ofreduced viscosity of the polymer complex solution, which is explained by the implementation of a large number of ionic interactions.
Figure 3. Dependence of the electrostatic binding parameter (0) on pH value of the medium
However, after the ratio of 0.01 the increase of albendazole content in the polymer complex leads to the increase of its reduced viscosity and, eventually, it gels
after the ratio of0.15. This can probably be explained by the fact that hydrophobic interactions begin to prevail in the albendazole-pectin system. And eventually the polymer chains of pectin bind due to the presence of molecules between them, on the one hand, bound to the macromolecule by salt bonds, and on the other -hydrophobic to each other and the polymer.
The interaction of pectin with albendazole was also studied by infrared spectroscopy (Fig. 5). Comparison of infrared spectra of Alpec and initial components (albendazole, pectin) shows significant frequency changes in the area of double bonds and hydroxyl groups (3200-3500 cm -1). A low-frequency shift of absorption bands of OH-groups of pectin (3424 cm-1)in the Alpec complex (3400 cm-1), by 24 cm-1. There is observed also a high frequency shift of C=O pectin bands (1739 cm-1), in Alpec (1752 cm-1), by 13 cm-1. In addition, the albendazole absorption band at (1268 cm-1), due to C-N bond vibrations, shifts in Alpec by 20 cm-1 h and is detected at (1248 cm-1).
The results obtained clearly indicate the formation of albendazole polymer complex with pectin, which we call "Alpec".
Figure 4. Dependence of the reduced viscosity of water-alcohol (1:1) solutions of albendazole polymer complex with pectin on the ratio. ^=[Albendazole]/[Pectin]. T = 303 °C
4000.0 3600 3200 2800 2400
2000 1800 1600 1400 1200 1000 800 600 400.0
cm-1
Figure 5. IR spectra of albendazole, pectin-GENU, albendazole complex with pectin (Alpec)
The formation of polymer complex as a result of interaction of albendazole with pectin was also studied by X-ray phase analysis (Fig. 6).
5 10 15 20 25 30 35 40 45 50
2 theta
Figure 6. Comparison of X-ray diffractograms of Alpec (1) with pectin (2) and albendazole (3)
%T
On the diffractogram of the pectin complex with albendazole one can see new diffraction peaks at 4.840, 9.73°, 10.48°, 11.60°, 12.42° and other 2-0 angles, at which diffraction peaks of initial albendazole and pectin are not observed, that testifies to the formation of a crystal lattice of the complex, which differs from albendazole and pectin. It is also seen that the amorphous peak of pectin with the center at 13.68° is not observed on the diffractogram of the complex, which means that the amorphous part of
the pectin passes into the crystal structure of the
complex.
Thus, the comparative X-ray phase analysis of albendazole, pectin and Alpec confirms the formation ofAlpec polymer complex with a new crystal lattice.
Biological studies of anthelminthic activity of albendazole polymer complex with pectin Alpec were
carried out on an experimental model of aspiculuro-sis. The results obtained indicate that Alpec has the pronounced antinematodetic aaction against mature stage of A. tetraptera already in a dose 2 times lower than the albendazole itself.
The study of the protozoic activity of the giardiasis model of white mice also revealed a pronounced an-tigirardiasis effect of Alpec, which is superior to that of albendazole, too.
The increased biological activity of Alpec can probably be explained by the good water solubility of Alpec unlike albendazole, which, as a consequence, leads to increased bioavailability of the preparation.
On the experimental model of aspicululosis the administration of drugs at the sexually mature stage A. tetraptera showed the expressed activity of albendazole complex with pectin (Table 1).
Table 1. - Results of the study on the efficacy of alpec efficiency on the semiadult stage of A. tetraptera development (in comparison with albendazole)
Drug Dose mgkg n = 6, once The number of parasites after autopsy Intense Efficiency,%
2.5 5.2 ± 0.38*,** 91.4
"Alpec" 5 4.0 ± 0.58*,** 93.4
10 3.6 ± 0.8*** 94.1
2.5 15.6 ± 1.2*,** 74.2
Albendazole 5 6.0 ± 0.36* 90.1
10 5.0 ± 0.44* 91.7
Control - 60.8 ± 2.2 -
Note: * - accurate with control; ** - accurate with comparative drug effect (p < 0.05)
The dose ofAlpec 5 mgkg 1 was 93.4% effective, Alpec remained almost unchanged (94.1%), but it while albendazole in this dose was 90.1%. With the was higher than that of albendazole (91.7%) in the increase of the dose up to 10 mgkg 1 the efficacy of same dose (Table 1).
Table 2.- Results of the study on the antigirardiasis effect of Alpec (in comparison with albendazole)
Drug Dose mgkg n=6, for 5 days The number of parasites after autopsy. Intense Efficiency,%
1 2 3 4
2.5 402.3 ± 2.2* 81.0
Alpec 5 324.7 ± 3.4*,** 84.7
10 315.5 ± 1.2*,** 85.2
1 2 3 4
2.5 784 ± 29.2*,** 6.1
Albendazole 5 412.6 ± 2.1* 80.6
10 400.4 ± 2.3* 81.2
Control - 2125.8 ± 121.2 -
Note: * - accurate with control; ** - accurate with comparative drug effect (p < 0.05)
Therefore, Alpec has an antinomatose effect on A. tetrapetra in a dosage 2 times lower than that of albendazole itself.
The antiprotozoic effect of Alpec was studied on the model of giardiasis of white mice (vegetative forms of Lambliamuris and their cysts).
The evident antigirardiasis effect of albendazole complex with pectin as the studied drug was observed when administered for 5 days in a dose of 5 mgkg 1 with IE = 84.7% (table 2).
The result of a 2-fold increase in dose (10 mgkg -1) - resulted in a slight change of 85.2%, respectively. The dose of 2.5 mgkg 1 can also be considered quite effective - 81.0%. All this data indicates the high antiprotozoic activity of the test Albendazole complex with pectin in an optimal dose of 5 mgkg-1. Comparison of Alpec with the initial albendazole showed that it is significantly more active than the latter latter as the reference drug in the same dose.
The study of acute toxicity ofAlpec was carried out in experiments on white pedigree rats weighing 210250 grams. The average lethal dose of Alpec (LD50),
-i
was determined, it is - 680 (601.8-768.4) mgkg (LD50 of albendazole - 400.2-450.4) mgkg -1), i.e. the polymeric complex ofAlpec is classified as moderately dangerous in terms of parameters of acute toxicity for intragastric use. 4. Conclusion
The interaction of albendazole with pectin was investigated by potentiometry, viscometry, infrared spectroscopy and X-ray phase analysis methods. It was shown that the interaction takes place with the help of salt and hydrophobic bonds to form a polymer complex (Alpec). High antiparasitic (more pronounced antinematodetic than antigirardiasis) activity ofAlpec as compared to the original albendazole was detected, which is probably explained by an increase of the solubility of the drug and, consequently, by its increased bioavailability. It was found that in terms of "acute" toxicity index, the "Alpec" complex exceeds the initial albendazole by almost 1.5 times.
The obtained polymeric complex Alpec can be recommended for the use in medical and veterinary practice.
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