Ukrainian biopharmaceutical journal

Determination of clemastine by the HPLC method in the blood

V.I. Kabachny — 2021-04-12

Topicality. Сlemastine fumarate (tavegil)-1-methyl-2 [2-α-methyl-p-chlorobenzhydryloxy)-ethyl]-pyrrolidine fumarate is the first generation H₁-histamine receptor blocker. Сlemastine fumarate selectively inhibits histamine H₁ receptors and reduces capillary permeability. The drug has a pronounced anti-allergic and antipruritic effect. Clemastine prevents the development of vasodilation and the smooth muscle contraction induced by histamine. Сlemastine fumarate has an isignificant anticholinergic activity, causes sedation. The drug is used to treat pruritus in psoriasis, multiple sclerosis and optic neuritis. Clemastine is characterized by the following side effects: increased fatigue, drowsiness, sedation, weakness, lethargy, impaired coordination of movements; nausea, vomiting, decreased blood pressure, palpitations, hemolytic anemia, skin rash, anaphylactic shock. In case of an overdose, the drug has a neurotoxic effect, which manifests itself in impaired consciousness with the development of generalized anticholinergic convulsive syndrome. The urgent task for monitoring the treatment effectiveness of the population with сlemastine fumarate and diagnosis of drug intoxication is the choice of highly sensitive and selective research methods of its analysis in pharmaceuticals and biological matrices during the treatment.

Aim. To develop an algorithm for directed analysis of clemastine in biological extracts from the blood using a unified method of the HPLC research.

Materials and methods. The extraction of clemastine was performed with chloroform at Ph 9.0. The extracts were purified from impurities by a combination of TLC and extraction with hexane. The TLC purification and identification of clemastine were carried out under optimal conditions: the system of organic solvents – methanol – 25 % solution of ammonium hydroxide (100 : 1.5) and chromatographic plates – Sorbfil PTLC-AF-A, R_{f сlemastine} = 0.60 ± 0.03. To detect clemastine, the most sensitive location reagents were used –UV light (λ = 254 nm) and Dragendorff’s reagent modified by Mounier. The chromatographic analysis was performed on a “Milichrome A-02” microcolumn liquid chromatograph (EkoNova, Closed Joint-Stock Company, Russia) under standardized HPLC conditions: the reversed-phase variant using a metal column with a non-polar absorbent Prontosil 120-5C 18 AQ, 5 μm; the mobile phase in the linear gradient mode – from eluent А (5 % acetonitrile and 95 % buffer solution – 0.2 М solution of lithium perchlorate in 0.005 М solution of perchloric acid) to eluent B (100 % acetonitrile) for 40 min. Regeneration of the column was conducted for 2 min with the mixture of solvents; the flow rate of the mobile phase was 100 μl/min, the injection volume – 4 μl. The multichannel detection of the substance was performed using a two-beam multi-wave UV spectrophotometer at 8 wavelengths of 210, 220, 230, 240, 250, 260, 280, and 300 nm; the optimal value of the column temperature – 37-40 °С and the pump pressure – 2.8-3.2 MPa.

Results and discussion. Isolation of clemastine from the blood was performed according to the method developed, including the extraction with chloroform at pH 9.0; the extraction purification of extracts with hexane from impurities; the TLC purification and identification of clemastine. Using the unified HPLC method clemastine was identified by retention parameters and spectral ratios. For the quantitative determination, a calibration graph or the straight line equation corresponding to this graph were used. The results obtained indicated the reliability and reproducibility of the method. It was found that the relative uncertainty of the average result in the analysis of clemastine in the blood was ε = ± 4.63 %, the relative standard deviation of the average result was RSDx = 1.67 %.

Conclusions. Clemastine was extracted with chloroform at pH 9.0 from the blood. Purification of extracts from co-extractive compounds was performed by combining TLC and extraction with hexane. It has been found that when isolating сlemastine from the blood according to the methods developed it is possible to determine 36.05-39.55 % of the substance (ε = ± 4.63 %, RSDx = 1.67 %). The method of TLC purification and identification of сlemastine in biogenic extracts was tested under the optimal conditions: the system of organic solvents – methanol – 25 % solution of ammonium hydroxide (100 : 1.5), the use of reagents – UV light, Dragendorff’s reagent modified by Mounier, R_{f сlemastine} = 0.60 ± 0.03 (Sorbfil PTLC-AF-A). The unified HPLC method for identification and quantification of сlemastine was tested in biogenic extracts from the blood according to the algorithm of the directed analysis developed. It has been found that сlemastine can be identified by the retention time – 25.997-26.011 min; the retention volume – 2599.7-2601.1 μl; spectral ratios – 0.741; 0.536; 0.096; 0.023; 0.027; 0.005; 0.003. The сlemastine content was determined by the equation S = 0.15 · 10^-3 С + 0.14 · 10^-3; the correlation coefficient was equal to 0.9998. Chromatographic methods can be recommended for implementation in practice of the Bureau of Forensic Medical Examination, poison control centers, clinical laboratories regarding the study of medicinal substances in biological objects.

Study of chlorophylls and carotenoids in Sansevieria hyacinthoides leaves

V. Velma — 2021-04-12

Topicality. Sansevieria hyacinthoides (L.) Druce belongs to the Asparagaceae family. The plant is native to African countries. It has been introduced and naturalized in some countries of America, Asia, Australia and Europe. This species is grown as an ornamental plant in many countries, and as a medicinal plant in Africa.

Aim. To determine the quantitative content of chlorophylls a and b and the amount of carotenoids in Sansevieria hyacinthoides leaves freshly collected and dried.

Materials and methods. The quantitative content was determined by the spectrophotometric method. This method allows determining the amount of chlorophyll a and b and carotenoids by the characteristics of the absorption spectra without their prior separation.

Results and discussion. The amount of chlorophyll a and b in the dried leaves was almost 7 times higher than in the freshly collected ones (1.82 ± 0.036 mg/g vs. 0.25 ± 0.005 mg/g, and 2.08 ± 0.038 mg/g vs. 0.29 ± 0.006 mg/g, respectively). The quantitative content of the amount of carotenoids differed by 3.6 times (0.05 ± 0.001 mg/g in freshly collected leaves, and 0.18 ± 0.004 mg/g in the dried raw material).

Conclusions. The quantitative content of the amount of carotenoids, chlorophyll a and chlorophyll b in freshly collected and dried leaves of Sansevieria hyacinthoides has been determined by the spectrophotometric method. The dried plant raw material of Sansevieria leaves has a higher content of biologically active substances: chlorophyll a – 1.82 ± 0.036 mg/g, chlorophyll b – 2.08 ± 0.038 mg/g, and the amount of carotenoids – 0.18 ± 0.004 mg/g.

The study of the cytotoxic activity of the dry extract and the anthocyanin complex of Gebu Dahlia variety flowers

L. Maloshtan — 2021-04-12

Topicality. For today, the search for promising plants with a high content of anthocyanins to create safe herbal medicines is an urgent problem of modern science.

Aim. To study the presence and severity of the cytotoxic activity of the dry extract of Gebu Dahlia variety flowers, the qualitative analysis and quantify the amount of anthocyanins in flowers of this variety.

Materials and methods. For the study, the dry extract of Gebu flowers was obtained. The cytotoxicity analysis of 1 %; 0.5 %; 0.25 %; 0.125 %; 0.0625 % solutions of the extract were performed by microscopy on red bone marrow (RBM) cells of rats at an exposure of 15, 45, 90 minutes. The spectrophotometric study and high-performance liquid chromatography were used to assess the quantitative and qualitative content of the amount of anthocyanins.

Results and discussion. The results obtained indicate that the viability of RBM cells is affected by the concentration of the extract and exposure: in the concentrations of 0.063-0.125 % no toxic effect of the extract was detected, while 0.25-1 % solutions were able to show cytostatic properties. The content of anthocyanin amount in the flowers was determined (1.8 % ± 0.02); 18 substances were identified, among them substances from the groups of cyanidin (54.7 %) and delphinidin (28 %) predominated.

Conclusions. The first stage of testing of the dry extract from Gebu Dahlia variety flowers for potential toxicity has been carried out: the extract is potentially non-toxic in the lowest of the concentrations studied. For the first time, the content of anthocyanins in flowers of Gebu Dahlia variety has been determined, 18 substances from the groups of delphinidin, cyanidin, petunidine, peonidine, malvidin have been identified.

The study of the antioxidant activity of polyphenol extracts from cranberry leaves on the model of insulin resistance

O. Krasilnikova — 2021-04-12

Topicality. In Ukraine, the number of patients with diabetes is growing rapidly, it ranks the 4th in the structure of diseases of the endocrine system, and it is one of the main causes of incapacitation and disability among the population and takes the second place by the number of deaths. Polyphenolic extracts from leaves of the heather (Ericaceae) family, in particular blueberries, cranberries and bearberry, in the previous studies demonstrated the presence of antioxidant, hypoglycemic and lipotropic properties. However, the pharmacological activity of extracts from cranberry leaves has not been sufficiently studied.

Aim. To study the effect of polyphenolic extracts from cranberry leaves on the processes of LPO and the state of the antioxidant defense in the liver and serum in rats with the experimental insulin resistance.

Materials and methods. The study was performed on white outbred male rats weighing 180 ± 15 g, kept in the vivarium of the NUPh. Insulin resistance was induced by keeping animals on a high fructose diet for 6 weeks according to the recommendations. The extracts were administered intragastrically in the dose of 200 μg/100 g of the body weight from the fourth week of the experiment for 14 days. The POL activity was assessed by the level of TBA reactants, diene conjugates and conjugated trienes in the liver and serum. The state of the antioxidant defense was assessed by the level of reduced glutathione, as well as the activity of catalase and superoxide dismutase. Indicators were determined by the spectrophotometric method.

Results and discussion. The development of the experimental resistance was accompanied by an increase in LPS activity in the liver and serum of rats. In the serum of animals with IR the content of primary products of LPO increased in 1.75 times, the content of triene conjugates in 1.7 times, respectively, while the content of TBA reactants in 1.61 times. The introduction of the polyphenolic concentrate from cranberry leaves was accompanied by a sharp decrease in the products of lipid peroxidation both in the eserum and liver to almost ascending levels. There was normalization of the activity of enzymes of the first line of the antioxidant defense compared to animals with insulin resistance due to the presence of plant flavonoids in the extract.

Conclusions. Thus, the data obtained indicate that the polyphenolic extract from cranberry leaves exhibits antioxidant properties in the liver and serum of rats with the experimental insulin resistance. The polyphenolic extract also restored the antioxidant defense of cells and the body as a whole. The data obtained indicate the need for further research to create a new herbal medicine for use in the treatment of insulin resistance and diabetes.

The study of the antidepressant and actoprotective activity of a synthetic analog of the terminal region of neuropeptide Y

I. O. Havrylov — 2021-04-12

Topicality. Neuropeptide Y (NPY), which is significantly widespread in the human body, is responsible for various physiological processes, in particular it is involved in the regulation of the emotional behavior, memory and metabolism. The modified short fragment of NPY proposed – NP9 nonapeptide for intranasal administration – shows certain biological properties of the native peptide, in particular the anxiolytic and antiamnesic action. The effect of NP9 on affective disorders of depressive nature and physical endurance remains unknown.

Aim. To study the antidepressant and actoprotective activity of NP9 in models of tail suspension, reserpine-induced depression, and forced weighted swimming.

Materials and methods. The antidepressant properties of NP9 were studied in the immobilization tail suspension test in mice and on a model of reserpine-induced depression with registration of the body temperature, ptosis, and behavior in the Porsolt forced swimming tests and the open field test. The actoprotective properties of NP9 were studied in the swimming test with a load (10 % of the weight of the animal).

Results and discussion. In the tail suspension test, NP9 significantly increased the latent time of the first immobilization compared to the indicators of intact animals (3.2 times) and the imipramine group (2.7 times). NP9 did not significantly affect the manifestations of reserpine-induced depression. These results indicate the weak antidepressant properties of NP9. Nonapeptide significantly increased the swimming time to exhaustion by 2.5 times, indicating actoprotective properties, according to which NP9 exceeded the reference drug Semax.

Conclusions. In the tail suspension test, nonapeptide NP9 exhibits dose-dependent antidepressant properties after the first administration, but this effect is practically not manifested in the rigid reserpine-induced model of depression. NP9 increases the physical endurance of animals in the forced weighted swimming test and is superior to the reference drug Semax by its effectiveness.

A comparative study of the hepatotrophic properties of non-steroidal anti-inflammatory drugs

К. Г. Щекіна — 2021-04-12

Topicality. Recently, more and more works on the hepatotoxicity of NSAIDs have appeared in the literature. Presumably, all NSAIDs have hepatotoxicity, but the degree of adverse effects on the liver in different drugs is variable.

Aim. To study the effect of different generations of NSAIDs on the functional state of the liver of the experimental animals.

Materials and methods. Diclofenac sodium, piroxicam, indomethacin, meloxicam and celecocosib were selected for the comparative study. Changes in the functional state of the liver of intact rats during subchronic administration of the drugs selected, as well as their impact on the course of the model hepatitis were determined. The state of the liver was determined by the following indicators: the mass coefficient of the liver, the activity of alanine aminotransferase, alkaline phosphatase, the content of total protein, urea, cholesterol in the blood serum, the level of TBA-active products, diene conjugates, reduced glutathione, catalase and glycogen in the liver homogenate.

Results and discussion. It was found that diclofenac, piroxicam, indomethacin in the doses of ED₅₀ by the antiexudative activity when used for 14 days adversely affected the liver of intact animals, as well as worsened the course of the model hepatitis, i.e. had a pronounced hepatotoxic effect. Meloxicam and celecoxib did not show a pronounced adverse effect in the carbon tetrachloride hepatitis, but contributed to the deterioration of the functional state of the liver of intact rats, i.e. had a moderate hepatotoxic effect.

Conclusions. By the level of hepatotoxicity the drugs studied can be arranged as follows: diclofenac > indomethacin > piroxicam > meloxicam > celecoxib.

Dynamics of connective tissue metabolites in the blood of rats of different age under hypokinesia

D. Morozenko — 2021-04-12

Topicality. Hypokinesia is a condition of insufficient motor activity of the body with limited pace and volume of movement, and is currently the fourth leading cause of endemic death in the world. It is known that hypokinesia can affect the metabolism of components of bone and cartilage, which is the basis for the development of pathological processes, but there are no biochemical markers of this process in the literature. Thus, currently, there is insufficient number of experimental scientific works devoted to the study of biochemical parameters characterizing the state of the connective tissue in the experiment to assess the impact of hypokinesia on the body, and this fact determines the relevance of the study.

Aim. To study the dynamics of the main metabolites characterizing the state of the connective tissue in the blood serum in hypokinesia in rats of different ages.

Materials and methods. The studies were performed on 42 white male rats aged 3 and 12 months, kept in the vivarium of the Sytenko Institute of Spine and Joint Pathology National Academy of Medical Sciences of Ukraine, Kharkiv. The conditions of hypokinesia were reproduced using a specially designed device that limited the mobility of the animal without disturbing the ventilation of the body. Animals were removed from the experiment by decapitation in Day 7 and 30 of immobilization under thiopental anesthesia. The content of sialic acids in the serum was determined by the Hess method, chondroitin sulfate – by Nemeth-Csoka method modified by L. I. Slutsky, the fractional composition of glycosaminoglycans (GAG) – by the reaction with resoquine.

Results and discussion. In the study of the rat serum, it was found that in intact animals of 3 and 12 months of age the content of sialic acids was different; it was 2.94 ± 0.16 and 1.98 ± 0.09 mmol/L, respectively. After 7 days of hypokinesia, their concentration in young animals did not change, but in older animals, this figure increased. By Day 30, the serum sialic acid levels suddenly increased compared to intact animals. In animals of 12 months of age, the concentration of sialic acids in the blood serum increased by both Day 7 and Day 30 of hypokinesia, especially at the end of the experiment. Hypokinesia also showed a significant increase in serum concentrations of chondroitin sulfates on Day 7 of the experiment in animals of both age groups. At the same time, the level of these metabolites on Day 30 was at the same level. In young rats on Day 7 the fraction of GAG remained unchanged, but on Day 30 there was a significant decrease. During this period, the second fraction of GAG increased. In animals aged 12 months on Day 7 and 30, the content of most GAG fractions was lower than the values characterizing the fractional composition of GAG in intact rats.

Conclusions. Thus, the determination of sialic acids, GAG fractions and chondroitin sulfates in the serum can be presented as a set of biochemical tests to assess disorders of the connective tissue metabolism while limiting the motor activity of the body. It has been found that the 30-day hypokinesia causes significant disorders of the connective tissue, and it is reflected in changes in the biochemical parameters of the serum of experimental rats.

Prospects of creating a new medicine based on biologically active substances of bearberry leaves for correction of the metabolic syndrome

N. Chaika — 2021-04-12

Topicality. The metabolic syndrome is pathogenetically interrelated metabolic disorders in the condition of a sick person. A large set of factors is involved in its occurrence. Risk factors include genetic predisposition, overeating, insulin resistance, obesity, bad habits, hypodynamics, stress and unfavorable environmental conditions. First of all, defects of the lipid and carbohydrate metabolism provoke a cascade of genetic, metabolic, hormonal, nervous, inflammatory and other reactions and disorders in cells, tissues and organs, causing the metabolic syndrome and associated diseases, such as diabetes, kidney and gallstone disease, hypertension, platelet hyperaggregation, etc. Therefore, the rational use of synthetic and herbal medicines in the complex correction of these disorders can slow down the development of the metabolic syndrome.

Aim. To develop the method for obtaining a dry modified extract from bearberry, study its chemical composition, hypoglycemic and hypolipidemic activity in order to determine the prospects of its use for the correction of the metabolic syndrome.

Materials and methods. The study object was a dry extract of bearberry leaves modified with cysteine. HPLC and spectrophotometry were used to analyze the extract obtained. The hypoglycemic and hypolipidemic activity of dry extracts of bearberry was studied in rats with insulin resistance.

Results and discussion. The method for obtaining a dry modified extract from bearberry leaves was developed by adding cysteine. Phenologlycoside (arbutin), 2 phenolic acids (gallic and ellagic), 6 flavonoids, 8 saponins were identified in the extract, and their quantitative content was determined. Hyperoside and catechin were dominant among flavonoids, and ursolic acid, uvaol, and lupeol prevailed among saponins. The content of the main groups of phenolic compounds was determined in the extract by spectrophotometry. The introduction of the dry extract from bearberry leaves modified with cysteine has a normalizing effect on metabolic disorders on the background of a high-fructose diet; therefore, it can be a promising agent for the correction of the metabolic syndrome.

Conclusions. As a result of the research conducted, a new dry extract from bearberry leaves modified with cysteine has been created. The phytochemical composition, hypoglycemic and hypolipidemic activities of the extract have been studied, indicating the prospects for its use to correct the metabolic syndrome.

The choice of excipients to obtain tablets based on the Primula denticulate Smith. Extract by wet granulation

A. Yu. Shyrko — 2021-04-12

Introduction. Herbal medicines are increasingly being used in the treatment of various diseases. Significant number of resources, high-level availability and the possibility of cultivation have provided high prospects of plant raw materials in the development of new herbal medicines. Primula denticulate Smith. is one of the most interesting medicinal plant raw materials source in modern medicine and pharmacy with the wide spectrum of pharmacotherapeutic action, that is mainly used only in traditional medicine. Thus, nowadays the development of new and effective medicines based on Primula denticulate Smith. in tablet dosage form is the topical task of pharmacy.

The aim of the work. To study the influence of excipients on the pharmacotechnological parameters of tablets with Primula denticulate Smith, are obtained by wet granulation method and to substantiate the choosing of the best excipients by the method of mathematical planning of the experiment.

Materials and Methods. It was used a self-prepared dense extract of Primula denticulate Smith. and excipients, which are complied with the requirements of State Pharmacopoeia of Ukraine in terms of pharmaco-technological quality parameters, for developing tablets. The hyper Greek-Latin square 4x4 was used to study the four qualitative factors.

Results and Discussion. In the course of the experiment, it was determined the dependence of pharmacotechnological parameters (friability, resistance to crushing and disintegration of tablets) on type of excipients. It was selected excipients with the optimal pharmaco-technological parameters for further research the tablets quality.

Conclusions.

According to the method of mathematical planning of the experiment, it’s chosen the optimal excipients for obtaining tablets based on dense extract of Primula denticulate Smith. Considering the investigated pharmaco-technological properties (friability, resistance to crushing and disintegration of tablets) the qualitative composition of excipients for obtaining tablets by the method of wet granulation was determined: Avicel PH - 105 and MCC 101, croscarmellose sodium, colloidal anhydrous silicon dioxide, magnesium stearate and Prosolv 90.

The substantiation of the choice of a gel former for developing the gel composition with hyaluronic acid and decametoxin

G. Melnyik — 2021-04-12

Topicality. Bedsores are an urgent problem of modern medicine both at the outpatient and inpatient stage. For the treatment of infected bedsores it is advisable to use drugs that have a complex effect focused on various parts of the pathological process. For the treatment of chronic wounds and bedsores together with the inhibition of the growth of microorganisms the drug must provide the optimal conditions for the growth of granulation, have a reparative and anti-inflammatory effect.

Aim. To experimentally substantiate the composition of the gel base with hyaluronic acid and decamethoxine.

Materials and methods. To select the structure-forming component in the gel base, the possibility of using “Ultrez 10 NF” carbomer, “Aristoflex AVC” (copolymer of acrylamidomethylpropanesulfonic acid and vinyl pyrrolidone) from Clariant Surfactants company, “Structure XL” (hydropropylated corn starch) as a gelling agent was studied. Organoleptic, physicochemical, structural and mechanical parameters of gel model samples were determined according to the methods of the State Pharmacopoeia of Ukraine. The statistical processing of the results was performed using the Statistica 6.0 program.

Results and discussion. The choice of the optimal gelling agent was performed taking into account the results of the study of organoleptic, physicochemical, structural and mechanical parameters of gel model samples. The experimental studies conducted allowed to choose the optimal gelling agent – “Aristoflex AVC” in the concentration of 1 %. It is easily dispersed with water and does not require the addition of a neutralizer. It has been proven that the base selected has the optimal consumer, structural-mechanical, and physicochemical properties.

Conclusions. Based on the organoleptic, physicochemical, structural-mechanical studies the composition of the gel base with hyaluronic acid and decamethoxine has been experimentally substantiated.