A HPLC-MS/MS METHOD DEVELOPMENT AND VALIDATION FOR THE SIMULTANEOUS DETERMINATION OF BISOPROLOL AND ENALAPRIL IN THE PRESENT OF ENALAPRILAT IN HUMAN PLASMA

Objective: A highly specific, sensitive and rapid HPLC-MS/MS method has been developed and validated for the simultaneous quantification of bisoprolol and enalapril in the present of enalaprilat in human plasma.Methods: Analytes were extracted from plasma using a protein precipitation extraction method. Chromatography was achieved on Discovery C18, 50 × 2.1 mm, 5 μm column. Samples were chromatographed in a gradient mode (eluent A (acetonitrile-water–formic acid, 5: 95: 0.1 v/v), eluent B (acetonitrile–formic acid, 100: 0.1 v/v)). The initial content of the eluent B is 0%, which increases linearly by 1.0 min to 100% and to 1.01 min returns to the initial 0%. The mobile phase was delivered at a flow rate of 0.400 ml/min into the mass spectrometer ESI chamber. The sample volume was 5 μl.Results: The total chromatographic run time was 2.0 min and the elution of bisoprolol, enalapril, enalaprilat and IS (verapamil) occurred at ~1.01, 1.03, 0.96 and 1.09 min, respectively. A linear response function was established at 0.5-50 ng/ml for bisoprolol fumarate, 2-200 ng/ml for enalapril maleate, 1-100 ng/ml for enalaprilat dehydrate in human plasma. The intraday and interday accuracy and precisions were in the range of 0.311 %-0.647 % and 0.364 %-0.572 % for bisoprolol, 0.321 %-0.747 % and 0.390 %-0.673 % for enalapril, 0.221 %-0.547 % and 0.264 %-0.773 % for enalaprilat, respectively.Conclusion: A new rapid method was developed for simultaneous determination of bisoprolol and enalapril in the present of enalaprilat in human plasma. The method was strictly validated according to the ICH guidelines. The information thus obtained from the study can be used for the full pharmacokinetic profiling in individuals.Â

Immunomodulatory effect of melatonin supplementation in experimental diabetes

Aim: To investigate the effect of melatonin on the immunomodulatory response in experimental type 1 and 2 diabetes mellitus. Methods: Experiments were performed on male rats (180–200 g), purchased from the Experimental Animal Holding,. Animals were maintained in standard diet conditions. Two pathological states were simulated on male rats: experimental type 1 and type 2 diabetes. Melatonin was introduced from 14 to 23 days of experiment intraperitoneally. Levels of immunoglobulin classes A, M and G (Ig A, M, G), circulating immune complexes (CIC), interleukin 1β (EE), interleukin 6 (IL-6), and tumor necrosis factor (TNF-a) were measured. Results: We demonstrated that melatonin in case of immune hyperactivity, can, provide a suppressive effect and is able to enhance immune reactivity under conditions of its limitation, indicating the immunostimulating activity. Furthermore, we found that administration of melatonin decreased inflammatory responses by mediating the levels of immunomodulatory factors, including TNF-α, IL-1β and IL-6. Conclusion: Melatonin is a positive regulator of immune system, may be a potential therapeutic agent, it has no reported side effects

Вивчення амінокислотного складу Epilobium angustifolium L. методом ВЕРХ

The use of plant raw materials is one of the areas of modern pharmaceutical science in the production of herbal drugs. The genus Epilobium counts more than 200 species, many species of which are used in traditional medicine. Among the Epilobium species, Epilobium angustifolium is one of the well-known medicinal plants which have been used worldwide in habitual medicine. There is insufficient information in the literature on the biologically active substances of Epilobium angustifolium L. The presence of three major polyphenol groups: phenolic acids, flavonoids, and ellagitannins were identified in E. angustifolium extracts. Traditionally, the infusion of leaves of this plant could be useful for headaches, cold and gastrointestinal disorder. The Epilobium angustifolium L. as an insufficiently studied plant is a promising object of study, including amino acids composition. To assess the relationship between the production of primary metabolites and their possible therapeutic properties, we analyzed the amino acid profile of the plant Epilobium angustifolium used in traditional medicine. The study of compounds generated by plants as a result of defense mechanisms permits an understanding of the molecular mechanism involved in their medicinal properties. The aim. Thus, the aim of the study was to conduct an HPLC analysis of the amino acids of E. angustifolium to establish the prospects for the use of the raw materials in medical and pharmaceutical practice. The results of the current study will be used in further breeding programs aimed to obtain an industrial form of E. angustifolium suitable for pharmaceutical and food applications. Materials and methods. The determination of amino acids composition of Epilobium angustifolium was conducted using Agilent 1200 (Agilent Technologies, USA). Results. The HPLC method identified sixteen free amino acids and seventeen bound amino acids in the Epilobium angustifolium herb. The studies have shown that Epilobium angustifolium L. herb is mainly composed of free amino acids such as L-phenylalanine (1.65 µg/mg), L-glutamic acid (1.51 µg/mg), L-arginine (1.24 µg/mg), L-alanine (0.98 µg/mg) and L-aspartic acid (0.57 µg/mg), which were presents in the greatest amount. The dominant bound amino acids in the studied raw material were L-glutamic acid, L-aspartic acid, L-leucine, and L-alanine, the content of which was 32.37 µg/mg, 10.59 µg/mg, 8.70 µg/mg, and 6.22 µg/mg respectively. Conclusions. Using the HPLC method determined the amino acids in the herb of Epilobium angustifolium L. The concentrations of L-aspartic acid, L-glutamic acid, L-arginine, L-alanine and L-phenylalanine are predominate among free and bound amino acids in the Epilobium angustifolium L. herb. The result shows that Epilobium angustifolium L. is the source of amino acids, so the use of this plant raw material for new remedies is possible in the futureИспользование растительного сырья является одним из направлений современной фармацевтической науки в производстве препаратов на растительной основе. Род Epilobium насчитывает более 200 видов, многие виды которых используются в традиционной медицине. Среди видов Epilobium, одной из известных лекарственных растений, которые используются во всем мире в традиционной медицине есть Epilobium angustifolium. В литературе недостаточно информации о биологически активных веществах Epilobium angustifolium L. Показано наличие трех основных полифенольных групп: фенольных кислот, флавоноидов и еллаготанинив в экстрактах E. angustifolium. Традиционно настой листьев этого растения может быть полезным при головных болях, простуде и желудочно-кишечных расстройствах. Epilobium angustifolium L. как недостаточно изученое растительное сырье является перспективным объектом исследования, включая исследования аминокислотного состава. Для оценки связи между продукцией первичных метаболитов и возможными терапевтическими свойствами мы проанализировали аминокислотный профиль растения Epilobium angustifolium, используемого в традиционной медицине. Изучение соединений, образуемых растениями в результате защитных механизмов позволяет понять молекулярные механизмы, участвующие в их лечебных свойствах. Цель. Таким образом, целью исследования было проведение ВЭЖХ-анализа аминокислотного содержания E. angustifolium для установления перспектив использования сырья в медицинской и фармацевтической практике. Результаты текущего исследования будут использованы в последующих селекционных программах, направленных на получение промышленной формы E. angustifolium, пригодной для фармацевтического и пищевого применения. Материалы и методы. Определение аминокислотного состава Epilobium angustifolium проводили с помощью Agilent 1200 (Agilent Technologies, США). Результаты. Методом ВЭЖХ в траве Epilobium angustifolium было обнаружено шестнадцать свободных и семнадцать связанных аминокислот. Исследования показали, что трава Epilobium angustifolium L. в основном состоит из свободных аминокислот, таких как L-фенилаланин (1,65 мкг / мг), L-глутаминовая кислота (1,51 мкг / мг), L-аргинин (1,24 мкг / мг), L-аланин (0,98 мкг / мг) и L-аспарагиновая кислота (0,57 мкг / мг), содержание которых представлено в наибольшем количестве. Доминирующими связанными аминокислотами в исследуемой сырье были L-глутаминовая кислота, L-аспарагиновая кислота, L-лейцин и L-аланин, содержание которых составил 32,37 мкг / мг, 10,59 мкг / мг, 8,70 мкг / мг и 6,22 мкг / мг, соответственно. Выводы. С помощью метода ВЭЖХ было определено аминокислотный состав травы Epilobium angustifolium L. Установлено, что среди свободных и связанных аминокислот в траве Epilobium angustifolium преобладают концентрации L-аспарагиновой кислоты, L-глутаминовой кислоты, L-аргинина, L-аланина и L- фенилаланина. Результат показывает, что трава Epilobium angustifolium L. может быть источником аминокислот, поэтому перспективным является использование этого растительного сырья для получения новых лекарственных средств в будущемВикористання рослинної сировини є одним із напрямків сучасної фармацевтичної науки у виробництві препаратів на рослинній основі. Рід Epilobium нараховує понад 200 видів, багато видів яких використовуються в традиційній медицині. Серед видів Epilobium, однією з відомих лікарських рослин, які використовуються у всьому світі у традиційній медицині є Epilobium angustifolium. У літературі недостатньо інформації про біологічно активні речовини Epilobium angustifolium L. Вказано на наявність трьох основних поліфенольних груп: фенольних кислот, флавоноїдів та еллаготанінів в екстрактах E. angustifolium. Традиційно настій листя цієї рослини може бути корисним при головних болях, застуді та шлунково-кишкових розладах. Epilobium angustifolium L. як недостатньо вивчена рослина є перспективним об'єктом для дослідження, включаючи дослідження амінокислотного складу. Для оцінки зв’язку між продукцією первинних метаболітів та їх можливими терапевтичними властивостями ми проаналізували амінокислотний профіль рослини Epilobium angustifolium, що використовується в традиційній медицині. Вивчення сполук, що утворюються рослинами в результаті захисних механізмів, дозволяє зрозуміти молекулярні механізми, які беруть участь у їхніх лікувальних властивостях. Мета. Таким чином, метою дослідження було проведення ВЕРХ-аналізу амінокислотного вмісту E. angustifolium для встановлення перспективи використання сировини у медичній та фармацевтичній практиці. Результати поточного дослідження будуть використані в подальших селекційних програмах, спрямованих на отримання промислової форми E. angustifolium, придатної для фармацевтичного та харчового застосування. Матеріали і методи. Визначення амінокислотного складу Epilobium angustifolium проводили за допомогою Agilent 1200 (Agilent Technologies, США). Результати. Методом ВЕРХ у траві Epilobium angustifolium було виявлено шістнадцять вільних та сімнадцять зв’язаних амінокислот. Дослідження показали, що трава Epilobium angustifolium L. в основному складається з вільних амінокислот, таких як L-фенілаланін (1,65 мкг / мг), L-глутамінова кислота (1,51 мкг / мг), L-аргінін (1,24 мкг / мг), L-аланін (0,98 мкг / мг) та L-аспарагінова кислота (0,57 мкг / мг), вміст яких представлено в найбільшій кількості. Домінуючими зв’язаними амінокислотами у досліджуваній сировині були L-глутамінова кислота, L-аспарагінова кислота, L-лейцин та L-аланін, вміст яких становив 32,37 мкг / мг, 10,59 мкг / мг, 8,70 мкг / мг та 6,22 мкг / мг, відповідно. Висновки. За допомогою методу ВЕРХ було визначено амінокислотний склад трави Epilobium angustifolium L. Встановлено, що серед вільних та зв’язаних амінокислот у траві Epilobium angustifolium переважають концентрації L-аспарагінової кислоти, L-глутамінової кислоти, L-аргініну, L-аланіну та L-фенілаланіну. Результат показує, що трава Epilobium angustifolium L. може бути джерелом амінокислот, тому перспективним є використання цієї рослинної сировини для одержання нових лікарських засобів у майбутньом

Concepts for a New Rapid and Simple HPLC Method for Simultaneous Determination of Metoprolol and Meldonium in Pharmaceutical Dosage Forms

Simultaneous determination of the tandem of drugs, like meldonium and metoprolol, with enormous polarity differences between them, requires thorough research and careful selection of chromatographic conditions. The three different CN-cyano groups with link-based particle columns, LiChrospher CN, Waters Spherisorb CNRP, Zorbax CN SB stationary phases, were tested, in an isocratic elution system, with a running mobile phase containing various concepts of composition contents. They were first with buffering salts which included acetonitrile and ammonium phosphate in one group, and then without buffering salts but with diluted acids, composed of acetonitrile and diluted acids as the second group. We can conclude that the most optimal concepts, in terms of expressiveness and environmental friendliness, were concepts using of column Zorbax CN SB (4.6 mm i.d. × 250 mm, 5 μm) and mobile phase ACN—0.15% NH4H2PO4 (50:50 and 60:40, v/v). There are very poor available data about ideas and usable information about the development of methods for simultaneous determination of these two active substances with polarity differences between them. We suggest that our work offered detailed and successful solutions for the mentioned aim using less sophisticated equipment for quality control and a lab for routine manufacturing control

Epilobium angustifolium L.: A medicinal plant with therapeutic properties

Abstract Epilobium angustifolium L. is a medicinal plant belonging to the Onagraceae family, which includes more than 200 different species from all over the world. Traditional medicinal applications include treatment of prostate, gastrointestinal, menstrual disorders and recently it has been used for its analgesic and anti-inflammatory activity. In this investigation E. angustifolium was collected in Ternopil region of Ukraine. The obtained data demonstrated that E. angustifolium herb extract, rich in polyphenolic compounds such as flavonoids and tannins, display high antioxidant properties. In addition the potential anticancer activity has been investigated in vitro on human hepatocellular carcinoma cells (HepG2). Furthermore the cytotoxic and genotoxic effects of E. angustifolium have been investigated respectively by MTT and Comet assay. Results showed that at low concentration, up to 25 μg/mL, the cytotoxic effect was not observed. Increasing concentration from 50 to 75 μg/mL reduced significantly cell viability and induced an important DNA damage in hepatocellular carcinoma. These promising data were also confirmed with mitochondrial potential test. It is possible to conclude that E. angustifolium has beneficial properties in low concentration, in term of antioxidant activity, and it could be a potential antitumoral natural product if it will be used at high concentratio

Natural Ingredients to Improve Immunity

The immune system protects the body from infectious agents such as bacteria, viruses, or fungi. Once encountered with pathogens or antigens, the innate and adaptive arms of the immune system trigger a strong immune response to eliminate them from the system and protect the body. Thus, well-balanced immunity is pivotal for maintaining human health, as an insufficient level of immune defense leads to infections and tumors. In contrast, the excessive functioning of the immune system causes the development of autoimmune diseases and allergies. Strong immunity requires adequate nutrition, dietary interventions, and sufficient intake of certain vitamins (vitamin C, vitamin D, and folic acid) and minerals (magnesium, zinc, and selenium). Therefore, nutritional and micronutrient deficiencies lead to compromised immunity. Several natural ingredients have shown potent immunomodulatory properties. The immune-enhancing properties of many plants and fungi are due to containing bioactive phytoconstituents such as polyphenols, terpenoids, β-glucans, vitamins, etc. Probiotics and prebiotics can be used as innovative tools to reduce intestinal inflammation and downregulate hypersensitivity reactions. Plant sources of melatonin, a multifunctional molecule with proven anti-inflammatory and immunomodulatory properties, have been discovered relatively recently. The bioactive compounds augment the immune response by directly increasing the cytotoxic activity of natural killer cells, macrophages, and neutrophils. Many phytoconstituents prevent cell damage due to their powerful antimicrobial, antioxidant, and anti-inflammatory properties. The present review attempts to understand the molecular mechanisms underlying the immune-enhancing properties of some bioactive compounds from plants, fungi, animals, microorganisms, and other natural sources

Metformin Attenuates Postinfarction Myocardial Fibrosis and Inflammation in Mice

International audienceDiabetes is a major risk factor for the development of cardiovascular disease with a higher incidence of myocardial infarction. This study explores the role of metformin, a first-line antihyperglycemic agent, in postinfarction fibrotic and inflammatory remodeling in mice. Three-month-old C57BI/6J mice were submitted to 30 min cardiac ischemia followed by reperfusion for 14 days. Intraperitoneal treatment with metformin (5 mg/kg) was initiated 15 min after the onset of reperfusion and maintained for 14 days. Real-time PCR was used to determine the levels of COL3A1, αSMA, CD68, TNF-α and IL-6. Increased collagen deposition and infiltration of macrophages in heart tissues are associated with upregulation of the inflammation-associated genes in mice after 14 days of reperfusion. Metformin treatment markedly reduced postinfarction fibrotic remodeling and CD68-positive cell population in mice. Moreover, metformin resulted in reduced expression of COL3A1, αSMA and CD68 after 14 days of reperfusion. Taken together, these results open new perspectives for the use of metformin as a drug that counteracts adverse myocardial fibroticand inflammatory remodeling after MI

Nitric oxide-mediated effects of L-ornithine-L-aspartate in acute toxic liver injury

This study was aimed to investigate nitric oxide-dependent mechanisms of L-ornithine-L-aspartate (LOLA) action in acute toxic liver injury in rats. Acute hepatitis was induced in Wistar rats using 50% oil solution of tetrachloromethane (CCl4) intragastrically (2 g/kg) twice in a 24 hour interval. Intraperitoneal treatment with LOLA (200 mg/kg) was started 6 hours after the second CCl4 administration and maintained for 3 consecutive days. L-Nω-Nitroarginine Methyl Ester (L-NAME) was used intraperitoneally (10 mg/kg). In CCl4-induced hepatitis, LOLA restores the structure of hepatocytes and prevents aminotransferases, alkaline phosphatase and gamma-glutamyl transferase elevation. It decreases total bilirubin concentration but does not affect increased cholesterol level. LOLA augments urea concentration, total protein level in blood and liver as well as serum and liver content of nitrite anions. LOLA enhances activity of catalase, glutathione S-transferase, manganese superoxide dismutase, increases reduced glutathione level and total antioxidant capacity and decreases thiobarbituric acid reactive substances level. The concomitant use of L-NAME inhibits the action of LOLA to enhance nitrite anions synthesis both in serum and liver, to delay the recovery of hepatocytes, to counteract LOLA effect against blood total protein reduction, to prevent the decline in aminotransferases, alkaline phosphatase,, gamma-glutamyl transferase and glutathione S-transferase activity and to reduce catalase activity and reduced glutathione level. Therefore, in CCl4-induced hepatitis, LOLA effectively prevents cytolysis and cholestasis, improves liver metabolism and protects against oxidative stress. Partially, these changes occur in nitric oxide-mediated mechanism since the use of L-NAME declines most of LOLA effects

Nitric oxide-mediated effects of L-ornithine-L-aspartate in acute toxic liver injury

This study was aimed to investigate nitric oxide-dependent mechanisms of L-ornithine-L-aspartate (LOLA) action in acute toxic liver injury in rats. Acute hepatitis was induced in Wistar rats using 50% oil solution of tetrachloromethane (CCl4) intragastrically (2 g/kg) twice in a 24 hour interval. Intraperitoneal treatment with LOLA (200 mg/kg) was started 6 hours after the second CCl4 administration and maintained for 3 consecutive days. L-Nω-Nitroarginine Methyl Ester (L-NAME) was used intraperitoneally (10 mg/kg). In CCl4-induced hepatitis, LOLA restores the structure of hepatocytes and prevents aminotransferases, alkaline phosphatase and gamma-glutamyl transferase elevation. It decreases total bilirubin concentration but does not affect increased cholesterol level. LOLA augments urea concentration, total protein level in blood and liver as well as serum and liver content of nitrite anions. LOLA enhances activity of catalase, glutathione S-transferase, manganese superoxide dismutase, increases reduced glutathione level and total antioxidant capacity and decreases thiobarbituric acid reactive substances level. The concomitant use of L-NAME inhibits the action of LOLA to enhance nitrite anions synthesis both in serum and liver, to delay the recovery of hepatocytes, to counteract LOLA effect against blood total protein reduction, to prevent the decline in aminotransferases, alkaline phosphatase,, gamma-glutamyl transferase and glutathione S-transferase activity and to reduce catalase activity and reduced glutathione level. Therefore, in CCl4-induced hepatitis, LOLA effectively prevents cytolysis and cholestasis, improves liver metabolism and protects against oxidative stress. Partially, these changes occur in nitric oxide-mediated mechanism since the use of L-NAME declines most of LOLA effects