Method for assessing the athero-protective activity of biologically active substances

• Main Authors: ŞVEŢ Inna, PANTEA Valeriana, POPA Veaceslav, ANDRONACHE Lilia, TAGADIUC Olga, GUDUMAC Valentin
• Format: Patent
• Language: English; Romanian; Russian
• Published: 31.03.2020
• Subjects: BEER; BIOCHEMISTRY; CHEMISTRY; COMPOSITIONS OR TEST PAPERS THEREFOR; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL ORENZYMOLOGICAL PROCESSES; ENZYMOLOGY; INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIRCHEMICAL OR PHYSICAL PROPERTIES; MEASURING; MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEICACIDS OR MICROORGANISMS; METALLURGY; MICROBIOLOGY; MUTATION OR GENETIC ENGINEERING; PHYSICS; PROCESSES OF PREPARING SUCH COMPOSITIONS; SPIRITS; TESTING; VINEGAR; WINE

The invention relates to medicine and biochemistry and can be used for assessing the athero-protective activity of biologically active substances.Summary of the invention consists in that biologically active substances in various concentrations are mixed with a solution comprising 20.8...41.6 IU/l of PON1/arylesterase in 0.05 M phosphate buffer solution with the pH 7.4 (final concentration 10.4...20.8 IU/l), then incubated at 37°C for 5...10 min, after which is added a reaction medium comprising 1.5...2.5 mM of p-nitrophenyl acetate (final concentration 1.37...2.28 mM/l), 1.0...2.0 mM/l of CaCl2 (final concentration 0.91...1.82 mM/l) and 10.0...20.0 µM/l of chloramine T (final concentration 9.1...18.3 µM/l) in 0.05 M phosphate buffer solution with the pH 7.4 to obtain the test sample, the control sample is prepared identically as the test sample, but the test substance is replaced with an equivalent amount of 0.05 M buffer phosphate solution with the pH 7.4, and the blank sample is prepared identically as the control sample, but the reaction medium does not contain the PON1/arylesterase enzyme, then is determined the initial absorption of A1 at 405...410 nm, after which the samples are incubated at 37°C, for 30 min and is re-determined the absorption of A2 at 405...410 nm, then is calculated the percentage of activation of PON1/arylesterase of the test substances, at the same time, the higher the percentage of activation of the corresponding concentration of the test substances the higher the athero-protective activity. Invenţia se referă la medicină şi biochimie şi poate fi folosită pentru aprecierea activităţii atero-protective a substanţelor biologic active.Esenţa invenţiei constă în aceea că substanţele biologic active în diferite concentraţii se amestecă cu o soluţie ce conţine 20,8...41,6 UI/l de PON1/arilesterază în 0,05 M soluţie tampon fosfat cu pH-ul 7,4 (concentraţia finală 10,4...20,8 UI/l), apoi se incubează la temperatura de 37°C, timp de 5...10 min, după care se adaugă un mediu de reacţie ce conţine 1,5...2,5 mM de p-nitrofenil acetat (concentraţia finală 1,37...2,28 mM/l), 1,0...2,0 mM/l de CaCl2 (concentraţia finală 0,91...1,82 mM/l) şi 10,0...20,0 µM/l de cloramină T (concentraţia finală 9,1...18,3 µM/l) în 0,05 M soluţie tampon fosfat cu pH-ul 7,4 cu obţinerea probei de cercetat, proba de control se pregăteşte identic ca proba de cercetat, dar substanţa de testat se înlocuieşte cu o cantitate echivalentă de soluţie de 0,05 M tampon fosfat cu pH-ul 7,4, iar proba blanc se pregăteşte identic ca proba de control, dar mediul de reacţie nu conţine enzima PON1/arilesterază, apoi se determină absorbanţa iniţială A1 la 405...410 nm, după care probele se incubează la temperatura de 37°C, timp de 30 min şi se măsoară repetat absorbanţa A2 la 405...410 nm, apoi se calculează procentul de activare a PON 1/arilesterazei substanţelor cercetate, totodată, cu cât este mai mare procentul de activare a concentraţiei corespunzătoare a substanţelor cercetate, cu atât activitatea atero-protectivă este mai mare. Изобретение относится к медицине и биохимии, и может быть использовано для оценки атеро-протекторной активности биологически активных веществ.Сущность изобретения состоит в том, что биологически активные вещества в различных концентрациях смешивают с раствором, содержащим 20,8...41,6 МЕ/л PON1/арилэстеразы в 0,05 М раствора фосфатного буфера с pH 7,4 (конечная концентрация 10,4...20,8 МЕ/л), затем инкубируют при температуре 37°С, в течение 5...10 мин, после чего добавляют реакционную среду, содержащую 1,5...2,5 мМ п-нитрофенил ацетата (конечная концентрация 1,37...2,28 мМ/л), 1,0...2,0 мМ/л CaCl2 (конечная концентрация 0,91...1,82 мМ/л) и 10,0...20,0 мкМ/л хлорамина Т (конечная концентрация 9,1...18,3 мкМ/л) в 0,05 М раствора фосфатного буфера с рН 7,4 для получения исследуемой пробы, контрольную пробу готовят идентично как исследуемая проба, но испытуемое вещество заменяют эквивалентным количеством 0,05 М раствора буферного фосфата с pH 7,4, a пробу бланк готовят идентично как контрольная проба, но реакционная среда не содержит фермента PON1/арилэстеразы, затем определяют начальную абсорбцию A1 при 405...410 нм, после чего пробы инкубируют при температуре 37°С, в течение 30 мин и повторно определяют абсорбцию A2 при 405...410 нм, затем рассчитывают процент активации PON 1/арилэстеразы исследуемых веществ, причем чем выше процент активации соответствующей концентрации исследуемых веществ, тем выше атеро-протекторная активность.