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دراسة التاثيرات الكيموحيوية لمستخلصات نبات الريحان على الارانب المصابه بسمية الكبد من خلال نظام الاكسده - مضادات الاكسده == A Study of Biochemical Effects of Ocimum sanctum L. Extracts on Hepatotoxicity in Rabbits Via Oxidant - Antioxidant System

Author name: سارة صالح مهدي التميمي
Supervisor name: لمياء عبد المجيد المشهدي
General topic: Chemistry
Specific topic: Analytical Biochemistry
Degree: Master
Language: English
University location: Babylon
Abstract: تضمن البحث دراسة المكونات الفعالة في المستخلصات المائية(الباردة والحارة) والكحولية (70%) لنبات الريحان الذي جمع من الاسواق المحلية في مدينة الحلة. من خلال التقدير النوعي والكمي لمستخلصات نبات الريحان حيث بينت نتائج التقدير النوعي ان المستخلص المائي الحار والكحولي يحتويان مكونات فعالة اكثر مثل الفلافونيدات ,القلويات, الصابونيات ,التانينات ,التربينات , الكلايكوسيدات والاحماض الامينية او الامينات الاولية والثانوية مقارنة بالمستخلص المائي البارد. | اتضح من التقدير الكمي للمستخلصات ان المستخلص المائي الحار والكحولي يحتويان مكونات فعالة مثل الفلافونيدات ,القلويات, الصابونيات والتانينات بنسب مختلفه مقارنة بالمستخلص البارد. و تضمنت الدراسه تقدير الفعالية البايولوجيه للمستخلصات المائيه والكحولية لنبات الريحان باستخدام اربعة انواع مختلفة من السلالات البكتيرية موجبة لصبغة كرام(Staphylococcus aures, Streptococcus pyogenes) وسالبة لصبغة كرام (Salmonella typhi and Klebsiella) باستخدام طريقة الانتشار في الاكار وبتراكيز مختلفة من المستخلصات ( 200,100 , 50)ملغم/مل وبينت النتائج ان المستخلص الكحولي يمتلك اكبر قطر للتثبيط ضد ثلاثة انواع بكتريا هي (Staphylococcus aures, Streptococcus Pyogenes and Klebsiella) عند التركيز 200ملغم/مل مقارنة مع المستخلصات المائية التي تعطي نتائج تثبيط اقل .من هذه النتائج يتضح ان الاستخلاص بالايثانول ( (70%اثبت فعاليته في عزل المكونات الفعالة (نوعيا وكميا) وان التركيز 200ملغم للمستخلص الكحولي هو افضل تركيز مقارنة بالتركيز(100 , 50) ملغم من نفس المستخلص كونه يعطي افضل قطر للتثبيط ضد مختلف انواع البكتريا لذلك اعتبرنا ان الجرعة الفعالة هي 200ملغم/كغم من وزن الجسم حيث استندنا على نتائج التقدير النوعي والكمي والفعالية البايولوجيه في الحصول على الجرعة المؤثرة والتي تم استخدامها بعد ذلك كجرعة مؤثره لعلاج الحيوانات المختبريه المصابة بسمية الكبد. | ولتحديد الزمن الفعال تم تقسيم (30)ارنب والتي تتراوح اوزانهم بين (900-1500)غم الى خمسة مجاميع حيث تحتوي كل مجموعه على ستة ارانب وهذه المجاميع هي : ( (G1 مجموعه السيطرة, ( (G2المجموعه المستحث فيها سمية الكبد بواسطة CCl4)), ( (G3 المجموعة المعالجه ,(G4)المجموعه الوقائية و (G5)هي المجموعة التي تعامل بالمستخلص الكحولي فقط (وتعتبر مجموعة سيطره ثانيه) . وبعدها تم اعطاء المستخلص | | الكحولي لحيوانات المجموعه الثالثة ,الرابعة والخامسة وتم قياس انزيمات الكبد مثل (AST, ALT, ALP , TP and Alb) في اوقات مختلفة لمعرفة الزمن الفعال الذي يؤدي الى اكبر تاثير في وظائف الكبد ,حيث اتضح ان الزمن اربع ساعات هو الزمن المؤثر والفعال والذي تم الاستناد عليه في التجارب اللاحقة. | ولدراسة تاثير المستخلص الكحولي العلاجي والوقائي لنبات الريحان ضد سمية الكبد المستحثة بواسطة رابع كلوريد الكاربون (CCl4) تم قياس انزيمات الكبد مثل AST, ALT, ALP, TP and Alb لهذه المجاميع وبينت النتائج ان مستويات انزيماتAST, ALT and ALP سوف تزداد بينما مستويات انزيمات ((TP and Alb سوف تنخفض بالنسبة للارانب المصابة بسمية الكبد بواسطة رابع كلوريد الكاربون لمدة اسبوع وان معالجة الارانب بالمستخلص الكحولي لنبات الريحان قللت من مستويات انزيمات الكبد لتصبح قريبة من القيم الطبيعية للمجموعة الاولى(مجموعة السيطره) وهذه النتائج توضح الطبيعة الوقائية لنبات الريحان ضد ماده رابع كلوريد الكاربون والتي تسبب تلف الكبد. | اضافه الى ذلك فان دراسة المقاطع النسيجية لكبد الارانب المختبريه تدعم النتائج التي تم الحصول عليها من خلال قياس انزيمات الكبد والتي تبين التاثير الفعال للمستخلص الكحولي لنبات الريحان وذلك يعود الى كون نبات الريحان هو احد النباتات الطبية التي تمتلك خواص مضادات الاكسده. | == The research included the phytochemical study of aqueous(cold and hot)and ethanolic extracts (70%) of Ocimum sanctum L. that were collected from local markets in Hilla. The investigation of phytochemical constituents involved the qualitative and quantitative analysis. The results for qualitative analysis explained that the aqueous hot and ethanolic extracts contained more amounts of active components such as flavonoids, saponins, tannins, alkaloids, terpenoids, glycosides and amino acids or primary and secondary amines rather than cold extract ,while the quantitative analysis illustrated that the aqueous hot and ethanolic extracts containing different percentages of flavonoids, saponins, tannins and alkaloids more than cold extract. | Antibacterial activity was also analyzed for the aqueous and ethanolic extracts of Ocimum sanctum L. with different concentrations (50,100,200) mg/ml using four different bacterial strains which are gram positive : Staphylococcus aures, Streptococcus pyogenes and gram negative : Salmonella typhi and Klebsiella using agar diffusion method and the results showed that ethanolic extract had the biggest zones of inhibition at the concentration 200mg against Staphylococcus aures, Streptococcus pyogenes and Klebsiella compared with aqueous extracts that gave less results of zones of inhibition. | From these results which illustrated that extraction with ethanol (70%) proved its effectiveness in the isolation of active components and the concentration 200mg was the best concentration for ethanolic extract which given the best results of inhibition zones against different types of bacteria. Therefore, the active dose was 200mg/kg body weight based on the results of qualitative, quantitative and antibacterial activity to get the active dose that was used in the treatment of experimental animals induced hepatotoxicity. | To determine the active time , thirty rabbits were divided into five groups for six rabbits in each group with body weight of (900-1500)gm and these groups were : (G1) is the control group that received normal saline, (G2) is the toxic control that received CCl4 for one week, (G3)is the treated group, (G4) is the preventive group and (G5)that treated with ethanolic extract only(it's considered as a second control group).Then ethanolic extract was given to the rabbits of (G3, G4 and G5) and the liver function enzymes like(AST, ALT, ALP, TP and Alb)were measured for these groups in different times to detect the active time which leads to the greater impact in liver function and it became clear that four hours is the active time that was used in the next experiments. | Then to study the hepatoprotective effect of ethanolic extract of Ocimum Sanctum L. on hepatotoxic rabbits that induced by CCl4 , the liver function enzymes such as AST ,ALT ,ALP ,TP and Albumin were measured for these groups and the results indicated that the levels of AST ,ALT and ALP were significantly increased while the levels of TP and Albumin were decreased in rabbits that received CCl4 for one week. Post treatment with Ocimum sanctum L. extract for two weeks made the levels of liver enzymes become nearer to the normal values of G1.These findings demonstrated the protective nature of Ocimum sanctum L. against CCl4 that induced liver damage. In addition, the histopathological studies of liver sections support the results that were obtained from serum liver enzymes which appeared the hepatoprotective effect of ethanolic extract and this was due to the antioxidant properties that have been found in Ocimum sanctum L. plant. |

فصل انزيم اللايبوكسيجينيز من مصل الرجال الاصحاء ومعاملة خارجية للانزيم في مرضى الربو == Separation of Lipoxygenase from Sera of Healthy men and in Vitro Treatment of Enzyme in Asthmatic Patients

Author name: احمد جياد عباس البرقعاوي
Supervisor name: عودة مزعل ياسر الزاملي
General topic: Chemistry
Specific topic: Analytical Biochemistry
Degree: Doctorate
Language: English
University location: Babylon
First pages:
Abstract: This study is concerned with asthma. Sixty seven asthmatic male patients were examined and tested , their age ranged from 10 - 65 years and they previously diagnosed as asthmatic patient under the physician supervision in AL - Hilla center of Allergy and Asthma . They have the disease for more than five years. Forty patients were examined during the period between July2015 to October 2015, and twenty seven patients were examined during period between June to August 2016, also the control group consisted of sixty seven healthy males ranged from 10 - 65 years.In this study asthmatic patients were divided into three groups according to medication , the first group consisted of (17) asthmatic patients taking antihistamines such as loratadine , citrizine and allermine , while the second group included (24) asthmatic patients taking montelukast , and the third group included (26) asthmatic patients who were on different drugs other than antihistamines and montelukast such as steroids and beta agonists .Five milliliters of blood were obtained from each patient, serum collected absolutely free from hemolysis stored at - 20 °C until uses for measurement of the concentrations of Glutathione, Malondialdehyde, Catalase, Histamine, Serotonin, Lipoxygenase - 5, Leukotriene A4 Hydrolase, total Immunoglobulin E and specific immunoglobulin E.The results revealed that the mean of IgE level (measured by ELISA system)in serum of asthmatic group was (368.16 ±1.772 ng/ml) versus the control group (138.9±2.515 ng/ml) with a significant difference,)P < 0.001(. Asthmatic patients and patients treated with pharmacological drugs such as montelukast and antihistamines showed high sensitivity to some food, animals, plant, palm pollen, fungal and mite allergen and they showed different values between patients and control. The study showed that there is no association between the increased sensitization to food, animal, plant and fungal allergens with age of patients.The present study showed the relationship of count blood cells (WBC and RBC) in studied groups. In all studied groups, WBC values higher than control group while the RBC values showed little differences among all studied groups.According to levels of some variables (histamine, serotonin, LOX - 5 and LTA4H) in asthmatic patients, the results showed a higher mean value of serotonin in asthmatic patients (0.023± 0.004 ng /ml) compared with control group (0.002± 0.001 ng/ml, p˂0.001) In addition the value of histamine was observed high in asthmatic patients (8.511± 0.410 ng /ml)while the control group was (2.565 ± 0.140 ng/ ml) with a significant difference of the mean value of histamine between asthmatic patients and control group (p˂0.001). LOX - 5 level in serum of asthmatic patients was higher (11.080 ± 0. 190 ng/ml) than in control group (9.821 ± 0.203 ng/ml, p˂0.001.The data revealed significant high mean value of LTA4H in asthmatic patients (24.148 ± 0 .450 ng/ml) compared with control group (18.912 ± 0.459 ng/ml, p˂0.001).The result showed that histamine level was increased in the group (>40 years) with a mean value (9.67 ± 4.25 ng /ml ) . While LOX - 5 showed high mean value (11.532 ± 1.274) in the group 20 - 40 years . Also LTA4H enzyme showed high mean value (25.54 ± 3.504 ng/ml)in the group 20 - 40 years , while serotonin appeared with high mean value (0.027± 0.015 ng/ml) in the group (< 20 years) with a significant difference between levels of these variables in each age group (p ˂ 0.001)The mean concentration of an antioxidant, Glutathione(GSH), and Malondialdehyde (MDA) in serum of asthmatic patient and control groups. The mean concentration of MDA increased in asthmatic patients (11.317 ± 1.096 μmol/l) more than control group(8.733 ± 3.756 μmol/l)with a significant difference between these groups (p < 0.05). The mean concentration of GSH increased was in the control (5.106 ± 3.197 μmol/l) more than asthmatic patients(3.905 ± 1.638 μmol/l, p < 0.05).According to the results of the present study, the mean activity of catalase(CAT) was higher (1.555±0.982 U/l) in asthmatic patients when compared with control group(1.173 ± 0.502 U/l, p < 0.05).The current study also concerned with the extrection of lipoxygenase from serum of asthmatic patient and healthy men by taking 15 ml serum from ten asthmatic patients men and 15 ml from serum of ten healthy men. And these quantities of serum were precipitated by ammonium sulfate, The best ratio for precipitating serum and extraction of enzyme is 60%. The anion exchange chromatography was used for extraction of the enzyme. The molecular weight of the extracted enzyme was determined by SDS - PAGE . In this procedure one subunit of the extracted LOXs enzyme showed a molecular weight of 35KD for one chain while a recombinant pure human LOX - 5 showed a molecular weight of 38 KD. The current study used NATIVEN technique for purification of the extracted enzyme obtained from ion exchange chromatography.The results revealed the optimum pH and temperature for LOXs activity and they were 8 and 40°C, respectively.The velocity constant and maximum velocity of the extracted Lipoxygenase enzyme was detected by using PHYWE SYSTEME GMBH SYSTEM, this system had the ability to obtain the relationship between conductivity and concentration (an increase in concentration lead to an increase in conductivity).The extracted lipoxygenase enzyme Km was 2.2mM and Vmax= 140mM/min in asthmatic patients and Km was 2.7mM and Vmax= 135mM/min in controls (by applying Michaelis - Menten equation).While the enzyme Km= 5.5mM and Vmax= 238mM/min in asthmatic patients and Km= 5 mM and Vmax= 208 mM/min in controls (by applying Lineweaver - Burk equation).The extracted LOXs from asthmatic patients and controls was treated with montelukast. The Km and Vmax appeared to have low values Km 3.6mM and Vmax 153mM/min and Km 3.7mM and Vmax 147mM/min, respectively. Also the extracted LOXs from asthmatic patients and controls was treated with molsiodomine, showed low values Km 4.4mM and Vmax 182 mM/min and Km 4.3mM and Vmax 166 mM/min, respectively.