استخلاص وتنقية وتوصيف عقار التاكسول من عزلة محلية للفطر Schizophyllum radiatum وتقييم فعالياته البايولوجية والمضادة لسرطان المبايض

الباحث

حنين صالح سالم كردي

المشرف

شمال يونس عبد الهادي

الجامعة

جامعة الموصل

الكلية / القسم

كلية التربية للعلوم الصرفة / قسم علوم الحياة

الشهادة

ماجستير

السنة

2023

لغة الرسالة

عربي

الكلمات المفتاحية

التاكسول ; الفطر Schizophyllum ; سرطان المبايض ; الفعالية البايولوجية

A Due to the environment’s richness in medical plants and mushrooms with great biodiversity, that produce various anti-cancer metabolite active compounds, such as Taxol, this study was conducted and filled with thorough details. Several scientific trips were done, including Sulaymaniyah and Mosul cities, and finding 65 samples, 40 species of the medical plants that represent endophytes for many fungal types, in addition to 25 species of fruiting bodies that belong to ascomycetes. The results of the initial molecular screening of the obtained fungi indicated that six isolates were obtained containing the gene responsible for the Taxol production. Two of which are endophytes fungal isolates, with scientific symbols HP-15, HP-37 and four isolates belong to macrofungi, with scientific symbols, HM-43, HM-55, HM-57, HM-58 respectively. In order to make the initial result more reliable, unbiased secondary screening was carried out using six different cultured medium, it was clear that the isolate, with scientific symbol HM-58, was the most productive in Taxol, with range of 0.85 gm/lit. when grown in the cultured medium MID, and based on this result, it was chosen to complete the research requirements. Polymerase Chain Reaction (PCR) and by using Internal Transcribed Spacer (ITS), led to identifying the fungal isolate to a type level, and it was proved that it belongs to Schizophyllum radiatum, which was registered in the US genbank for the first time in Iraq, with serial number OQ709247. The research outcomes of the cultured conditions synthesis when growing the selected fungal isolate in the cultures revealed its needs to 18 days of incubation to produce a larger amount of taxol (0.96) gm/lit, and the sucrose of concentration 3% as a carbonic source to get 1.04 gm/lit of B taxol. The yeast extract in concentration of 1 gm/lit to produce 1.23/gm/lit. Whereas the ideal temperature to produce taxol reached 28 C, with amount of 0.98 gm/lit and at a pH 6.5 and speed of 160/ circle/min. The idea of enriching the growth medium with Vitamin B12 at concentration 100 microgram/ lit. increased the amount of the produced Taxol which reached to 1.38 gm/lit also adding Salicylic acid has become a milestone for the growth medium in which the production amount of taxol has increased to 1.48 gm/lit . In order to complete what we have started, the extracted taxol was purified from the fungal culture filtrate with a chromatographic separation column. The taxol was recovered with a mixture of chloroform and acetonitrile with gradual concentration. The recovery process revealed 10 parts, part Fraction 6 has activity when detected by the spectrophotometer, which reached 1.42 gm/lit . The step of detecting the purity of the purified taxol using high performance liquid chromatography, by obtaining one peak of taxol at holding time of 19.16 min. which is similar to the time of the standard taxol 19.22 min . The process of transferring the purified taxol on a silica gel plate, after the appearance of one unique spot, with value Rf, reaching 0.56, which is the same Rf value for the standard taxol . As we are facing the spread of plants diseases, the results of evaluating the effectiveness of purified taxol against Oomycetes, especially Pythium ultimum, which caused damping off disease for a big number of economically important plants, made an incomparable successful. In which taxol was able to inhibit the mushroom growth, the inhibition aura reached 65 mm compared with the commercial taxol in which the diameter of the diameter aura reached 46 mm. The results proved that taxol has anti-tumor activity on the potato, and inhibition of bacterial activity, Agrobacterium tumefaciens, which is responsible for tumor formation after 21 after 21 of treatment. The purified taxol overcame the problem of antibiotic resistance of pathogenic bacteria by inhibiting their growth with both used concentration 50, 100 microgram/ml, and was the most effective on Staphylococcus aureus bacteria, in which the inhibition diameter reached 48.2 mm. at concentration of 100 microgram/ml compared with the commercial counter Gentamycin, its diameter inhibition aura reached 11 mm. The purified taxol proved to have anti-free radical’s activity after 30 minutes of treatment with taxol, which increased as the concentration increased, reaching the highest value 78% at concentration 100 microgram/ ml, with significant difference with the standard ascorbic acid of 66% . The experiment of revealing cytotoxicity of taxol on the ovarian cancer cell lines SKOV-3, highest inhibition rate 82.67% and 15% on the natural cell lines MCT-10 at concentration 200 microgram/ ml. While the inhibitory half lethal concentration IC50, and reached 24 microgram/ ml for the ovarian cancer cell lines in which its value is 73377.06 microgram/ml on the natural cell lines . It was resulted from the treatment of ovarian cancer cells with half lethal inhibitory concentration after it was dyed with crystal violet dye, for 24 hours, various apparent changes, in which the natural cells did not undergo any apparent changes. In order to detect the occurrence of programmed cell death, a double dye, Acridine Orange, was utilized. After treating cells with taxol, dyed for 24 hours, examined using inverted fluorescent microscope, the treated cells were dyed with red color, and spaces that are free of cells, this indicates the cells death, while the untreated cells were green . D In the flow cytometry test for mitochondrial proteins through the membrane and treating the cancer cells with RH123 dye, one peak of ovarian cancer cells shifted to the left compared with untreated cells that have appeared with one peak and shifted to the right . The screening of the flow cytometry for proteins P53, Caspase 8, and they were two of the most important indicators of programmed cell death, the production of active oxygen molecule ROS in the ovarian cancer cells treated with taxol, and produce Death inducing signaling complex (DISC), and the appearance of one peak and shifted to the left. The final findings have come to reveal the comet assay technique, with concrete evidence of the damage that the taxol has made on the genetic material of treated ovarian cancer cells,The genetic material showed an increase in the tail length, which is 51.93 micro/lit., whereas the genetic material appeared in a form of centered aura around the cell nucleus, and the tail length was 2.3 ml . --- page 180 [OCR+] --- Extraction, Purification, and Characterization of Taxol Drug from the Local Isolate Mushroom Schizophyllum radiatum and Evaluating Its Biological Activites and Anti-Ovarian Cancer Activity A Thesis Submitted By Haneen Salih Salim Al-kurdy To The Council of College of Education for Pure Science University of Mosul In Partial Fulfillment of Requirements for the Degree of M. Sc. In Biology Supervised by Prof. Dr. Shimal Younis Abdul-Hadi 2123 A.D 1445 A.H [OCR of page] Extraction, Purification, and Characterization of Taxol Drug from the Local Isolate Mushroom Schizophyllum radiatum and Evaluating Its Biological Activites and Anti-Ovarian Cancer Activity A Thesis Submitted By Haneen Salih Salim Al-kurdy To The Council of College of Education for Pure Science University of Mosul In Partial Fulfillment of Requirements for the Degree of M. Sc. In Biology Supervised by Prof. Dr. Shimal Younis Abdul-Hadi 2023 A.D 1445 A.H --- page 181 [OCR+] --- University of Mosul College of Education for Pure Science Extraction, Purification, and Characterization of Taxol Drug from the Local Isolate Mushroom Schizophyllum radiatum and Evaluating Its Biological Activites and Anti-Ovarian Cancer Activity Haneen Salih Salim Al-kurdy M. Sc. Thesis Biology Supervised by Prof. Dr. Shimal Younis Abdul-Hadi 2123 A.D 1445 A.H [OCR of page] University of Mosul 5 College of Education 1 7 for Pure Science ده Extraction, Purification, and Characterization of Taxol Drug from the Local Isolate Mushroom Schizophyllum radiatum and Evaluating Its Biological Activites and Anti-Ovarian Cancer Activity Haneen Salih Salim Al-kurdy M. Sc. Thesis Biology Supervised by Prof. Dr. Shimal Younis Abdul-Hadi 2023 A.D 1445 A.H

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