Muğla ili çevresinde yetişen narcissus tazetta L. subsp. tazetta L. bitkisi üzerinde farmakognozik çalışmalar
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Tarih
2018
Yazarlar
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Cilt Başlığı
Yayıncı
Ege Üniversitesi, Sağlık Bilimleri Enstitüsü
Erişim Hakkı
info:eu-repo/semantics/openAccess
Özet
Amaryllidaceae alkaloitleri biyoaktivitesi yönünden kapsamlı olarak araştırılan ve çeşitli hastalıkların tedavisinde kullanım potansiyeli olan sekonder metabolit grubudur. Amaryllidaceae familyasına ait olan Narcissus cinsi, doğal ilaç kaynağı olarak değerlendirilip, pek çok tür ve alt türü alkaloit profili açısından araştırılmaktadır. Bu tez çalışmasında ülkemizde Muğla ve çevresinde doğal olarak yetişen Narcissus tazetta subsp. tazetta L. alt türü üzerinde herhangi bir çalışma yapılmamış olması nedeniyle çiçekli dönemde toplanmış toprak üstü kısımları ve soğanları üzerinde fitokimyasal çalışmalar gerçekleştirilmiştir. Drog örnekleri üzerinde, Avrupa Farmakopesi'ne göre kurutmada kayıp, bütün kül, sülfat külü, hidroklorik asitte çözünmeyen kül miktarı deneylerini içeren kalite kontrol çalışmaları yapılmıştır. Bu deneylerin sonuçları % değerler olarak verilmiştir. Ayrıca drog örnekleri üzerinde, total alkaloit miktar tayini titrimetrik bir yöntemle gerçekleştirilmiştir. Amaryllidaceae familyasının biyolojik aktivite açısından önemli olan alkaloitleri galantamin ve likorin, Narcissus tazetta subsp. tazetta bitkisinden hazırlanan toprak üstü ve soğan örneklerinde yüksek performanslı sıvı kromatografisi (HPLC) kullanılarak kalitatif ve kantitatif olarak araştırılmıştır. Ek olarak, yine bu iki farklı drog örneğinin alkaloit profilini belirlemek amacıyla kapiler gaz kromatografisi-kütle spektroskopisi (GC-MS) yöntemi kullanılmıştır. Bunun sonucunda, Narcissus tazetta subsp. tazetta bitkisinin 23 adet alkaloit içerdiği saptanmıştır. İzolasyon ve yapı aydınlatma çalışmalarında, oda sıcaklığında kurutulmuş ve toz edilmiş total bitki materyalinden hazırlanan total alkaloit ekstresinin kolon kromatografisiyle fraksiyonlandırılması sonucu elde edilen fraksiyonlar, preparatif sütun kromatografisi, preparatif ince tabaka kromatografisi ve kristallendirme gibi farklı yöntemler kullanılarak saflaştırılmıştır. Elde edilen bileşiklerin yapıları modern spektroskopik yöntemlerle (1D, 2D NMR, MS) aydınlatılmıştır. Bu çalışmaların sonucunda, Narcissus tazetta subsp. tazetta bitkisinden, Amaryllidaceae alkaloitlerinin likorin alt grubundan (-)-likorin, (-)-psödolikorin, (-)-9-O-metilpsödolikorin, (+)-11-hidroksigalantin, (-)-narsissidin; homolikorin alt grubundan (+)-9-O-demetil-2-α-hidroksihomolikorin; montanin alt grubundan (-)-pankratinin-C ve galantamin alt grubundan (-)-galantamin adlı bileşikler izole edilmiştir. İzole edilen bileşiklerden daha önce in vitro Ellman metodu ile biyoaktivite çalışması yapılmamış olanlarda bu yöntemle mikroplaka deneyi uygulanarak asetilkolinesteraz ve bütirilkolinesteraz inhibitör aktiviteleri araştırılmıştır. Aktif olan tüm bileşiklerin IC50 değerleri hesaplanmıştır. Ayrıca izole edilen bileşikler için moleküler doking çalışması yapılarak kolinesteraz enzimlerinin aktif yöreleri içerisindeki bağlanma yönelimleri incelenmiştir.
Amaryllidaceae alkaloids are a group of secondary metabolites which have therapeutic potential for the treatment of various diseases and have been deeply searched in terms of bioactivity. The genus Narcissus, belonging to the Amaryllidaceae family, evaluated as a valuable source of natural products, has been investigated for its alkaloidal profile in terms of its species and subspecies. This thesis consists of phytochemical studies on bulbs and aerial parts of Narcissus tazetta subsp. tazetta L. collected from its natural habitat in Mugla/Turkey as there have been no study carried out on this subspecies. Quality-control studies including, loss on drying, total ash, sulphated ash and hydrochloric acid-insoluble ash have been performed according to European Pharmacopoeia on drug specimens. The results were given as percentage values. Additionally, total alkaloidal contents of the drug specimens were determined by a titrimetric method. Galanthamine and lycorine, biologically important alkaloids of Amaryllidaceae family, in two different drug specimens prepared from Narcissus tazetta subsp. tazetta L, were qualitatively and quantitatively investigated using high pressure liquid chromatography (HPLC). Furthermore, capillary gas chromatography-mass spectrometry (GC-MS) was used to determine the alkaloidal profile of two different drug specimens. As a result, 23 alkaloids were detected in Narcissus tazetta subsp. tazetta. In the studies, where isolation and structure elucidation investigations were carried out, total alkaloidal extract, prepared from powdered whole plant material dried at room temperature, was fractionated by column chromatography. The obtained fractions have been purified by different methods such as preparative column chromatography, preparative thin layer chromatography and crystallization. The structures of the obtained compounds were elucidated by modern spectroscopic methods (1D, 2D NMR, MS). As a result of these studies, the Amaryllidaceae alkaloids, (-)-lycorine, (-)-pseudolycorine, (-)-9-O-methylpseudolycorine, (+)-11-hydroxygalanthine, (-)-narcissidine from the lycorine subgroup; (+)-9-O-demethyl-2-α-hydroxyhomolycorine from the homolycorine subgroup; (-)-pancratinine-C from the montanine subgroup and (-)-galanthamine from the galanthamine subgroup were isolated from Narcissus tazetta subsp. tazetta. All compounds are reported for the first time in the title subspecies collected from Turkey. Acetylcholinesterase ve butyrylcholinesterase inhibitory activities of isolated compounds which are not studied before, were determined by using a microplate assay based on in vitro Ellman's method. The IC50 values of all active compounds were calculated. Also, molecular docking studies were performed to evaluate the binding orientations of the isolated compounds inside the active sites of cholinesterase enzymes.
Amaryllidaceae alkaloids are a group of secondary metabolites which have therapeutic potential for the treatment of various diseases and have been deeply searched in terms of bioactivity. The genus Narcissus, belonging to the Amaryllidaceae family, evaluated as a valuable source of natural products, has been investigated for its alkaloidal profile in terms of its species and subspecies. This thesis consists of phytochemical studies on bulbs and aerial parts of Narcissus tazetta subsp. tazetta L. collected from its natural habitat in Mugla/Turkey as there have been no study carried out on this subspecies. Quality-control studies including, loss on drying, total ash, sulphated ash and hydrochloric acid-insoluble ash have been performed according to European Pharmacopoeia on drug specimens. The results were given as percentage values. Additionally, total alkaloidal contents of the drug specimens were determined by a titrimetric method. Galanthamine and lycorine, biologically important alkaloids of Amaryllidaceae family, in two different drug specimens prepared from Narcissus tazetta subsp. tazetta L, were qualitatively and quantitatively investigated using high pressure liquid chromatography (HPLC). Furthermore, capillary gas chromatography-mass spectrometry (GC-MS) was used to determine the alkaloidal profile of two different drug specimens. As a result, 23 alkaloids were detected in Narcissus tazetta subsp. tazetta. In the studies, where isolation and structure elucidation investigations were carried out, total alkaloidal extract, prepared from powdered whole plant material dried at room temperature, was fractionated by column chromatography. The obtained fractions have been purified by different methods such as preparative column chromatography, preparative thin layer chromatography and crystallization. The structures of the obtained compounds were elucidated by modern spectroscopic methods (1D, 2D NMR, MS). As a result of these studies, the Amaryllidaceae alkaloids, (-)-lycorine, (-)-pseudolycorine, (-)-9-O-methylpseudolycorine, (+)-11-hydroxygalanthine, (-)-narcissidine from the lycorine subgroup; (+)-9-O-demethyl-2-α-hydroxyhomolycorine from the homolycorine subgroup; (-)-pancratinine-C from the montanine subgroup and (-)-galanthamine from the galanthamine subgroup were isolated from Narcissus tazetta subsp. tazetta. All compounds are reported for the first time in the title subspecies collected from Turkey. Acetylcholinesterase ve butyrylcholinesterase inhibitory activities of isolated compounds which are not studied before, were determined by using a microplate assay based on in vitro Ellman's method. The IC50 values of all active compounds were calculated. Also, molecular docking studies were performed to evaluate the binding orientations of the isolated compounds inside the active sites of cholinesterase enzymes.
Açıklama
Anahtar Kelimeler
Narcissus Tazetta Subsp. Tazetta, Antikolinesteraz Aktivite, Izolasyon Ve Yapı Aydınlatma Çalışmaları, Kalite Kontrol Çalışmaları, Antcholinesterase Activity, Isolation, Quality Control Experiments, Structure Elucidation Studies
