Ahmet ONAY
(adres yazılmamıs)
Abdulselam ERTAŞ
(adres yazılmamıs)
Mustafa Abdullah YILMAZ
(adres yazılmamıs)
Hilal SURMUŞ ASAN
(Dicle Universty, Science Faculty, Department of Biology, Diyarbakir, Turkey)
Proje Grubu: TÜBİTAK KBAG ProjeSayfa Sayısı: 244Proje No: 114Z842Proje Bitiş Tarihi: 15.04.2018Türkçe

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Pistacia lentiscus L.’un In Vitro Sürgün, Kallus ve Hücre Süspansiyon Kültürlerinde Antikanser Aktivite Gösteren Kimyasal Bileşenlerin Üretilmesi
Sekonder metabolitler, bitki savunma sisteminde önemli işlevleri olan, aynı zamanda insan sağlığı açısından da birtakım biyolojik aktivite ve koruyucu işlevlere (antimikrobiyal, antifungal, anti-kanser, anti-inflamatuar, antiaterojenik, antitümöral, antiproliferatif ve proapoptotik aktivite) sahip organik kimyasallardır. Projemizin amacını, sakız ağacı (P. lentiscus L.)’nın jüvenil (kök, gövde ve yaprak) ve olgun (dişi ve erkek) gövde ve yapraklarından elde edilen kallus ve hücre süspansiyon kültürlerinin optimizasyonu için protokollerin geliştirilmesi, sürgün ile optimize edilmiş kallus ve hücre süspansiyon kültürlerine faklı fiziksel ve kimyasal elisitör uygulamaları ile bitkinin çeşitli kısımlarında bulunan triterpen yapıdaki antikanser bileşenlerin miktar ve çeşitlerinin arttırılması, bu kısımlardan hazırlanan ekstrelerin kanserli hücre hatları üzerindeki etkilerinin ortaya çıkarılması ve biyolojik aktivitelerinin belirlenmesi oluşturmaktadır. Bu bağlamda öncelikle sakız ağacına ait tohumlar başarılı bir şekilde çimlendirilmiş, jüvenil ve olgun dişi ve erkek sürgünlerin proliferasyonu ise 1 mg/l BAP, 0.5 mg/l GA3 ile destekli MS besi ortamında gerçekleştirilmiştir. Aksenik stok materyallerden, yaklaşık 1 cm uzunluğunda alınan sürgünler, biyotik ve abiyotik olmak üzere 25 farklı elisitör uygulamasına tabi tutulmuştur. Test edilen elisitasyon çalışmalarında 4 mg/l AgNO3 uygulamasının Ursolik Asit (2.915 ppm) gibi triterpenoitlerin miktarlarında belirgin derecede artış sağladığından ve 1 mg/l MeJA uygulamasının ise kontrol grubunda tespit edilemeyen farklı triterpenoitlerin oluşumuna (Oleonolik Asit ve Mastikadienolik Asit) yol açtığından, bu iki elisitör optimizasyon protokolleri tanımlanmış jüvenil ve olgun kallus ve süspansiyon kültürlerine de uygulanmıştır. Kallus kültürlerinin optimizasyon çalışmalarında farklı BBD, karbon tipi ve konsantrasyonları, farklı pH, farklı ışık yoğunluğu, farklı sıcaklık ile farklı besiyeri tiplerinin test edildiği çalışmada 1mg/l KIN ve 1 mg/l 2,4-D içeren 1/1 kuvvetinde MS besi ortamının optimum kallus oluşumu ve gelişimi için en iyi sonuçlar verdiği tespit edilmiştir. Kallus kültürleri optimizasyonu çalışmalarında test edilen tüm parametrelere ilave olarak farklı çalkalama hızlarının etkisinin de test edildiği süspansiyon kültürlerinin optimizasyonu çalışmalarında ise, sonuç olarak en yüksek PHH, taze ve kuru ağırlık sonuçları; 25°C sıcaklık, 95 rpm çalkalama hızı, pH 5.8 ile 30 gr/l sükroz destekli MS besi ortamından elde edilmiştir. Optimize edilmiş kallus ve süspansiyon kültürlerine en başarılı elisitör tip ve konsantrasyonları (1 mg/l MeJA ve 4 mg/l AgNO3) uygulanmış ve sürgün kültürlerine benzer olarak triterpen içeriği ve çeşidinin arttığı tespit edilmiştir. Kalitatif ve kantitatif analizler sonucunda ise, projemiz kapsamında ilk kez sürgün, kallus ve süspansiyon kültürlerine uygulanan elisitör uygulamaları arasından en iyi sonuç veren ekstrelere ait antioksidan (CUPRAC, DPPH ve ABTS) ve Antikolinesteraz (Asetil ve Bütirilkolinesteraz) enzim aktiviteleri bakımından sırasıyla α-TOC, BHT ve Galantaminden yüksek sonuçların bulunduğu tespit edilmiştir. MTT bakımından ise en iyi sonucu, 4 mg/l AgNO3 uygulanmış sürgün kültürü kökenli gövde ekstreleri ve kültürün 7. gününde 48 saat süresince 1 mg/l MeJA uygulanmış kök kökenli süspansiyon hücre ekstrelerinin verdiği tespit edilmiştir. İleri hücre testleri sonuçlarına göre test edilen örneklerin genel anlamda MCF7 göğüs kanser hücre hatları üzerinde farklı aktiviteler sergilediği, ancak örneklerin kendi aralarında değerlendirildiğinde ise, test edilen tüm örnekler arasında en iyi sonucu, 4 mg/l AgNO3 uygulanmış sürgün kültürü kökenli gövde ekstreleri ve kültürün 7. gününde 48 saat süresince 1 mg/l MeJA uygulanmış kök kökenli süspansiyon hücre ekstrelerinin verdiği tespit edilmiştir.
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