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Proje Grubu: KBAG Sayfa Sayısı: 244 Proje No: 114Z842 Proje Bitiş Tarihi: 15.04.2018 Metin Dili: Türkçe İndeks Tarihi: 04-12-2018

Pistacia lentiscus L.’un In Vitro Sürgün, Kallus ve Hücre Süspansiyon Kültürlerinde Antikanser Aktivite Gösteren Kimyasal Bileşenlerin Üretilmesi

Öz:
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.
Anahtar Kelime: MTT biyolojik aktivite elisitör süspansiyon kallus P. lentiscus L.

Konular: Biyoloji Kimya, Tıbbi Onkoloji

Pistacia lentiscus L.’un In Vitro Sürgün, Kallus ve Hücre Süspansiyon Kültürlerinde Antikanser Aktivite Gösteren Kimyasal Bileşenlerin Üretilmesi

Öz:
Secondary metabolites are organic chemicals with important functions in the plant defense system and also with a number of biological activities and protective functions (antimicrobial, antifungal, anti-cancer, anti-inflammatory, antiatherogenic, antitumoral, antiproliferative and proapoptotic activity) in terms of human health. The aim of our project is to develop protocols for the optimization of callus and cell suspension cultures from the juvenile (root, stem and leaf) and mature (female and male) shoots and leaves, to increase the amount and variety of anticancer components in the triterpenic structure in various parts of the plant by application of physical and chemical elicitors on shoot-optimized callus and cell suspension cultures and prepared from these parts by the determining the effect of the extracts on the cancerous cell lines and determinate qualitative and quantitative of biological activity of the mastic tree (P. lentiscus L.). In this context, seeds of mastic were successfully germinated and proliferation of juvenile and mature female and male shoots was carried out in MS medium supplemented with 1 mg/l BAP, 0.5 mg/l GA3. From axenic shoots, about 1 cm long, biotic and abiotic 25 different elicitor applications were applied. In the elicitation studies, 4 mg/l AgNO3 application resulted in a significant increase in the amount of triterpenoids such as Ursolic Acid (2.915 ppm) and 1 mg/l MeJA application provided the formation of triterpenoids (Oleonolic Acid and Masticadienolic Acid) of mature callus and suspension cultures. In studies in which different nutrient types were tested with different BBD, carbon type and concentrations, different pH, different light intensity, different temperature in the callus cultures' optimization studies, 1/1 strength MS media containing 1 mg/l KIN and 1 mg/l 2,4-D have been found to give the best results for optimum callus formation and development. Optimization studies of suspension cultures in which callus culturing conditions were tested as well as different shaking speeds were obtained from the MS medium supplemented with 30 g/l sucrose with the highest PCV, fresh and dry weight results at 25°C, 95 rpm shaking speed, pH 5.8. The most successful elicitor types (MeJA and AgNO3) and concentrations (1-4 mg/l) were applied to callus and suspension cultures and triterpene contents similar to shoot cultures were determined. As a result of the qualitative and quantitative analyzes, it was found that the highest antioxidant (CUPRAC, DPPH and ABTS) and Anticholinesterase (Acetyl and Butyrylcholinesterase) enzyme activities was highest from α-TOC, BHT and Galantamine respectively, which applied the best results among the elicitors applied to the shoot, callus and suspension cultures for the first time. The best result in terms of MTT was found to be stem culture with 4 mg/l AgNO3, and stem originated cell suspension extracts administered with 1 mg/l MeJA for 48 hours on culture day 7. When the samples tested according to the results of the advanced cell tests exhibited different activities on MCF7 breast cancer cell lines in general, but when the samples were evaluated among themselves, the best result among all the samples tested was shoot culture derived stem cultures and cultures with 4 mg /l AgNO3 cells were administered with 1 mg/l MeJA for 48 hours.
Anahtar Kelime:

Konular: Biyoloji Kimya, Tıbbi Onkoloji
Erişim Türü: Erişime Açık
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APA Tilkat E, ONAY A, Ertaş A, YILMAZ M, SURMUŞ ASAN H (2018). Pistacia lentiscus L.’un In Vitro Sürgün, Kallus ve Hücre Süspansiyon Kültürlerinde Antikanser Aktivite Gösteren Kimyasal Bileşenlerin Üretilmesi. , 1 - 244.
Chicago Tilkat Engin,ONAY Ahmet,Ertaş Abdulselam,YILMAZ Mustafa Abdullah,SURMUŞ ASAN Hilal Pistacia lentiscus L.’un In Vitro Sürgün, Kallus ve Hücre Süspansiyon Kültürlerinde Antikanser Aktivite Gösteren Kimyasal Bileşenlerin Üretilmesi. (2018): 1 - 244.
MLA Tilkat Engin,ONAY Ahmet,Ertaş Abdulselam,YILMAZ Mustafa Abdullah,SURMUŞ ASAN Hilal Pistacia lentiscus L.’un In Vitro Sürgün, Kallus ve Hücre Süspansiyon Kültürlerinde Antikanser Aktivite Gösteren Kimyasal Bileşenlerin Üretilmesi. , 2018, ss.1 - 244.
AMA Tilkat E,ONAY A,Ertaş A,YILMAZ M,SURMUŞ ASAN H Pistacia lentiscus L.’un In Vitro Sürgün, Kallus ve Hücre Süspansiyon Kültürlerinde Antikanser Aktivite Gösteren Kimyasal Bileşenlerin Üretilmesi. . 2018; 1 - 244.
Vancouver Tilkat E,ONAY A,Ertaş A,YILMAZ M,SURMUŞ ASAN H Pistacia lentiscus L.’un In Vitro Sürgün, Kallus ve Hücre Süspansiyon Kültürlerinde Antikanser Aktivite Gösteren Kimyasal Bileşenlerin Üretilmesi. . 2018; 1 - 244.
IEEE Tilkat E,ONAY A,Ertaş A,YILMAZ M,SURMUŞ ASAN H "Pistacia lentiscus L.’un In Vitro Sürgün, Kallus ve Hücre Süspansiyon Kültürlerinde Antikanser Aktivite Gösteren Kimyasal Bileşenlerin Üretilmesi." , ss.1 - 244, 2018.
ISNAD Tilkat, Engin vd. "Pistacia lentiscus L.’un In Vitro Sürgün, Kallus ve Hücre Süspansiyon Kültürlerinde Antikanser Aktivite Gösteren Kimyasal Bileşenlerin Üretilmesi". (2018), 1-244.
APA Tilkat E, ONAY A, Ertaş A, YILMAZ M, SURMUŞ ASAN H (2018). Pistacia lentiscus L.’un In Vitro Sürgün, Kallus ve Hücre Süspansiyon Kültürlerinde Antikanser Aktivite Gösteren Kimyasal Bileşenlerin Üretilmesi. , 1 - 244.
Chicago Tilkat Engin,ONAY Ahmet,Ertaş Abdulselam,YILMAZ Mustafa Abdullah,SURMUŞ ASAN Hilal Pistacia lentiscus L.’un In Vitro Sürgün, Kallus ve Hücre Süspansiyon Kültürlerinde Antikanser Aktivite Gösteren Kimyasal Bileşenlerin Üretilmesi. (2018): 1 - 244.
MLA Tilkat Engin,ONAY Ahmet,Ertaş Abdulselam,YILMAZ Mustafa Abdullah,SURMUŞ ASAN Hilal Pistacia lentiscus L.’un In Vitro Sürgün, Kallus ve Hücre Süspansiyon Kültürlerinde Antikanser Aktivite Gösteren Kimyasal Bileşenlerin Üretilmesi. , 2018, ss.1 - 244.
AMA Tilkat E,ONAY A,Ertaş A,YILMAZ M,SURMUŞ ASAN H Pistacia lentiscus L.’un In Vitro Sürgün, Kallus ve Hücre Süspansiyon Kültürlerinde Antikanser Aktivite Gösteren Kimyasal Bileşenlerin Üretilmesi. . 2018; 1 - 244.
Vancouver Tilkat E,ONAY A,Ertaş A,YILMAZ M,SURMUŞ ASAN H Pistacia lentiscus L.’un In Vitro Sürgün, Kallus ve Hücre Süspansiyon Kültürlerinde Antikanser Aktivite Gösteren Kimyasal Bileşenlerin Üretilmesi. . 2018; 1 - 244.
IEEE Tilkat E,ONAY A,Ertaş A,YILMAZ M,SURMUŞ ASAN H "Pistacia lentiscus L.’un In Vitro Sürgün, Kallus ve Hücre Süspansiyon Kültürlerinde Antikanser Aktivite Gösteren Kimyasal Bileşenlerin Üretilmesi." , ss.1 - 244, 2018.
ISNAD Tilkat, Engin vd. "Pistacia lentiscus L.’un In Vitro Sürgün, Kallus ve Hücre Süspansiyon Kültürlerinde Antikanser Aktivite Gösteren Kimyasal Bileşenlerin Üretilmesi". (2018), 1-244.