In vitro oksidatif hasar oluşturulmuş DNA üzerine nar kabuğu, tetra ve zeytin yaprağı ekstraktlarının etkisinin incelenmesi
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Dosyalar
Tarih
2022
Yazarlar
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Yayıncı
Trakya Üniversitesi Fen Bilimleri Enstitüsü
Erişim Hakkı
info:eu-repo/semantics/openAccess
Özet
Çalışmanın amacı besin olarak kullanılmayan nar (Hicaz türü) kabuğu, zeytin yaprağı (Domat ve Gemlik türleri) ve tetra yaprağından (Cotinus coggygria) elde edilen ekstraktların antioksidan aktivitelerine katkı sağlayan fitobileşenlerce analizlenmesi, bu bitki örneklerinin DNA üzerinde oluşturulan in vitro oksidatif hasar üzerinde koruyucu etkisinin, karaciğer sağlıklı ve kanser hücre hattı üzerinde sitotoksik etkisinin olup olmadığının incelenmesidir. Bitkisel kaynaklardan fenolik bileşence zengin ekstrakt elde etmek için farklı ekstraksiyon teknikleri kullanılmaktadır. Nar kabuğu, tetra ve zeytin yapraklarının %70’lik etanol çözücüsüyle çalkalamalı su banyosu kullanılarak geleneksel bir metod ile, ve ultrasonik destekli ekstraksiyon yöntemi kullanılarak modern bir metod ile ekstraksiyonları yapıldı. Ultrasonik destekli ekstraksiyondan elde edilen ekstraktlarda genel olarak ekstrakt verimi, spektrofotometrik yöntem ile yapılan toplam fenolik ve flavonoid madde içeriği ve serbest radikal giderme gücünün daha yüksek olduğu görüldü. DPPH ve ABTS metodlarında tetra yaprağı ve nar kabuğu düşük EC50 değerlerine sahip olarak serbest radikalleri gidermede etkili oldular. AML12 (normal karaciğer) ve Hep3B (karaciğer kanseri) hücre hatları kullanılarak MTT testi uygulandı. Farklı konsantrasyonlardaki ekstraktlar hücreler ile 24 saat ve 48 saat muamele edildi. Tetra yaprağı, Hicaz türü nar kabuğu, Gemlik zeytin yaprağı ve Mix (nar kabuğu+tetra yaptağı+Domat türü zeytin yaprağı karışımı) ekstraktlarının AML12 hücrelerinde doza bağımlı koruyucu etki gösterdiler, Domat zeytin yaprağı ekstraktı ise çalışılan yüksek konsantrasyonlarında canlılıkta azalmaya neden oldu. Hep3B hücrelerinde doza bağımlı sitotoksik etki gözlendi, 24 saatlik inkübasyon sonrasında Domat örneği dışında, başta Tetra yaprağı ve Mix ekstraktı olmak üzere tüm ekstraktlar antiproliferatif etki gösterdiler. Antioksidanlar özellikle serbest radikallerin oluşumunu ve zararlı etkilerini önleyen ve bunun sonucunda oksidatif hasara karşı DNA ve diğer biyomolekülleri koruyan bileşikler veya bitkisel kaynaklardır. 8-hidroksi-2'-deoksi guanozin (8-OH-2dG) ve/veya 8-hidroksiguanin (8OHGua) ölçümü DNA’daki oksidatif hasarın belirtecidir. Çalışmada, buzağı timus DNA’sı Fenton reaksiyonuyla oluşturulan hidroksil radikali ile oksidatif hasara uğratıldı ve oluşan 8-OH2dG miktarları LC-MS/MS cihazı ile ölçüldü. Nar kabuğu, tetra, Domat ve Gemlik türü zeytin yaprakları ekstraktlarının ve Mix ekstraktının oluşturulan hasarı önleme kapasitesi değerlendirildi. Oksidatif DNA hasarını en yüksek oranda Hicaz türü nar kabuğu, tetra yaprağı ve Mix ekstraktları engelledi. Gıda olarak tüketilmeyen bitkisel ürünlerin veya tarımsal atık ürünlerin biyoaktif bileşik kaynağı olarak kullanılması ekonomi ve geri kazanım açısından önem arzetmektedir. Çalışmada LC-MS/MS cihazı ile bu tür bitkisel kaynaklardan alınan ekstraktların fenolik bileşenlerinin türü ve miktar tayinleri yapıldı. Hicaz türü nar kabuğunda ana bileşen olarak ellajik asit başta olmak üzere absisik asit, etil gallat, gallik asit; tetra yaprağında en fazla verbaskosit, gallik asit, protokateşik asit, mirisetin, etil gallat ve şiringik asit; zeytin yapraklarında oleuropein başta olmak üzere klorojenik asit, verbaskosit, tirozinol ve rutin bulundu. Bitkilerin ikincil metabolitleri olan bu biyoaktif bileşikler ekstraktların gösterdikleri antioksidan, sitotoksik ve oksidatif DNA hasarını önleme aktivitelerinden sorumlu olabilirler.
This study aims to investigate the extracts obtained from pomegranate peel (Hicaz gentype), olive leaves (Domat ve Gemlik gentypes) and smoke tree leaf (Cotinus coggygria), which are not used as nutrients, by phytocomponents that contribute to their antioxidant activities, and to determine whether these plant samples have protective effect against oxidative DNA damage. Different extraction techniques are used to obtain phenolic-rich extract from plant sources. Both conventional extraction technique using a shaking water bath and ultrasoundassisted extraction as a modern method were performed with 70% ethanol solvent for extractions of pomegranate peel, smoke tree and olive leaves. It was observed that the extracts obtained from ultrasonic assisted extraction (USE) generally had higher extract yield, free radical scavenging capacity and, total phenolic and flavonoid compounds carried out by spectrophotometric method. In DPPH and ABTS methods, smoke tree leaf and pomegranate peel had low EC50 values which indicates the strong free radicals scavenging activities. MTT assay was applied to the extracts using AML12 (normal liver) and Hep3B (liver cancer) cell lines. Cells were treated with extracts of different concentrations during 24 hours and 48 hours. The extracts of smoke tree leaf, Hicaz type pomegranate peel, Gemlik olive leaf and Mix (pomegranate peel + smoke tree leaf + Domat type olive leaf mixture) showed a dosedependent protective effect on AML12 cells. Domat olive leaf extract, on the other hand, caused a decrease in viability at the high concentrations studied. A dose-dependent cytotoxic effect was observed in Hep3B cells, after 24 hours of incubation, all extracts, especially smoke tree leaf and Mix extract, showed antiproliferative effects, except for Domat sample. Antioxidants are compounds or herbal sources that prevent the formation and harmful effects of free radicals and, as a result, protect DNA and other biomolecules against oxidative stress. Measurement of 8-hydroxy 2'-deoxyguanosine (8-OH-2dG) and/or 8-hydroxyguanine (8OHGua) are markers of oxidative damage in DNA. In the study, the hydroxyl radical formed by Fenton reaction damaged calf thymus DNA oxidatively and, the amounts of 8-OH-2dG were measured by LC-MS/MS. The capacity of pomegranate peel, smoke tree leaf, Domat and Gemlik olive leaf extracts and the Mix extract to prevent this damage was evaluated. Hicaz pomegranate peel, smoke tree leaf and Mix extracts prevented oxidative DNA damage at the highest rate. The use of plant products that are not consumed as food or agricultural waste products as a source of bioactive compounds is important in terms of economic and recovery. The phenolic compounds and quantity determinations of the plants used in the study were made by LCMS/MS. The Hicaz pomegranate peel contains ellagic acid as major compound, abscisic acid, ethyl gallate, gallic acid. Smoke tree leaf contains the most verbascoside, gallic acid, protocatechic acid, myricetin, ethyl gallate and syringic acid. In olive leaves, mainly oleuropein, chlorogenic acid, verbascoside, tyrosinol and rutin were found. These bioactive compounds, which are secondary metabolites of plants, may be responsible for their antioxidant, cytotoxic and oxidative DNA damage prevention activities.
This study aims to investigate the extracts obtained from pomegranate peel (Hicaz gentype), olive leaves (Domat ve Gemlik gentypes) and smoke tree leaf (Cotinus coggygria), which are not used as nutrients, by phytocomponents that contribute to their antioxidant activities, and to determine whether these plant samples have protective effect against oxidative DNA damage. Different extraction techniques are used to obtain phenolic-rich extract from plant sources. Both conventional extraction technique using a shaking water bath and ultrasoundassisted extraction as a modern method were performed with 70% ethanol solvent for extractions of pomegranate peel, smoke tree and olive leaves. It was observed that the extracts obtained from ultrasonic assisted extraction (USE) generally had higher extract yield, free radical scavenging capacity and, total phenolic and flavonoid compounds carried out by spectrophotometric method. In DPPH and ABTS methods, smoke tree leaf and pomegranate peel had low EC50 values which indicates the strong free radicals scavenging activities. MTT assay was applied to the extracts using AML12 (normal liver) and Hep3B (liver cancer) cell lines. Cells were treated with extracts of different concentrations during 24 hours and 48 hours. The extracts of smoke tree leaf, Hicaz type pomegranate peel, Gemlik olive leaf and Mix (pomegranate peel + smoke tree leaf + Domat type olive leaf mixture) showed a dosedependent protective effect on AML12 cells. Domat olive leaf extract, on the other hand, caused a decrease in viability at the high concentrations studied. A dose-dependent cytotoxic effect was observed in Hep3B cells, after 24 hours of incubation, all extracts, especially smoke tree leaf and Mix extract, showed antiproliferative effects, except for Domat sample. Antioxidants are compounds or herbal sources that prevent the formation and harmful effects of free radicals and, as a result, protect DNA and other biomolecules against oxidative stress. Measurement of 8-hydroxy 2'-deoxyguanosine (8-OH-2dG) and/or 8-hydroxyguanine (8OHGua) are markers of oxidative damage in DNA. In the study, the hydroxyl radical formed by Fenton reaction damaged calf thymus DNA oxidatively and, the amounts of 8-OH-2dG were measured by LC-MS/MS. The capacity of pomegranate peel, smoke tree leaf, Domat and Gemlik olive leaf extracts and the Mix extract to prevent this damage was evaluated. Hicaz pomegranate peel, smoke tree leaf and Mix extracts prevented oxidative DNA damage at the highest rate. The use of plant products that are not consumed as food or agricultural waste products as a source of bioactive compounds is important in terms of economic and recovery. The phenolic compounds and quantity determinations of the plants used in the study were made by LCMS/MS. The Hicaz pomegranate peel contains ellagic acid as major compound, abscisic acid, ethyl gallate, gallic acid. Smoke tree leaf contains the most verbascoside, gallic acid, protocatechic acid, myricetin, ethyl gallate and syringic acid. In olive leaves, mainly oleuropein, chlorogenic acid, verbascoside, tyrosinol and rutin were found. These bioactive compounds, which are secondary metabolites of plants, may be responsible for their antioxidant, cytotoxic and oxidative DNA damage prevention activities.
Açıklama
Anahtar Kelimeler
8-OH-2'dG, Fenolik madde, LC-MS/MS, MTT testi, Oksidatif DNA hasarı, Radikal giderme, Phenolic compound, MTT test, Oxidative DNA damage, Radical scavenging
