Lignoselülozik ksilooligosakkaritlerin kolon kanseri hücre hattı üzerinde moleküler etkilerinin belirlenmesi
Yükleniyor...
Tarih
2017-04
Yazarlar
Dergi Başlığı
Dergi ISSN
Cilt Başlığı
Yayıncı
Selçuk Üniversitesi Fen Bilimleri Enstitüsü
Erişim Hakkı
info:eu-repo/semantics/openAccess
Özet
Fonksiyonel gıdalar beslenme üzerinde sağlık yararları sağlayan gıda veya gıda bileşenleridir. Ksilooligosakkaritler (XOS), fonksiyonel gıdalarda aktif bileşen olarak yer almaktadırlarlar. Ksilooligosakkaritlerin gıdalara eklenmesi kolon kanseri ve kardiyovasküler hastalıkları önleme, kalsiyum emiliminde artış, diş çürümelerinde azalma, bağırsak fonksiyonlarında iyileşme gibi olumlu pek çok fayda sağlayabilmektedir. Bu araştırmanın amacı doğal kaynaklardan biyoteknolojik yollarla elde edilen ksiloolgosakkaritlerin insan sağlıklı epitel kolon hücresine model hücre CoN ve kolon kanserine model hücre Caco-2 üzerinde sitotoksik ve moleküler düzeyde etkilerini araştırmaktır. Bu amaçla birinci aşamada kenevir kıymıklarından elde edilen ksilooligosakkaritlerden furfural ve hidroksimetil furfural (HMF)' nin uzaklaştırılması için nanofiltrasyon (NF), aktif karbon (AK) (Meck AC) adsorbsiyonu ve iyon değişim reçineleri (DOW-XUS 43578, Diaion CR11, Dowex-HCR W2, Purolite C160) kullanılmıştır. Ayrıca NF-AK ve NF-iyon değişim reçineleri hibrit sistemi de kullanılmıştır. Ksilooligosakkaritlerin antioksidan aktivitesini belirlemek için DPPH (2,2-diphenyl-1-picrylhydrazyl) metodu kullanılmıştır. DPPH ölçümleri 517 nm dalga boyunda ELISA okuyucu ile gerçekleştirilmiştir. İkinci aşamada ise gerçek zamanlı hücre analiz sistemi xCELLigence kullanılarak sitotoksisite analizleri yapılmıştır. Ayrıca XOS' ların moleküler etkilerinin incelenmesi için farklı miktarlarda XOS (10 mg/ml, 5 mg/ml, 1.25 mg/ml) ile muamele edilmiş sağlıklı ve kanserli kolon hücrelerinden RNA izole edilmiş ve XOS'ların RT-PCR (Geri transkriptaz-PZR) yöntemiyle oksidatif stres ile ilgili NFE2L2 geninin ifadesinde etkisi olup olmadığı araştırılmıştır. Nanofiltrasyon prosesinin HMF gidermede etkili olmadığı, ancak furfuralı neredeyse tamamen uzaklaştırdığı belirlenmiştir. HMF ve furfural uzaklaştırmada reçine ve aktif karbon uygulamalarının etkili olduğu tespit edilmiştir. Purolite C160 ve Dowex-HCRW2 reçinelerinin HMF ve furfural uzaklaştırmada etkili olmadığı, DOW XUS 43578, Diaion CR11 ve Merck aktif karbon ise verimli olduğu belirlenmiştir. Nanofiltrasyon ve aktif karbon proseslerinin birlikte düşünüldüğü hibrid sistem kullanımı ile HMF % 99.3 ve furfural % 100 oranında uzaklaştırılmıştır. Ksilooligosakkaritlerin DPPH süpürücü etkisi L-Askorbik asit ile karşılaştırılmıştır. XOS ve L-Askorbik asit için IC50 değerleri sırasıyla 122.29 μl/ml ve 12.01 µg/ml olarak ölçülmüştür. CoN ve Caco-2 hücre modelleri ile yapılan hücre kültürü çalışmalarında XOS' un 5 mg/ml' den daha az olan konsantrasyonları sitotoksisite ve dublikasyon süresi bakımından güvenli bulunmuşlardır. XOS'ların artan doz miktarına göre CoN/NFE2L2 ve Caco-2/NFE2L2 gen ekspresyon seviyelerinde anlamlı artış ve azalışlar bulunmamıştır.
Functional foods are food or food ingredients that provide health benefits over nutrition. Xylooligosaccharides (XOS) are present as active ingredients in functional foods. The addition of xylooligosaccharides to foods may provide many positive benefits such as prevention of colon cancer and cardiovascular diseases, increase in calcium absorption, decrease in tooth decay, improvement in bowel function. The aim of this research is to investigate the effects of xylooligosaccharides biotechnologically obtained from natural sources on model cell CoN to human healthy epithelial colon cell and on model cell Caco-2 to colon cancer cell at cytotoxic and molecular levels. For this purpose, in the first step, nanofiltration (NF), activated carbon (AC) (Merck AC) adsorption and ion exchange resins (DOW-XUS 43578, Diaion CR11, Dowex-HCR W2, Purolite C160) were used to remove furfural and hydroxymethyl furfural (HMF) from xylooligosaccharides obtained from cannabis splinters. The hybrid system of NF-AC and NF-ion exchange resins were also used. The DPPH (2,2-diphenyl-1-picrylhydrazyl) method was used to determine the antioxidant activity of xylooligosaccharides. DPPH measurements were performed with an ELISA reader at a wavelength of 517 nm. In the second step, cytotoxicity analyzes were performed using real-time cell analysis system Xcelligence. In addition, RNA was isolated from healthy and cancerous colon cells treated with different amounts of XOS (10 mg/ml, 5 mg/ml, 1.25 mg/ml) to examine the molecular effects of XOS and the effect of XOS on the expression of NFE2L2 gene related to oxidative stress.was investigated by RT-PCR (Reverse transcriptase-PCR) method. The nanofiltration process was not effective for the removal of HMF but was effective for completely removal of furfural. Resin and activated carbon applications were effective for the removal of HMF and furfural. Purolite C160 and Dowex-HCR W2 resins were not effective for the removal of HMF and furfural. However, DOW XUS 43578, Diaion CR11 and Merck activated carbon were effective for removal of HMF and furfural. With the utilization of the hybrid system, in which nanofiltration and activated carbon processes were considered together, HMF was removed by 99.3% and furfural by 100%. The DPPH scavenging activity of xylooligosaccharides was compared with L-Ascorbic acid. The IC50 values for XOS and L-Ascorbic acid were 122.29 μL/mL and 12.01 μg/mL, respectively. Concentrations of XOS less than 5 mg/ml in cell culture studies with CoN and Caco-2 cell models were found to be safe in terms of cytotoxicity and duration of duplication. There was no significant increase or decrease in the expression levels of CoN / NFE2L2 and Caco-2 / NFE2L2 gene according to the increasing doses of XOS.
Functional foods are food or food ingredients that provide health benefits over nutrition. Xylooligosaccharides (XOS) are present as active ingredients in functional foods. The addition of xylooligosaccharides to foods may provide many positive benefits such as prevention of colon cancer and cardiovascular diseases, increase in calcium absorption, decrease in tooth decay, improvement in bowel function. The aim of this research is to investigate the effects of xylooligosaccharides biotechnologically obtained from natural sources on model cell CoN to human healthy epithelial colon cell and on model cell Caco-2 to colon cancer cell at cytotoxic and molecular levels. For this purpose, in the first step, nanofiltration (NF), activated carbon (AC) (Merck AC) adsorption and ion exchange resins (DOW-XUS 43578, Diaion CR11, Dowex-HCR W2, Purolite C160) were used to remove furfural and hydroxymethyl furfural (HMF) from xylooligosaccharides obtained from cannabis splinters. The hybrid system of NF-AC and NF-ion exchange resins were also used. The DPPH (2,2-diphenyl-1-picrylhydrazyl) method was used to determine the antioxidant activity of xylooligosaccharides. DPPH measurements were performed with an ELISA reader at a wavelength of 517 nm. In the second step, cytotoxicity analyzes were performed using real-time cell analysis system Xcelligence. In addition, RNA was isolated from healthy and cancerous colon cells treated with different amounts of XOS (10 mg/ml, 5 mg/ml, 1.25 mg/ml) to examine the molecular effects of XOS and the effect of XOS on the expression of NFE2L2 gene related to oxidative stress.was investigated by RT-PCR (Reverse transcriptase-PCR) method. The nanofiltration process was not effective for the removal of HMF but was effective for completely removal of furfural. Resin and activated carbon applications were effective for the removal of HMF and furfural. Purolite C160 and Dowex-HCR W2 resins were not effective for the removal of HMF and furfural. However, DOW XUS 43578, Diaion CR11 and Merck activated carbon were effective for removal of HMF and furfural. With the utilization of the hybrid system, in which nanofiltration and activated carbon processes were considered together, HMF was removed by 99.3% and furfural by 100%. The DPPH scavenging activity of xylooligosaccharides was compared with L-Ascorbic acid. The IC50 values for XOS and L-Ascorbic acid were 122.29 μL/mL and 12.01 μg/mL, respectively. Concentrations of XOS less than 5 mg/ml in cell culture studies with CoN and Caco-2 cell models were found to be safe in terms of cytotoxicity and duration of duplication. There was no significant increase or decrease in the expression levels of CoN / NFE2L2 and Caco-2 / NFE2L2 gene according to the increasing doses of XOS.
Açıklama
Anahtar Kelimeler
Fonksiyonel gıda, Functional food, İyon değişim reçinesi, Ion-exchange resin, Pürifikasyon, Purification
Kaynak
WoS Q Değeri
Scopus Q Değeri
Cilt
Sayı
Künye
Aykül, F. (2017). Lignoselülozik ksilooligosakkaritlerin kolon kanseri hücre hattı üzerinde moleküler etkilerinin belirlenmesi. Selçuk Üniversitesi, Yayımlanmış yüksek lisans tezi, Konya.
