Bazı bitki ekstratlarının theileria annulata'ya karşı in vitro etkisi
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Date
2008-10-16
Authors
Girişgin, Oya
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Publisher
Uludağ Üniversitesi
Abstract
Bitki ekstratları paraziter enfeksiyonların tedavisi açısından önemli potansiyele sahiptirler. Naftakinonlar ve artemisinin veteriner ve tıp hekimliğinde kan parazitlerine karşı kullanılan bitkisel orijinli moleküllerdir. Ülkemizin birçok bölgesinde doğal olarak yetişen Arnebia densiflora (A.densiflora) içerdiği naftakinonlar nedeni ile, ve Artemisia annua (A.annua) içerdiği artemisinin nedeni ile tıbbi öneme haiz bitkilerdir. Bu çalışmada söz konusu bitkilerden elde edilen ekstratların Theileria annulata (T.annulata) schizontları ile enfekte Peripheral Blood Mononuclear (PBM) hücrelere karşı etkinlikleri araştırılmıştır. Bu amaçla, Eskişehir yöresinden toplanan A.densiflora bitkisinin kökleri, Bursa yöresinden toplanan A.annua bitkisinin topraküstü kısımları açık havada ve gölgede kurutulmuş, mekanik olarak parçalanmıştır. A.densiflora’nın n-hekzan ekstresi, A.annua’nın petrol eteri ve metanol ekstreleri çıkarılmıştır. A.annua ekstrelerinin içerdikleri Artemisinin miktarını belirlemek amacı ile Gas Chromatographic analizleri yapılmıştır. Ekstrelerin çözdürülmesinde 8 µl/0.5 ml medium konsantrasyonunda Dimethylsulfoksit (DMSO) kullanılmıştır. Sağlıklı ve enfekte PBM hücrelerinin kültivasyonu RPMI 1640 besi yeri ile sağlanmıştır. Hücreler denemelerin yapılacağı kuyucuklara 0.5 ml medium içinde 2x106 adet bulunacak şekilde dağıtılmışlardır. A.densiflora 125, 62.5, 15.625, 7.8125, 3.9062 µg/ml konsantrasyonlarda, A.annua 125, 62.5, 31.25, 15.625, 7.8125, 3.9062, 1.9531 µg/ml konsantrasyonlarda PBM hücreleri üzerinde denenmiştir. Kontrol grubu olarak ayrılan kuyucuklara sadece medium ilave edilmiştir. Canlı ve ölü hücrelerin ayrımı denemenin 24 ve 48. saatlerinde örnek hücreleri Trypan blue ile boyayarak yapılmıştır. Deneme grupları ile kontroller arasındaki farkların istatistiki analizleri için Mann Whitney U Test uygulanmış, p<0.05 ve üstü değerler anlamlı kabul edilmiştir. A.densiflora bitkisinde 90.5 g drogdan 4.12 g ekstre, A.annua bitkisinde 100 g drogdan petrol eteri ile 2.28 g, metanol ile 5.56 g ekstre elde edilmiştir. A.annua metanol ekstresinde 1.14 ± 0.07 mg/ 100 g, petrol eteri ekstresinde 4.54 ± 0.10 mg/100g düzeylerinde Artemisinin varlığı tespit edilmiştir. A.densiflora ekstresinin denenen tüm konsantrasyonlarda hem sağlıklı hem enfekte hücreleri kontrol gruplarındakilere kıyasla anlamlı düzeylerde (p<0.05) öldürdüğü gözlenmiştir. A.annua metanol ekstresinin sağlıklı hücreler üzerinde hiçbir olumsuz etkisi olmamış, enfekte hücreler üzerindeki etkisi ise yetersiz düzeyde olmasına rağmen sadece 125 µg/ml konsantrasyonda gözlenmiştir. A.annua petrol eteri ekstresi sağlıklı hücreler üzerinde olumsuz etki göstermemiş, enfekte hücreleri ise denemenin 24. saati itibarı ile 15.625 µg/ml ve üstü konsantrasyonlarda anlamlı düzeylerde (p<0.05) öldürmüştür. Denemenin 48. saatinde 62.5 µg/ml konsantrasyonda tüm enfekte hücreler ölmüştür. Sonuç olarak A.annua petrol eteri ekstresinin 15.625-62.5 µg/ml konsantrasyonlarda sağlıklı hücrelere zarar vermeden T.annulata schizontu ile enfekte PBM hücrelerini in vitro ortamda etkili şekilde yok ettiği ortaya konmuş, ekstrenin hastalar üzerinde denenmesi gerektiği kanısına varılmıştır.
Plant extracts have significant potentials for the treatment of parasitic infections. Naphtaquinones and artemisinin are plant based molecules used against blood parasites in veterinary and human medicine. Arnebia densiflora (A.densiflora) and Artemisia annua (A.annua) grow in most parts of our country and have medical value due to their naphtaquinone and artemisinin contents, respectively. In this study, effects of the extracts of these plants on peripheral blood mononuclear (PBM) cells infected with Theileria annulata schizonts were investigated. For this aim, roots of A. densiflora collected in Eskisehir province and over-soil parts of A.annua collected in Bursa province were shade-dried and grinned mechanically. A. densiflora’s n-hexane extract, and A. annua’s petroleum ether and methanol extract were prepared. Gas chromotography was performed to measure the artemisinin quantity in A.annua extracts. Dimethylsulfoxide (DMSO, 8 μl/0.5 ml medium) was used for dissolving the extracts. Cultivation of healthy and infected PBM cells was carried out with RPMI 1640 medium. Cells were put into trial wells as 2x106 cells in 0.5 ml medium per well. A. densiflora was added at concentrations of 125, 62.5, 15.625, 7.8125, 3.9062 μg/ml and A. annua was added at concentrations of 125, 62.5, 31.25, 15.625, 7.8125, 3.9062, 1.9531 μg/ml on PBM cells. In control wells medium was added only. Distinction of dead and alive cells was made by trypan blue staining of sampled cells at 24th and 48th hours of the trial. Mann Whitney U test was applied for the statistical analysis of trial and control groups; a p value of <0.05 and over was accepted significant. 4.12 g of extract was achieved from 90.5 g drog prepared from A.densiflora, while 5.56 g and 2.28 g of extract was achieved via methanol and petroleum ether extractions, respectively, from 100 g drog prepared from A.annua. 1.14 ± 0.07 mg/ 100 g and 4.54 ± 0.10 mg/100g of artemisinin was yielded after methanol and petroleum ether extraction of A.annua. A.densiflora extract killed both healthy and infected cells at all concentrations when compared with the control group (p<0.05). A.annua methanol extract did not have any negative effect on healthy cells. A mild affect was seen on infected cells at concentration of 125 μg/ml. A.annua petroleum ether extract had no negative effect on healthy cells, but killed infected cells at concentration of 15.625 μg/ml and over at 24th hour of trial (p<0.05). All infected cells died at concentration of 62.5 μg/ml at 48th hour. Consequently, it was proven that PBM cells infected with T. annulata schizonts are effectively destroyed in vitro by A. annua petroleum ether extract at concentrations of 15.625–62.5 μg/ml without damaging healthy cells. Trial of this extract on patients is suggested.
Plant extracts have significant potentials for the treatment of parasitic infections. Naphtaquinones and artemisinin are plant based molecules used against blood parasites in veterinary and human medicine. Arnebia densiflora (A.densiflora) and Artemisia annua (A.annua) grow in most parts of our country and have medical value due to their naphtaquinone and artemisinin contents, respectively. In this study, effects of the extracts of these plants on peripheral blood mononuclear (PBM) cells infected with Theileria annulata schizonts were investigated. For this aim, roots of A. densiflora collected in Eskisehir province and over-soil parts of A.annua collected in Bursa province were shade-dried and grinned mechanically. A. densiflora’s n-hexane extract, and A. annua’s petroleum ether and methanol extract were prepared. Gas chromotography was performed to measure the artemisinin quantity in A.annua extracts. Dimethylsulfoxide (DMSO, 8 μl/0.5 ml medium) was used for dissolving the extracts. Cultivation of healthy and infected PBM cells was carried out with RPMI 1640 medium. Cells were put into trial wells as 2x106 cells in 0.5 ml medium per well. A. densiflora was added at concentrations of 125, 62.5, 15.625, 7.8125, 3.9062 μg/ml and A. annua was added at concentrations of 125, 62.5, 31.25, 15.625, 7.8125, 3.9062, 1.9531 μg/ml on PBM cells. In control wells medium was added only. Distinction of dead and alive cells was made by trypan blue staining of sampled cells at 24th and 48th hours of the trial. Mann Whitney U test was applied for the statistical analysis of trial and control groups; a p value of <0.05 and over was accepted significant. 4.12 g of extract was achieved from 90.5 g drog prepared from A.densiflora, while 5.56 g and 2.28 g of extract was achieved via methanol and petroleum ether extractions, respectively, from 100 g drog prepared from A.annua. 1.14 ± 0.07 mg/ 100 g and 4.54 ± 0.10 mg/100g of artemisinin was yielded after methanol and petroleum ether extraction of A.annua. A.densiflora extract killed both healthy and infected cells at all concentrations when compared with the control group (p<0.05). A.annua methanol extract did not have any negative effect on healthy cells. A mild affect was seen on infected cells at concentration of 125 μg/ml. A.annua petroleum ether extract had no negative effect on healthy cells, but killed infected cells at concentration of 15.625 μg/ml and over at 24th hour of trial (p<0.05). All infected cells died at concentration of 62.5 μg/ml at 48th hour. Consequently, it was proven that PBM cells infected with T. annulata schizonts are effectively destroyed in vitro by A. annua petroleum ether extract at concentrations of 15.625–62.5 μg/ml without damaging healthy cells. Trial of this extract on patients is suggested.
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Keywords
Theileria annulata, Arnebia densiflora, Artemisia annua, Naftakinonlar, Artemisinin, Naphtaquinones
Citation
Girişgin, O. (2008). Bazı bitki ekstratlarının theileria annulata'ya karşı in vitro etkisi. Yayınlanmamış doktora tezi. Uludağ Üniversitesi Sağlık Bilimleri Enstitüsü.