Publication: Silybum marianum bitki ekstraktının antioksidan ve SARS-CoV-2'ye karşı antiviral aktivitelerinin in vitro modeller ile belirlenmesi
Abstract
COVID-19 pandemisi 700 milyon kişiyi etkilemiş, yaklaşık 7 milyon kişinin ölümüne sebep olmuştur. Yüksek bulaşıcılığa sahip SARS-CoV-2 virüsünün öldürücülüğü de yüksektir. Bu virüsün insanları enfekte etmesini engellemek için konakçı ile etkileşime girmesini ve virüsün konakçı içerisinde replikasyonunu inhibe etmek gerekmektedir. Virüsün konakçıya tutunduğu Spike proteini, konakçının ACE2 reseptörü, viral proliferasyonu sağlayan RdRp enzimi, fonksiyonel enzimlerin oluşmasını sağlayan Mpro enzimi kullanılan ilaçların inhibe etmesi amaçlanan hedeflerdir. Hastalığın tedavisi ve önlenmesi için ilaç araştırmaları halen devam etmekte olup kullanılan ilaçların etkinliğini artırmak için alternatif tedavi yöntemleri araştırılmaktadır. FDA tarafından acil kullanım izni çıkarılan aslen farklı virüslere karşı antiviral nitelikli bazı ilaçlardan tedavide yararlanılmaktadır. Alternatif tedavi yöntemi olarak geleneksel tıbba başvurulmaktadır. Bazı bitkilerin içeriklerinde bulunan fitokimyasallar viral proteinlerin inhibisyonunda etkinlik göstermektedirler. Deve dikeni (Silybum marianum) bitkisi de yüzyıllardır viral enfeksiyonlarda kullanılmış bir bitkidir. Silybum marianum bitkisinin Hepatit C, Chugunya, Mayarovirüs, SARS-CoV gibi diğer virüslere karşı etkinliği de in vitro ve klinilk çalışmalarla kanıtlanmıştır. Yapılan bilgisayar temelli çalışmalarda bu bitkiye özgü fitokimyasalların SARS-CoV-2’yi inhibe edebilecek özellikler taşıdığı saptanmıştır. Bu tez çalışmasının amacı Silybum marianum bitkisinin SARS-CoV-2 enfeksiyonuna karşı antiviral etkisinin araştırılmasıdır. Bitki ekstraktının biyoetkinlik çalışmaları i) in vitro hücre hatlarında sitotoksik etkiler - MTT kolorimetrik testi; ii) hücre morfolojine etkisi Hematoksilen – Eosin immünohistokimyasal boyama; iii) hücre apoptozu üzerine etkileri Annexin V/ PI immünofloresan boyama; ıv) antioksidan kapasitesi DCFDA floresan testi; v) ACE2-RBD bağlanma inhibisyonu – ELISA testi; vi) Mpro enzim inhibisyonu FRET testi; ve vii) SARS-CoV-2 enfeksiyonuna karşı antiviral potansiyeli in vitro pseudovirüs S1 nötralizasyon testi ile belirlenmiştir. Bitkilerden elde edilen doğal bileşenler; sentetik ilaçlara kıyasla daha az yan etkili, sürdürülebilir, kolay elde edilebilir ve biyouyumlu olmaları açısından tedaviye yardımcı önemli bir alternatif olmuşlardır. Bu tez çalışmasının günümüzde halen etkisini tam olarak yitirmemiş olan SARS-Cov-2 virüsünün bulaşını ve enfeksiyonunu engellemek adına katkıda bulunacağı düşünülmektedir. Bu tez çalışması sonucunda Silybum marianum bitkisinin antioksidan nitelik gösterdiği, RBD-ACE2 bağlanmasını 82.86 µg/ ml dozunda %50 inhibe ettiği, viral Mpro enziminin fonksiyonunu 100 µg/ ml dozunda %97.8 oranında inhibe ettiği belirlenmiştir. Bu sonuçlara dayanarak bu bitki ekstraktının SARS-CoV-2 virüsüne karşı kullanılacak etkin bir takviye olabileceği sonucuna varılmıştır.
The COVID-19 pandemic has affected 700 million people and caused approximately 7 million deaths. The lethality of the highly contagious SARS-CoV-2 virus is also high. To prevent this virus from infecting individuals, it is necessary to interfere with its interaction with the host and inhibit its replication within the host. Targets aimed to be inhibited by drugs include the Spike protein through which the virus attaches to the host, the host's ACE2 receptor, the RdRp enzyme responsible for viral proliferation, and the Mpro enzyme that facilitates the formation of functional enzymes. Research on drug treatment and prevention of the disease is still ongoing, with alternative treatment methods being explored to enhance the effectiveness of existing drugs. Some antiviral drugs originally approved by the FDA for emergency use against different viruses are being used in treatment. Traditional medicine is also being explored as an alternative treatment method. Phytochemicals found in some plants have shown efficacy in inhibiting viral proteins. Milk thistle (Silybum marianum) has been used for centuries in the treatment of viral infections. The efficacy of Silybum marianum against other viruses such as Hepatitis C, Chikungunya, Mayaro virus, and SARS-CoV has been demonstrated through in vitro and clinical studies. Computer-based studies have found that phytochemicals specific to this plant may possess properties capable of inhibiting SARS-CoV-2. The aim of this thesis is to investigate the antiviral effect of Silybum marianum against SARS-CoV-2 infection. The bioactivity studies of the plant extract include i) cytotoxic effects on in vitro cell lines - MTT colorimetric assay; ii) effects on cell morphology - Hematoxylin-Eosin immunohistochemical staining; iii) effects on cell apoptosis - Annexin V/ PI immunofluorescence staining; iv) antioxidant capacity - DCFDA fluorescence assay; v) ACE2-RBD binding inhibition - ELISA assay; vi) Mpro enzyme inhibition - FRET assay; and vii) antiviral potential against SARS-CoV-2 infection determined by in vitro pseudovirus S1 neutralization assay. Natural compounds derived from plants have emerged as significant alternatives to synthetic drugs due to their comparatively fewer side effects, sustainability, ease of access, and biocompatibility. It is believed that this thesis study will contribute to preventing the transmission and infection of the SARS-CoV-2 virus, which has not yet lost its full impact. The findings of this thesis demonstrate that the plant Silybum marianum exhibits antioxidant properties, inhibits the RBD-ACE2 binding by 50% at a dose of 82.86 µg/ ml, and inhibits the function of the viral Mpro enzyme by 97.8% at a dose of 100 µg/ ml. Based on these results, it is concluded that this plant extract could be an effective supplement against the SARS-CoV-2 virus.
The COVID-19 pandemic has affected 700 million people and caused approximately 7 million deaths. The lethality of the highly contagious SARS-CoV-2 virus is also high. To prevent this virus from infecting individuals, it is necessary to interfere with its interaction with the host and inhibit its replication within the host. Targets aimed to be inhibited by drugs include the Spike protein through which the virus attaches to the host, the host's ACE2 receptor, the RdRp enzyme responsible for viral proliferation, and the Mpro enzyme that facilitates the formation of functional enzymes. Research on drug treatment and prevention of the disease is still ongoing, with alternative treatment methods being explored to enhance the effectiveness of existing drugs. Some antiviral drugs originally approved by the FDA for emergency use against different viruses are being used in treatment. Traditional medicine is also being explored as an alternative treatment method. Phytochemicals found in some plants have shown efficacy in inhibiting viral proteins. Milk thistle (Silybum marianum) has been used for centuries in the treatment of viral infections. The efficacy of Silybum marianum against other viruses such as Hepatitis C, Chikungunya, Mayaro virus, and SARS-CoV has been demonstrated through in vitro and clinical studies. Computer-based studies have found that phytochemicals specific to this plant may possess properties capable of inhibiting SARS-CoV-2. The aim of this thesis is to investigate the antiviral effect of Silybum marianum against SARS-CoV-2 infection. The bioactivity studies of the plant extract include i) cytotoxic effects on in vitro cell lines - MTT colorimetric assay; ii) effects on cell morphology - Hematoxylin-Eosin immunohistochemical staining; iii) effects on cell apoptosis - Annexin V/ PI immunofluorescence staining; iv) antioxidant capacity - DCFDA fluorescence assay; v) ACE2-RBD binding inhibition - ELISA assay; vi) Mpro enzyme inhibition - FRET assay; and vii) antiviral potential against SARS-CoV-2 infection determined by in vitro pseudovirus S1 neutralization assay. Natural compounds derived from plants have emerged as significant alternatives to synthetic drugs due to their comparatively fewer side effects, sustainability, ease of access, and biocompatibility. It is believed that this thesis study will contribute to preventing the transmission and infection of the SARS-CoV-2 virus, which has not yet lost its full impact. The findings of this thesis demonstrate that the plant Silybum marianum exhibits antioxidant properties, inhibits the RBD-ACE2 binding by 50% at a dose of 82.86 µg/ ml, and inhibits the function of the viral Mpro enzyme by 97.8% at a dose of 100 µg/ ml. Based on these results, it is concluded that this plant extract could be an effective supplement against the SARS-CoV-2 virus.