Impact of protein binding on the analytical detectability and anticancer activity of thymoquinone |
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Authors: | Nahed El-Najjar Raimo A Ketola Teemu Nissil? Timo Mauriala Maxim Antopolsky Janne J?nis Hala Gali-Muhtasib Arto Urtti Heikki Vuorela |
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Institution: | (1) Division of Pharmaceutical Biology, Faculty of Pharmacy, University of Helsinki, Helsinki, Finland;(2) Centre for Drug Research, University of Helsinki, Helsinki, Finland;(3) Division of Pharmaceutical Chemistry, Faculty of Pharmacy, University of Helsinki, Helsinki, Finland;(4) Department of Chemistry, University of Eastern Finland, Joensuu, Finland;(5) Department of Biology, American University of Beirut, Beirut, Lebanon;(6) Division of Pharmaceutical Biology, Faculty of Pharmacy, University of Helsinki, P.O. Box 56, (Viikinkaari 5E), FI-00014 Helsinki, Finland; |
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Abstract: | Thymoquinone (TQ), an active component of Nigella sativa L., is known to have anti-cancer and anti-inflammatory effects; however, no studies on its analytical detection in serum
and its protein binding have been published. Using high performance liquid chromatography analysis, we show that the average
recovery of TQ from serum is 2.5% at 10 μg/ml of TQ and 72% at 100 μg/ml. The low recovery of TQ from serum is due to its
extensive binding to plasma proteins, as more than 99% of TQ was bound within 30 min of incubation. The binding of TQ to the
major plasma proteins, bovine serum albumin (BSA) and alpha −1 acid glycoprotein (AGP), was studied and found to be 94.5 ± 1.7%
for BSA and 99.1 ± 0.1% for AGP. Mass spectrometric analysis revealed that TQ was bound covalently to BSA, specifically on
Cyst-34. Using WST-1 proliferation assay, we showed that BSA plays a protective role against TQ-induced cell death; pre-incubation
with BSA prevented TQ from exerting its anti-proliferative effects against DLD-1 and HCT-116 human colon cancer cells. On
the other hand, binding of TQ to AGP did not alter its anti-proliferative activity against both cell lines. When TQ was pre-incubated
with AGP prior to the addition of BSA, the activity of TQ against DLD-1 was maintained, suggesting that AGP prevented the
binding of TQ to BSA. This is the first time the covalent binding and inhibitory effect of BSA on TQ is documented. These
data offer new grounds for TQ future pharmacokinetic analysis in vivo. |
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Keywords: | Thymoquinone Mass spectrometry Serum Protein binding Anticancer activity |
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