## 1 Introduction  
Serum albumin protein is the abundant constituent of the plasma and plays a pivotal role in transporting endogenous and/or exogenous ligands [1]. Ligands bind to the albumin, which influences both the pharmacodynamics and pharmacokinetics of the ligand [2, 3]. All the drugs reversibly bind to the plasma proteins, and their effect generally is dependent on the free drug fraction present in the systemic circulation. The binding capacity of the drug to plasma proteins is a major consideration for selecting the dosage of the drug. Thus, the free drug fraction present in the plasma is generally responsible for the drug's therapeutic effect [4, 5, 6]. The BSA is structurally homologous and presents a 75.6% sequence similarity to human serum albumin (HSA). Therefore, BSA was chosen in the present study as a substituent to HSA since it has the same ligand-binding properties and is readily available and cost-effective [7, 8]. BSA has three primary ligand-binding sites in subdomain IIA, IIIA, and IB [9, 10, 11, 12]. The intrinsic fluorescence of BSA, which is associated with two tryptophan amino acids (Trp-134 and Trp-213), is generally used for studies on drug-protein interactions. Drugs reversibly bind to the plasma proteins, and the free drug fraction present in the systemic circulation at a given time is considered responsible for its therapeutic effect. Therefore, a competition between two or more drugs can occur upon their coadministration for the same BSA-binding site. This phenomenon leads to the competition or displacement of bound drugs to their binding sites, resulting in altered pharmacokinetics [13, 14]. In addition, ligands binding to BSA might also cause conformational changes in the protein molecule, thus altering their ligand-binding phenomenon [15, 16]. Coadministration of drugs might lead to elevated plasma concentrations of the displaced drug because of competition or displacement from the binding site. Thus, interaction at the serum albumin-binding level might cause toxic effects in the case of narrow therapeutic index drugs. These interactions may also lead to a higher incidence of adverse events, higher volume of distribution, or a subtherapeutic response due to shorter elimination half-life [17]. Phenolic-compound flavonoids are commonly used as antioxidants in nutritional supplements [18]. A citrus flavonoid glycoside, RUT, is a potent antioxidant with anti-inflammatory activities [18, 19]. RTN is believed to have an anticancer potential and is recommended as a dietary supplement to prevent and manage cancer [20]. Another citrus flavonoid glycoside, NAR, which has high antioxidant potential, is also used as a dietary supplement [21] and has anticancer properties [22]. The hydroxcinamic-acid derivative sinapic acid (SIN) has shown anticancer activity by various mechanisms, including enzyme biotransformation (Figure 1) [23]. The ETB (Figure 1) is an ATP-competitive quinazoline derivative that works by binding to the epidermal growth factor receptor (EGFR), disabling its phosphorylation ability leading to apoptosis [24]. It is a tyrosine kinase inhibitor used to treat non-small-cell lung  
Figure 1: The chemical structures of (**A**): ETB; (**B**): RUT; (**C**): NAR; (**D**): SIN. cancer (NSCLC). In 2013, ETB was approved by FDA for use as a first-line treatment of NSCLC in patients whose tumors harbor EGFR exon-19 in-frame deletions or exon-21 (L858R) mutations. (20-b) [25]. Coadministering RTN, NAR, and SIN either as dietary supplements or as their presence in fruits or vegetables and ETB to treat NSCLC might cause some interactions, resulting in altered therapeutic benefit or higher incidence of adverse events to ETB [24; 26]. ETB's pharmacokinetics might be influenced by simultaneous administration of ETB along with these flavonoids [26; 27]. The present study investigates the effect of RUT, NAR, and SIN on the binding of ETB and BSA as a model protein.