Inclusion complexation between baicalein and β-cyclodextrin and the influence of β-cyclodextrin on the binding of baicalein with DNA: a spectroscopic approach

This work deals with the commonly studied cyclic oligosaccharide and gains importance as it is entered on a drug delivering carbohydrate and provides insight into the oligosaccharide complex–biomolecular interaction. The binding of a flavone, baicalein, to β-cyclodextrin and calf thymus DNA is studied. The binding of baicalein to calf thymus DNA in the presence of β-cyclodextrin is analysed using the UV–vis absorption and fluorescence spectroscopy. The mode of binding and structure of the baicalein–β-cyclodextrin complex are reported. The role of the structure and the stoichiometry of the inclusion complex of baicalein–β-cyclodextrin in its influence on DNA binding are analysed. Highlights • This paper deals with the binding of a flavone, baicalein to β-cyclodextrin and/or DNA. • The inclusion complexation between baicalein and β-cyclodextrin is analysed. • The stoichiometry and the binding strength of the inclusion complex is reported. • The role of β-cyclodextrin in tuning the binding of baicalein to DNA is emphasized. • Spectroscopic and docking analysis are used to articulate the results. Graphical abstract The influence of β-cyclodextrin on the binding of baicalein with DNA


Introduction
Flavones having hydroxyl group at the C-5 position are medicinally the most active amongst all flavones Sandip & Chabita, 2014;Wu et al., 2008;Zapata-Torres et al., 2004). Baicalein (5,6,7-trihydroxyflavone, BC) (Figure 1(a)) is such a flavonoid isolated from the root of Scutellaria baicalensis (Zhou, Hirotani, Yoshikawa, & Furuya, 1997). Owing to its medicinal properties, BC and several of its derivatives have been synthesized (Wu et al., 2008). The reported medicinal properties of BC are numerous. It is an antioxidant (Butković, Klasinc, & Bors, 2004), and it may reduce the oxidative stress and hence combat Alzheimer's disease as it prevents the cytotoxicity in amyloid β protein in PC 12 nerve cells (Heo, Kim, Choi, Shin, & Lee, 2004). It protects against the 6-OHDA-induced neurotoxicity through activation of Keap1/Nrf2/HO-1 and getting involved in PKCα and PI3 K/AKT signalling pathways (Zhang et al., 2012). It inhibits the production of reactive oxygen species and protects against endoplasmic reticulum stress-induced apoptosis (Choi et al., 2010). Synthetic BC derivatives cause apoptosis in human tumour cells (Ding et al., 2011). In a study of the pro-oxidant activities of flavonoids, including BC, in various cell-containing systems including human platelets, rat vascular smooth muscle cells, human umbilical vein endothelial cells, human THP-1 cells and fibroblast cells, only the BC generated hydroxyl radicals in human platelet suspension (Chou et al., 2007). According to a recent report on molecular effects of BC in HCT 116 cells and Caenorhabditis elegans, BC activates the transcriptional factor NF-E2-related factor 2 signalling pathway and prolongs lifespan (Havermann, Rohrig, Chovolou, Humpf, & Wätjen, 2013).
Cyclodextrins (CDs), which are cyclic oligosaccharides having a truncated cone structure, form inclusion complexes with several kinds of small molecules (Hingerty, Klar, Hardgrove, Betzel, & Saenger, 1984). The inclusion complexation occurs more frequently due to some lipophilic moiety of the molecule getting into the central cavity of CDs. The physicochemical properties of free molecules are different from those bound to CDs. Several approaches have been followed in improving the solubility of drugs, including complexation with CDs (Li & Purdy, 1992;. The effect of CDs on the antioxidant property of BC has been reported (Chao et al., 2011;Li, Han, & Chao, 2012). Amongst native CDs, β-cyclodextrin (β-CD, Figure 1(b)) has been used extensively to improve the solubility of drugs (Astray, Gonzalez-Barreiro, Mejuto, Rial-Otero, & Simal-Gándara, 2009;Qiuna, Xiaohui, Wei, Guixin, & Zhengtao, 2013;Sameena, Radhika, Enoch, & Murugesh, 2012). In this paper, we extend insight into the mode of binding of BC with β-CD using various spectroscopic approaches including fluorescence.
When small molecules bind to DNA, the topological modification of DNA gets inhibited (Raman, Jeyamurugan, Sudharsan, Karuppasamy, & Mitu, 2013). The apoptotic response is triggered by the formation of DNA adducts (Goldenber & Tannock, 1998). Due to the binding of small molecules, the functional property and the thermodynamic stability of DNA are altered (Lippard & Berg, 1994;Yu, Mikheikin, Streltsov, Zasedatelev, & Nabiev, 2001). CDs can influence the binding of flavonoids to DNA (Yousuf, Sudha, Murugesan, & Enoch, 2013;Sameena & Enoch, 2013). However, a detailed analysis of the effect of CDs on the binding of small molecules to DNA is scarce in the literature. Formation of inclusion complexes can achieve the (i) controlled release of drugs, (ii) targeting a specific receptor without undesired interactions at other sites (Allen & Cullis, 2004). Also, a partial encapsulation of molecules can stop their degradation (Marcolino, Zanin, Durrant, Benassi, & Matioli, 2011). Hence, this work was based on studying the exact mode of binding of BC to β-CD, and understanding how CD's encapsulation influenced the binding of BC to calf thymus DNA (ctDNA).

Materials and methods Materials
BC (Sigma-Aldrich, India) and β-cyclodextrin (Hi Media, India) were used as received. pH solutions were made with phosphate buffer, mixing phosphoric acid and sodium hydroxide (Products of Qualigens, India). For dilute aqueous solutions, pH scale is used. For super acid media, the Hammett acidity function, H 0 , and for super basic media its modified version H − is used. The Hammett-type acidity functions are defined in terms of a buffered medium containing a weak base B and its conjugate acid BH + as H 0 = pK a + log C B /C BH+ , where pK a is the dissociation constant of BH + . In solutions where the acidity was too great to allow a meaningful determination of pH, the ratio of (B) to (BH + ) could still be determined colorimetrically, and so define H 0 (Westheimer, 1997). In our experiment, a modified Hammett's acidity scale (Cave, Antesberger, Barbour, McKinlay, & Atwood, 2004) was used for the measurement of H 0 and pH below 2. All the solvents were the products of Merck (Spectral grade) and used as received. ctDNA was purchased from Merck (Genei), India. The purity of ctDNA was checked by optical measurements (A 260 /A 280 > 1.8, where A represents absorbance). ctDNA was dissolved in 50 mM NaCl prior to use. A 10-mM acetate buffer solution was used to adjust the pH value. Doubly distilled water was used throughout the course of the experiments and the solutions were stored at 0-4°C. All experiments were carried out at an ambient temperature of 27 ± 2°C.
The stock solution of BC was made in methanol due to its less solubility in water. The diluted test solutions were having the final concentration of methanol as 1%. The test solutions were prepared by adding microlitre amounts of the stock solution of BC, the required amount of aliquots of β-CD, acetate buffer solution and appropriate amount of ctDNA from its stock solution. The mixture was kept in dark to ensure the formation of a homogeneous solution. The measurements of absorption and fluorescence were made against the appropriate blank solution.

Preparation of BC-β-CD inclusion complexes
The inclusion complex of BC with β-CD was prepared by the co-precipitation of BC in methanol and β-CD in double-distilled water. In a typical procedure, the mixture (1:1) was stirred vigorously in an Ultra-sonicator and maintained for 30 min. The mixture was then warmed to 50°C for 10 min and kept at room temperature for two days. The homogenous solution was kept frozen until the solid separated out. They were allowed to defreeze and the solid complex precipitated out slowly from the homogeneous solution is separated by filtration. The solid was filtered and dried at room temperature. The obtained crude sample of BC was recrystallized in water-methanol mixture (90:10 ratio). The solid complexes were crushed to get a fine powder, stored at room temperature and characterized using Ultravioletvisible (UV-vis) absorption and Infrared (IR) spectroscopy. The morphology and the crystallite size of β-CD-bound and free forms of BC were analysed using Scanning electron microscope (SEM) and X-ray diffractometer (XRD). The structure of β-CD-bound BC was analysed using Nuclear magnetic resonance (NMR) and two-dimensional rotating frame nuclear overhauser effect spectroscopy (2D ROESY). The bond length and width of BC were viewed using Rasmol software to correlate the findings (Bernstein, 2011;Sameena et al., 2012).

Instruments
A Jasco-V 630 (Japan) double beam UV-vis spectrophotometer was used for absorption measurements using 1-cm path length cells. LS55 spectrofluorimeter (Perkin-Elmer, USA) equipped with a 120-W Xenon lamp for excitation served the measurement of fluorescence. Both the excitation and the emission band widths were set up at 2.5 nm. Time-resolved fluorescence measurements were done on a time-correlated single photon counting HORIBA spectrofluorimeter (Japan). Ultra-sonicator PCI 9L 250H (India) was used for sonication. Various pH solutions were prepared, adjusting the pH using Elico Li 120 pH meter, India. The surface topology of the samples was imaged using JEOL JSM 6360 SEM (Japan). The diffraction pattern of the samples was recorded using a Shimadzu XRD 6000 (Japan) operating with a monochromatic X-ray beam from CuKα radiation, at the voltage of 10 kV. IR spectra were recorded using KBr pellets on a Perkin-Elmer spectrometer RXI, USA. The 1 H NMR spectra were recorded on a Bruker 500 spectrometer operating at 500 MHz. The 2D ROESY spectra were recorded on a Bruker AV III instrument operating at 500 MHz. The chemical shift values were obtained downfield from Tetramethylsilane in parts per million (ppm). The mixing time for ROSEY spectra was 200 ms under the spin lock condition. The bond length and breadth of BC were calculated using Rasmol (Version 2.7.5.2) software (Bernstein, 2011). Molecular docking studies were carried out using the software Schrodinger, Glide 5.5 to determine the interaction between BC and DNA/β-CD by determining the Glide Score (G-Score). The B-DNA was used as the model for the theoretical studies. The structure of β-CD and duplex 5′-d(CCATTAATGG) 2-3 ′ were built and optimized by molecular mechanics (Lu, Wang, Lv, Zhang, & Liu, 2011;Sameena & Enoch, 2013). The ctDNA structure was downloaded from Protein data bank (ID, 3GJH).

Complexation of BC with β-CD
The UV-vis absorption spectra of BC with various added amounts of β-CD are shown in Figure 2 shows the absorption maximum at the wavelength of 273 and 322 nm in the aqueous solution. It may be because n-σ * and π-π * correspond to the 5,6,7-trihydroxy-4H-chromen-4-one and benzene moiety of BC molecule. The absorbance of BC increases at each addition of β-CD in increasing concentration. However, the shift of absorption band at 322 nm is not clearly visible as the band is not distinct at higher concentrations of β-CD. An approximately 4-nm hypsochromic shift of the 273 nm band is observed which occurs due to the migration of BC from the polar environment to the non-polar micro-environment, inside the cavity of β-CD. Hypsochromic and hyperchromic shifts mentioned above may be attributed to a complex formation between the fluorophore, BC and the β-CD. The inclusion complex formation between BC and β-CD can be represented in general by the following Equation (1).
The binding constant (K) as derived from Equation (1) can be represented as given in Equation (2).
here, A 0 is the absorbance of BC in water, A S is the absorbance at each concentration of β-CD; A′ is the absorbance at the highest concentration of β-CD and K is the binding constant. The fluorescence spectra of BC recorded in water and in aqueous β-CD solution with the excitation of BC of 322 nm are shown in Figure 2(b). The fluorescence maximum of BC is observed at 368 and 418 nm. The increase in the intensity of fluorescence is more pronounced than the increase in absorbance. The enhancement of fluorescence can be ascribed to the inclusion complex formation. On the addition of β-CD, the 418-nm fluorescence band is shifted to the blue region by 7 nm. This is due to the hydrophobic environment the BC molecule is in, as it shifts from the cage of water (solvent) molecules to the non-polar cavity of β-CD. Table 1 shows the absorption and fluorescence spectral data of BC-β-CD complex. The Benesi-Hildebrand plot (Enoch & Swaminathan, 2005;Lakowicz, 1999) plotted following Equation (4) is shown in the inset of Figure 2 in this equation, I is the intensity of fluorescence of BC at each concentration of β-CD, I′ is that at highest concentration of β-CD, I 0 is the intensity of fluorescence in water, K is the binding constant. The calculated K is of 291.88 mol −1 dm 3 (correlation coefficient: .97). The stoichiometry of the BC-β-CD complex is 1:1 as inferred from the linearity of the Benesi-Hildebrand plot. It is in accordance with the results of the absorption spectrometric titrations. The physical change being monitored can usually be the aggregate of the individual components and the K depends on the molecule in the excitation and emission determined by UV-vis absorption and fluorescence spectroscopy. These result in the variation in the binding constant. Figure 3 shows the time-resolved fluorescence spectra of BC in water and in β-CD. The fluorescence decay profile of BC in water is bi-exponential with two fluorescence lifetimes, viz. 1.47 ns and 6.39 ns. When β-CD is added, the decay profile becomes tri-exponential owing to the new β-CD-complexed species formed with a different fluorescence lifetime. The relative amplitude of the T1 and T2 states change with the addition of β-CD. This is due to the complexation of BC by β-CD. Time-resolved fluorescence spectral data are given in Table 2. The lifetime of the uncomplexed species only slightly decreased. The complex populations show an increase in lifetime due to the less probability of relaxation processes. The relative amplitude also changes. These support the complex formation. This is possible in the case of inclusion complex and less plausible in any non-inclusion complex. Figure 4(a) shows the absorption spectra of BC in water at various H 0 /pH values. A lowering of pH from 5 results in a hypochromic shift of absorbance at 254 nm, with a corresponding hyperchromic shift at around 320 nm. The shorter wavelength band vanishes at higher acidic conditions. There is an isosbestic point at 270 nm, due to the possible formation of cation of BC. The ground state pKa value for the neutral-mono-anion equilibrium was determined from the mid-point of absorption curve of the neutral form and it is 2.2. The effect of acid strength on the fluorescence of BC is shown in Figure 4(b) and (c). The addition of protons to BC causes a quenching of fluorescence at 415 nm. This may be a case of proton-induced fluorescence quenching. At a longer wavelength, there is formation of a new band which becomes more distinct below H 0 of -.84. The new band is centred at acidic conditions. This may be due to an exciplex formed between BC and the solvent in the more acidic condition. A decrease of acidity below H 0 of -1.85 does not fully quench the 415-nm band. Figure 5(a) shows the absorption spectra of BC at various H 0 /pH values, in aqueous β-CD. Between the pH range of 3.0-1.4 there is an isosbestic point observed in the absorption spectra (250 nm). Below pH 1.4, the absorption shows hypochromism at all wavelengths and the spectra do not pass through the isosbestic point. The calculated pK a for the protonated equilibrium is 2.0. The fluorescence spectra of BC with β-CD at various H 0 /pH values are shown in Figure 5(b) and (c). The 411-nm band is quenched in intensity due to the addition of protons. Unlike in water, the formation of a band at around 530 nm is not seen even at the H 0 of -1.85. There is observation of only a small red-shifted band at 435 nm. This band does not increase in intensity under further acidic conditions. Hence, these results imply that the formation of exciplex of BC with solvent is less likely when encapsulated in the β-CD cavity. The protonation of BC may require higher acidic conditions than H 0 of -1.85. However, under such conditions, the β-CD will be degraded by the high acidity. Hence, it is obvious that the fused ring containing hydroxyl groups of BC is encapsulated by the β-CD cavity and the phenyl ring remains free.
The solid β-CD inclusion complex of BC was analysed using the analysis of IR, UV-vis absorption and SEM. Supplementary Figure SI 1(a) and Table 3 show the comparison of the IR spectral data of BC and its β-CD inclusion complexes. Generally, the formation of the inclusion complex between a drug and β-CD results in a reduction in the transmittance of the corresponding bonds of the drug, due to the presence of hydrophobic interactions, Vander Waals forces and hydrogen bonds. Such a change is observed in the binding of some BC with β-CD. The absence of new peaks in the IR spectra of BC-β-CD complex, in comparison with the BC, rules out the chance of formation of any chemical bond in the inclusion complexes. An appreciable change in the char-   The crystallite size and strain of BC and its inclusion complex were calculated using XRD. The XRD pattern of BC-β-CD inclusion complex is given in Supplementary Figure SI 2(f). The crystalline material of BC produces main peaks (at 2θ of 10.1604, 11.3766 and 15.3335°) in the X-ray diffraction pattern. The diffraction pattern of a β-CD inclusion complex of BC produces differences in the diffraction patterns (at 2θ of 12.0147, 13.9700 and 18.0933°) of each individual phase of uncomplexed BC molecule. Using the wellknown Debye-Scherrer formula (as given in Equation 5), the calculated crystallite size of BC, β-CD and the BC-β-CD inclusion complex were 30, 18 and 32 nm, respectively. The crystallite size of BC-β-CD inclusion complex was very close to that of the physical size of the BC, which suggested that polycrystalline nanoparticles were not formed.
where D is the size of the crystal, λ is the wavelength of the radiation (=1.5418 Ǻ), θ is the diffraction angle, and β is the broadening factor (half width measured at half its maximum intensity). If the crystallite is strained then the d spacings will be changed (Fultz & Howe, 2013). The approximate relationship relating the mean inhomogeneous strain (ε) to the peak broadening produces βε, which is derived by differentiating Bragg's Law and relating the inhomogeneous strain to the differential δd/d. The lattice strains from displacements of the unit cells about their normal positions are often produced by domain  boundaries, dislocations, surfaces, etc. The peak broadening due to micro-strain (Bushroa, Rahbari, Masjuki, & Muhamad, 2012) will vary as given in Equation (6): where B and ε represent the peak width and strain, respectively. The strain in the BC, β-CD and BC-β-CD inclusion complex were calculated as .00028, .0011 and .00067, respectively. The structure of the BC to the β-CD complexes was characterized using the 1 H NMR and 2D-ROESY techniques. The 1 H NMR chemical shift values of the BC-β-CD complex are given in Table 4. The cross peaks of signals of BC protons and the protons of the β-CD are shown in Figure 6. An off-diagonal peak was found, which is due to the cross-correlation of the hydroxyl proton of BC (8.820 ppm) with H5 proton (3.644 ppm) of β-CD (Figure 1(b)). The H5 proton resides at the inner part of the lower rim of β-CD. The cross peaks observed for the hydroxyl protons of BC at 12.643 and 10.601 ppm cross-correlated with the H3 proton (3.298 ppm) signal of β-CD. Since the H3 proton exists in the wider rim of β-CD and facing towards the inner part of the hydrophobic cavity, it is highly possible that the spatial interaction of the hydroxyl protons of the chromenone moiety in the BC occurs with the β-CD host molecule. Hence, we conclude that the chromenone part of BC is encapsulated by β-CD. This view is supported by the molecular docking and the docking pose of BC and β-CD as shown in Figure 7(a)-(c). The proposed structure of 1:1 BC-β-CD inclusion complex is shown in Figure 8.

Binding of BC with ctDNA in the presence and the absence of β-CD
The strength of the BC-ctDNA binding was analysed in the presence of β-CD. The change in the absorbance of BC and BC-β-CD was followed by the addition of ctDNA. Figure 9(a) shows the presence of two main bands (λ max , 273 and 322 nm) in the absorption spectrum of BC. The presence of ctDNA (1.17 × 10 −5 , mol dm −3 ) results in the 2-and 3-nm blue shifts of both the absorption bands respectively. This shows the impact of ctDNA on chromenone and benzene moiety of BC. The possible interactions of BC with DNA were investigated by molecular docking. The glide score of the docking was −2.98 kcal mol −1 for BC-DNA binding. Molecular docking studies showed the score of phobic interaction of BC with DNA as 0 kcal mol −1 . The major interactions found were hydrogen and electrostatic bonding. Figure 7(d) shows the involvement of chromenone and benzene moiety in the binding by means of hydrogen and electrostatic interaction respectively. The O 2 oxygen of T5 of B-strand DNA interact with hydroxyl hydrogen of 5,6,7-trihydroxy-4H-chromen-4-one moiety by means  of hydrogen bonding (bond length, 1.974 Å). The occurrence of electrostatic interaction of BC with ctDNA was confirmed by means of a competitive binding study of ctDNA with methylene blue (MB) and BC (Figure 10(a)) (Ali, Reza, & Sara, 2009). The addition of BC did not result in any shift of absorption wavelength of MB-ctDNA complex (664 nm). This observation shows the existence of an electrostatic interaction also, along with the hydrogen bonding interactions. The K for BC-ctDNA was calculated as 2.22 × 10 3 mol −1 dm 3 (correlation coefficient, .99) by utilizing the following Equation (7) (Ibrahim, Shehatta, & Al-Nayeli, 2002;Yousuf, Sudha, Murugesan, & Enoch, 2013) (inset of Figure 9(a)).
where A 0 and A are the absorbance of the free quest and the apparent one, and ε G and ε H-G are their absorption coefficients, respectively. Figure 9(b) shows the influence of β-CD on the binding of BC-ctDNA. The absorption bands of the BC-β-CD inclusion complex were found at 269 and 322 nm. A 3-nm blue shift was observed for π-π * corresponding to the binding of benzene moiety of BC to DNA binding. The K for interaction between BC and ctDNA in the presence of β-CD was 1.50 × 10 4 , mol −1 dm 3 (correlation coefficient, .97) (inset of Figure 9(b)). The higher K might be due to the interaction of benzene part of BC with ctDNA after encapsulation of another moiety (chromenone) by β-CD. This is proved by following the absorption titration of BC-ctDNA complex, with the addition of β-CD solution in aliquots (Figure 10(b)). The small K (1.71 mol −1 dm 3 , inset of Figure 10(b), correlation coefficient, .99 denotes the insignificant accessibility of the strongly bound BC-DNA for the inclusion complexation by β-CD.
The fluorescence spectra of the binding of BC and BC-β-CD to ctDNA were recorded. Figure 11(a) shows the fluorescence quenching of BC with ctDNA. About 7-nm red shift from the fluorescence emission λ emi , 368 nm of BC was seen. This might be attributed to the hydrogen bonding between hydroxyl groups of BC with ctDNA. The presence of hydroxyl groups in chromenone moiety of BC can be seen. This observation is in line with the absorption measurements studies. The Stern-Volmer quenching constant, K SV (Lakowicz, 1999) was 9.55 × 10 4 mol −1 dm 3 (correlation coefficient: .99) (Figure 11(b)) obtained by utilizing the following Equation (8): where F 0 and F are the fluorescence intensities in the absence and presence of the quencher, respectively. K SV and [Q] are the Stern-Volmer quenching constant and the quencher concentration, respectively. The Equation (9) (Lu et al., 2011;Sameena & Enoch, 2013) was used for the plotting of double logarithm regression curve of log(F 0 -F/F) vs. log [Q] to find the binding affinity, K b and the binding site, "n" (of the interaction of BC with ctDNA). The number of "n" and the K b are .82 and 1.33 × 10 4 mol −1 dm 3 respectively (correlation coefficient: .99) (Figure 11(c)).
where F 0 and F are the fluorescence intensities in the absence and presence of the quencher respectively.
[Q] is the quencher concentration. The addition of ctDNA to BC-β-CD resulted in fluorescence quenching of BC-β-CD with insignificant shifts of spectrum (Figure 12(a)). The comparison of the fluo-  rescence intensity of BC with that of BC-β-CD showed nearly 53% increase in the fluorescence of BC in the presence of β-CD. The Stern-Volmer quenching constant K SV was found to as 1.11 × 104 mol −1 dm 3 (correlation coefficient: .98) (Figure 12(b)). The lowering of quenching constant of BC with ctDNA with insignificant blue shift shows either the extract of the interacted moiety or involvement of another moiety for binding with DNA. Utilization of Equation (9) yields K b and n as 4.26 mol -1 dm 3 and .26 (correlation coefficient: .97) (Figure 12(c)), respectively. The "n" and the K b of BC with DNA got much altered in the presence of β-CD, due to inclusion complexation of BC with β-CD through the chromenone moiety. Thus, the chromenone moiety has a preference for the interaction with β-CD and ctDNA over the benzene ring. In case of tri component system, BC binds strongly to β-CD and the remaining benzene moiety interacts with ctDNA, resulting in the decrease in binding site and affinity of BC with ctDNA.
The addition of β-CD resulted in the quenching of BC-ctDNA complex, which might be due to the formation of the BC-ctDNA-β-CD complex (Figure 10(c)). It was a non-linear, downward concave curve which might be due to the presence of two fluorophore populations, one of which was not accessible to the quencher. These molecules could have partially had the interaction with BC. This is in line with the discussion given in the previous section on absorption titration on BC-DNA vs. β-CD. Thus, the stronger binding of BC to DNA restricts the β-CD complexation of BC bound to DNA. In these cases, the Stern-Volmer constant of the accessible fraction (K a ) can be determined from Equation (10) (Lakowicz, 1999), where f a is the fraction of the initial fluorescence which remains accessible to the quencher. The modified form of the Stern-Volmer equation allowed f a and K a to be determined graphically as shown in Figure 10(d). A plot of F 0 /ΔF vs. 1/[Q] yields f a −1 as the intercept and (f a K a ) −1 as the slope. The calculated Stern-Volmer constant of the accessible fraction was 990 mol −1 dm 3 (correlation coefficient: .98). The results are in accordance with the results of the UV-vis absorption titration. The work on the binding study of BC to DNA based on the assessment of interaction of DNA with BC-β-CD inclusion complex and finding the rigidity of this inclusion complex when the binding of DNA. The question of whether the host, β-CD delivers BC to DNA or there is formation of a ternary species DNA: BC-β-CD is clarified. The accessibility of β-CD or DNA to bound-BC depends on the binding strength of BC-DNA and BC-β-CD. That's where the determination of the binding parameter K and stoichiometry for BC-DNA or BC-β-CD gain importance. Both the free BC and its β-CD inclusion complex bind to DNA. If β-CD transfers BC to DNA and there is not ternary formation, there cannot be change in the binding strength between BC and DNA. There is a variation in the binding strength and binding moiety for BC and β-CD-bound BC. There is access for BC-bound DNA for β-CD, in which case the non-linear fitting on the accessibility of BC-bound DNA by CD is analysed. The proposed model of the binding of BC to DNA in the absence and the presence of β-CD are schematically represented in Figure 13(a) and (b), respectively.

Conclusion
The stoichiometry of the baicalein-β-cyclodextrin inclusion complex is 1:1 and the binding constant is 1.17 × 10 2 and 291.88 mol −1 dm 3 as determined by UV-vis absorption and fluorescence spectroscopy, respectively. The ground and the excited state pKa values of the protonation equilibrium of baicalein are not significantly altered by β-CD, (pKa, 2.2 and 2.0) which may be due to a weaker non-covalent binding between β-CD and baicalein. The structure and the stoichiometry of the inclusion complex between baicalein and β-cyclodextrin plays a major role in DNA binding. The chromenone moiety of the baicalein is accommodated by the cyclodextrin cavity when forming the inclusion complex. The phenyl ring of baicalein stands outside host molecule and it gets bound to DNA due to its availability for binding. This work gains importance as baicalein is entered on a drug delivering carbohydrate and provides insight into the oligosaccharide complex-biomolecular interaction.