Interaction of Flavonoids from Woodwardia unigemmata with Bovine Serum Albumin (BSA): Application of Spectroscopic Techniques and Molecular Modeling Methods

Phytochemical investigation on the methanol extract of Woodwardia unigemmata resulted in the isolation of seven flavonoids, including one new flavonol acylglycoside (1). The structures of these compounds were elucidated on the basis of extensive spectroscopic analysis and comparison of literature data. The multidrug resistance (MDR) reversing activity was evaluated for the isolated compounds using doxorubicin-resistant K562/A02 cells model. Compound 6 showed comparable MDR reversing effect to verapamil. Furthermore, the interaction between compounds and bovine serum albumin (BSA) was investigated by spectroscopic methods, including steady-state fluorescence, synchronous fluorescence, circular dichroism (CD) spectroscopies, and molecular docking approach. The experimental results indicated that the seven flavonoids bind to BSA by static quenching mechanisms. The negative ΔH and ΔS values indicated that van der Waals interactions and hydrogen bonds contributed in the binding of compounds 2–6 to BSA. In the case of compounds 1 and 7 systems, the hydrophobic interactions play a major role. The binding of compounds to BSA causes slight changes in the secondary structure of BSA. There are two binding sites of compound 6 on BSA and site I is the main site according to the molecular docking studies and the site marker competitive binding assay.


Introduction
Multidrug resistance (MDR) renders the insensitivity of cancer cells to a diverse panel of anticancer agents, and it is the largest obstacle to cancer chemotherapy. A wide variety of natural products belonging to different classes have been found to have MDR-modulating activity [1,2]. Nevertheless, none of the MDR-reversal agents currently is available for clinical use, as they suffered from unpredictable pharmacokinetic behavior and toxicity [3]. Therefore, the exploration and development of potent and safe MDR reversal agents is still urgently needed.
Serum albumin (SA), a type of globular protein, is the most plentiful blood protein in mammals. The distribution of drugs is usually directed by SA, as most drugs circulate in plasma and reach the

Structure Elucidation of Compounds 1-7
The methanol extract of the rhizomes of W. japonica was fractionated and purified by repeated column chromatography as described in the experimental section, leading to the isolation of a new compound 1 along with six known compounds 2-7 ( Figure 1).
Compound 1 was obtained as an amorphous powder with the molecular formula C 33 H 36 O 17 , as determined by HR-ESI-MS (m/z 705.2027 [M + H] + ; calc. 705.2025). The 1 H-and 13 C-NMR (Table 1) spectra indicated that 1 was a flavonol with glycosidic and acyl moieties. The chemical shift and coupling constant data of the aromatic protons together with their corresponding 13 C-NMR chemical shifts obtained from Heteronuclear Single Quantum Correlation (HSQC) and Heteronuclear Multiple Bond Correlation (HMBC) experiments confirmed the identity of kaempferol as the aglycone [11]. The 1 H-NMR also indicated the presence of two sugar moieties with two anomeric protons at δH 5.64( 1 H, brs, H-1 ) and 5.58 ( 1 H, brs, H-1 ), which correspond to the carbon signals at δC 100. 5 and 98.1 in the HSQC spectrum. Thus, the glycosyl moiety of 1 consisted of two sugar units. Two rhamnosyl moieties were presumed by analysis of the 13 C-NMR data for monosaccharide. After sugar composition analysis, the presence of two L-rhamnose was confirmed [12,13]. The α-configuration of the rhamnose units were observed from the slight broadening of the appropriate H-1 and H-1 signals [14]. The presence of the HMBC correlations between the rhamnosyl anomericproton H-1 at δH 5.64 and the resonance of C-3 at δC 134.1, and between the rhamnosyl anomericproton H-1 at δH 5.58 and the resonance of C-7 at δC 130.6 suggested glycosidation at C-3 and C-7.     The remaining sub-structure of 1 was deduced by analysis of the 1 H-, 13 C-and 2D-NMR spectroscopic data to possess three acetoxy groups [δH 2.13 (3H, s), 2.05 (3H, s) and 1.99 (3H, s)]. The acetyl groups were determined to be linked at the C-3 , C-4 and C-4 due to the presence of the cross-peaks between in the HMBC spectrum ( Figure 2). Accordingly, the structure of 1 was elucidated as kaempferol3 The remaining sub-structure of 1 was deduced by analysis of the 1 H, 13 C and 2D NMR spectroscopic data to possess three acetoxy groups [δH 2.13 (3H, s), 2.05 (3H, s) and 1.99 (3H, s)]. The acetyl groups were determined to be linked at the C-3′′, C-4′′ and C-4′′′ due to the presence of the cross-peaks between in the HMBC spectrum ( Figure 2). Accordingly, the structure of 1 was elucidated as kaempferol3  (7) were identified on the basis of their spectroscopic profiles and comparison to the published data [15][16][17].

Pharmacological Studies
To determine the reversal effect of compounds 1-7 on resistant tumor cells, the cytotoxicity of the compounds towards K562, and their MDR variants K562/A02 cells was first measured by the MTT method. All compounds showed no cytotoxicity to both cell lines. The ability of compounds 1-7 to reverse MDR to doxorubicin (DOX) in cancer cells was investigated at a non-cytotoxic concentration (10 μM). Those compounds overcame the multidrug resistance (MDR) for doxorubicin-resistant K562/A02 cells, and the tested compounds showed the remarkable reversal effect (Table 2). Verapamil was a positive control. Those compounds are potential natural products that deserve more investigations to develop novel drugs against multifactorial drug-resistant cancers.

Pharmacological Studies
To determine the reversal effect of compounds 1-7 on resistant tumor cells, the cytotoxicity of the compounds towards K562, and their MDR variants K562/A02 cells was first measured by the MTT method. All compounds showed no cytotoxicity to both cell lines. The ability of compounds 1-7 to reverse MDR to doxorubicin (DOX) in cancer cells was investigated at a non-cytotoxic concentration (10 µM). Those compounds overcame the multidrug resistance (MDR) for doxorubicin-resistant K562/A02 cells, and the tested compounds showed the remarkable reversal effect ( Table 2). Verapamil was a positive control. Those compounds are potential natural products that deserve more investigations to develop novel drugs against multifactorial drug-resistant cancers. Fluorescence measurements were performed to investigate whether compounds 1-7 interact with BSA. The intrinsic fluorescence of BSA is caused by Trp, Tyr, and Phe residues. Trp residues dominate the fluorescence spectrum of BSA due to its high quantum yield and the ability to quench the emission of Tyr and Phe residues through energy transfer [6]. BSA intramolecular forces and conformation can be affected and changed by small molecule binding.
To correct the inner filter effects of compounds and BSA, fluorescence intensity was calculated as: where F cor and F obs are the corrected and observed fluorescence intensities, respectively, and A ex and A em are the absorption of the systems at the excitation and emission wavelength, respectively. All fluorescence data mentioned in this work is intensity corrected. The fluorescence quenching spectra of BSA at various concentrations of compounds are shown in Figure 3. The Figure 3A-G indicates the compounds' concentration dependent fluorescence quenching of BSA in the presence of 0-30 µM compounds. Despite the considerable fluorescence quenching in BSA, the emission maxima remained unchanged throughout the addition.

Fluorescence Quenching of BSA Induced by Compounds
Fluorescence measurements were performed to investigate whether compounds 1-7 interact with BSA. The intrinsic fluorescence of BSA is caused by Trp, Tyr, and Phe residues. Trp residues dominate the fluorescence spectrum of BSA due to its high quantum yield and the ability to quench the emission of Tyr and Phe residues through energy transfer [6]. BSA intramolecular forces and conformation can be affected and changed by small molecule binding.
To correct the inner filter effects of compounds and BSA, fluorescence intensity was calculated as: where Fcor and Fobs are the corrected and observed fluorescence intensities, respectively, and Aex and Aem are the absorption of the systems at the excitation and emission wavelength, respectively. All fluorescence data mentioned in this work is intensity corrected. The fluorescence quenching spectra of BSA at various concentrations of compounds are shown in Figure 3. The Figure 3A-G indicates the compounds' concentration dependent fluorescence quenching of BSA in the presence of 0-30 μM compounds. Despite the considerable fluorescence quenching in BSA, the emission maxima remained unchanged throughout the addition.   quenching is characterized by two types of mechanisms, usually classified as dynamic and static. Dynamic quenching refers to a process that involves the fluorophore and the quencher coming into contact during transient existence of the excited state, whereas static quenching refers to the formation of fluorophore-quencher complex [18]. Fluorescence quenching is described by the Stern-Volmer equation: where F 0 and F are the fluorescence intensities before and after the addition of the quencher, respectively, k q is the bimolecular quenching constant, τ 0 is the lifetime of the fluorophore in the absence of the quencher (τ 0 = 10 −8 s), [Q] is the concentration of the quencher, and K sv is the Stern-Volmer quenching constant. Hence, the Stern-Volmer equation was applied to determine K sv and k q by linear regression of F 0 /F sv [Q] [19]. K sv is shown in Table 3. Figure 4 shows the modified Stern-Volmer plots of the fluorescence quenching of BSA by compounds at different temperatures (289 K, 297 K and 307 K). Table 3. Binding and thermodynamic parameters for the interaction between compounds 1-7 and BSA at different temperatures, pH = 7.4. From the results of the Table 3 and Figure 4A-G, the quenching constant K sv of these systems are decreased with increase in temperature indicating a static quenching mechanism of the protein fluorescence. Furthermore, values of k q were calculated for the interaction of these compounds with BSA. k q values were found to be of the order of 10 12 , which are 100 times greater than maximum scatter collision quenching constant value 2.0 × 10 10 L mol −1 s −1 . It indicates that compounds interact with BSA in the static quenching manner [20]. Equation (3) is used to calculate the binding constant (K b ) and number of binding sites (n) [21]: where F and F 0 are fluorescence intensities with and without quencher, respectively. K b is binding constant, n is the number of binding sites per BSA molecule and [Q] is the concentration of compounds. Figure 5 shows the binding equilibrium plots for the fluorescence quenching of BSA by compounds 1-7 at 289, 297 K and 307 K. Values of K b and n gained from Equation (3) are given in Table 3. The number of binding sites in all systems approximates to 1 indicating that only one binding site in protein is reactive to the compounds 1-7. The binding constants K b of 2-6 are decreased with increasing temperature, which was in accordance with the change of K sv . The K b of compounds 1 and 7 are increased with the increasing temperature, which is denoted differently with respect to compounds 2-6.
compounds 1-7 at 289, 297 K and 307 K. Values of Kb and n gained from Equation (3) are given in Table 3. The number of binding sites in all systems approximates to 1 indicating that only one binding site in protein is reactive to the compounds 1-7. The binding constants Kb of 2-6 are decreased with increasing temperature, which was in accordance with the change of Ksv. The Kb of compounds 1 and 7 are increased with the increasing temperature, which is denoted differently with respect to compounds 2-6.   Table 3. The number of binding sites in all systems approximates to 1 indicating that only one binding site in protein is reactive to the compounds 1-7. The binding constants Kb of 2-6 are decreased with increasing temperature, which was in accordance with the change of Ksv. The Kb of compounds 1 and 7 are increased with the increasing temperature, which is denoted differently with respect to compounds 2-6.

Types of Interaction Forces between BSA and Compounds
The binding forces contributing to protein interactions with small molecular substrates often include van der Waals interactions, hydrophobic forces, electrostatic interactions, and hydrogen bonding. The signs and magnitudes of thermodynamic parameters, such as standard enthalpy change (∆H), standard entropy change (∆S) and free energy (∆G) of binding reaction provide evidence for binding mode between the small molecules and macromolecule. The thermodynamic parameters are evaluated using the the Van't Hoff e equations [22]: where K is the binding constant at the corresponding temperature, R is the gas constant, and T is absolute temperature. Due to the ∆H and ∆S signs of the system, the binding mechanism between a small molecule and protein can be determined. Accordingly, hydrophobic forces are dominant when ∆H > 0 and ∆S > 0, van der Waals interactions and hydrogen bonds contributed to binding when ∆H < 0 and ∆S < 0, and ∆H < 0 and ∆S > 0 are characteristics for electrostatic interactions [22,23]. The values of ∆H, ∆S and ∆G are listed in Table 3. The negative values of ∆G mean that the binding process is spontaneous. The negative ∆H and ∆S values indicate that hydrogen bonds and van der Waals forces played a major role in the interaction of the compounds 2-6 system. Both positive ∆S and ∆H in the case of compounds 1 and 7 suggest the presence of hydrophobic interactions.

Synchronous Fluorescence Spectroscopic Studies
Synchronous fluorescence spectroscopy is a very useful method to study the microenvironment of amino acid residues by measuring the emission wavelength shift. The synchronous fluorescence of protein at ∆λ = 15 nm and ∆λ = 60 nm are characteristic of Tyr and Trp residues, respectively. The ∆λ value represents the difference between excitation and emission wavelengths and is an important parameter [24]. The synchronous fluorescence spectra of BSA in the presence of different concentration of the compounds are shown in Figures 6 and 7. It can be seen that the intensities of synchronous fluorescence gradually decreased upon addition of compounds, which is consistent with the steady state fluorescence results. At the same time, the maximum emission wavelength almost does not change when the ∆λ was set at 15 nm. A slight red shift take place at the ∆λ = 60 nm. However, no shift was observed in fluorescence quenching experiments, suggesting a similar microenvironment surrounding Trp residues upon ligands binding.

Circular Dichroism Studies
In order to determine the effect of compounds binding on the secondary structure of BSA, a circular dichroism (CD) spectroscopic analysis was performed. As can be observed in Figure 8, the CD spectra of BSA exhibit two negative bands at 208 nm and 222 nm characteristic of α-helical structures in the protein to gain an understanding on the results of the CD spectra of BSA in the presence of compounds the α-helical content of BSA is evaluated from the following equations: where MRE 208 is mean residue ellipticity observed at 208 nm., C p is the molar concentration of the protein, n is the number of amino acid residues, and l is the path length of the cell. 4000 and 33,000 are the MRE values of a β-form with random coil conformation and a pure α-helix at 208 nm, respectively.

Circular Dichroism Studies
In order to determine the effect of compounds binding on the secondary structure of BSA, a circular dichroism (CD) spectroscopic analysis was performed. As can be observed in Figure 8, the CD spectra of BSA exhibit two negative bands at 208 nm and 222 nm characteristic of α-helical structures in the protein to gain an understanding on the results of the CD spectra of BSA in the presence of compounds the α-helical content of BSA is evaluated from the following equations: where MRE208 is mean residue ellipticity observed at 208 nm., Cp is the molar concentration of the protein, n is the number of amino acid residues, and l is the path length of the cell. 4000 and 33,000 are the MRE values of a β-form with random coil conformation and a pure α-helix at 208 nm, respectively. A molar ratio of 1:6 for BSA: compounds was used for the CD measurements. From the above equations, the α-helix contents of BSA for the compound-BSA complexes are 60 .12% for 1, 59.01% for  2, 60. 08% for 3, 59.28% for 4, 59.59% for 5, 64.17% for 6, and 60.49% for 7, which are slightly changed A molar ratio of 1:6 for BSA: compounds was used for the CD measurements. From the above equations, the α-helix contents of BSA for the compound-BSA complexes are 60.12% for 1, 59.01% for 2, 60. 08% for 3, 59.28% for 4, 59.59% for 5, 64.17% for 6, and 60.49% for 7, which are slightly changed compared with the native BSA value (58.61%). It can be seen from the data that the binding of compounds with BSA causes slight conformational change. These results are in agreement with those obtained from synchronous fluorescence spectra.

Energy Transfer from BSA to Compounds
Förster's non-radiative energy transfer theory is widely used to estimate the spatial distances between a biomolecule and a small molecule. The energy transfer efficiency (E) from the donor (BSA) to the acceptor (the compounds) can be determined by the following equation [25,26]: where E is the energy transfer efficiency, F and F 0 are the fluorescence intensities of BSA in the presence and absence of compounds, r is the distance between acceptor and donor and R 0 is the critical distance when the transfer efficiency is 50%. The R 0 can be calculated by Equation (10): where k 2 is the spatial orientation factor of the dipole with k 2 = 2/3, N is the refractive index of the medium and its value is 1.366, Φ is the fluorescence quantum yield of the donor and Φ = 0.118. J is the overlap integral of the fluorescence emission spectrum of the donor and the absorption spectrum of the acceptor [25]. J is given by Equation (7): where F(λ) is the fluorescence intensity of the fluorescent donor at wavelength, and ε(λ) is the molar absorption coefficient of the acceptor at wavelength. The overlap of the absorption spectra of compounds and the fluorescence emission spectra of BSA are shown in Figure 9 A-G. The calculated R 0 and r binding distances are showed in Table 4. A transfer of energy could take place through direct electrodynamic interaction between the primarily excited molecule and its neighbors, when the distance between the donor and the acceptor is approach in the range of 2-8 nm. As the distances between BSA and compounds are 2-8 nm scale and 0.5R 0 < r < 1.5R 0 , suggesting that the energy transfer from BSA to compounds occurs with high probability. The calculated r-value was larger than that of R 0 , further indicating that the fluorescence quenching of BSA induced by compounds 1-7 is the result of a static quenching mechanism [27,28].

The determination of Binding Sites of Compound 6 on BSA
The BSA has been previously reported to have two major specific drug-binding sites, which are defined as site I and site II, respectively. Binding site location for compound 6 was analyzed through competitive binding assay and molecular docking, since this compound has the strongest activity. Warfarin as the marker for site I and ibuprofen for site II was used in this experiment, respectively. The concentration of the markers and BSA were both 5 μM, while the concentration of the compound was 0-30 μM. Fluorescence emission spectra of the mixed solutions of BSA and site markers following a concentration increment of compound 6 were recorded and the results were presented in Table 5. It can be observed that the Kb and log Kb values of the compound with BSA decreased markedly in the presence of warfarin and ibuprofen, indicating that there are competitive interactions between compound 6 and the two site maker with BSA. Therefore, it can be deduced that the compound 6 may interact with BSA at both site I and site II. Nevertheless, the value of Kb is much lower in the presence of warfarin, suggesting that site I is the main binding site.  Figure 10). The detailed binding mode of compound 6 to the Site II binding site in subdomain IIIA of the BSA was shown in Figure 11. The acetyl group of compound 6 fit at the bottom

The determination of Binding Sites of Compound 6 on BSA
The BSA has been previously reported to have two major specific drug-binding sites, which are defined as site I and site II, respectively. Binding site location for compound 6 was analyzed through competitive binding assay and molecular docking, since this compound has the strongest activity. Warfarin as the marker for site I and ibuprofen for site II was used in this experiment, respectively. The concentration of the markers and BSA were both 5 µM, while the concentration of the compound was 0-30 µM. Fluorescence emission spectra of the mixed solutions of BSA and site markers following a concentration increment of compound 6 were recorded and the results were presented in Table 5. It can be observed that the K b and log K b values of the compound with BSA decreased markedly in the presence of warfarin and ibuprofen, indicating that there are competitive interactions between compound 6 and the two site maker with BSA. Therefore, it can be deduced that the compound 6 may interact with BSA at both site I and site II. Nevertheless, the value of K b is much lower in the presence of warfarin, suggesting that site I is the main binding site. Compound 6 was docked into the Site II (IIIA site) and Site I (IIA site) binding sites of the BSA, respectively ( Figure 10). The detailed binding mode of compound 6 to the Site II binding site in subdomain IIIA of the BSA was shown in Figure 11. The acetyl group of compound 6 fit at the bottom of the BSA pocket and made a high density of van der Waals contacts, whereas the rhamnopyranosyl group of compound 6 was positioned at the entrance of the pocket and makes only a few contacts. Detailed analysis showed that the acetyl group of compound 6 stretched into the hydrophobic pocket that consisted of Cys-391, Phe-402, Val-432 and Cys-437, while the 4-hydroxylphenyl group of compound 6 was located at another hydrophobic pocket, surrounded by the residues Pro-383, Leu-386, Ile-387 and Leu-452, forming a stable hydrophobic binding. In addition, cation-π interactions were observed between the kaempferol scaffold of compound 6 and the residues Arg-409, Lys-413 and Arg-484. Importantly, six hydrogen bond interactions were observed between the compound 6 and the residues Thr-448, Arg-409 and Tyr-410 of the BSA. of the BSA pocket and made a high density of van der Waals contacts, whereas the rhamnopyranosyl group of compound 6 was positioned at the entrance of the pocket and makes only a few contacts. Detailed analysis showed that the acetyl group of compound 6 stretched into the hydrophobic pocket that consisted of Cys-391, Phe-402, Val-432 and Cys-437, while the 4-hydroxylphenyl group of compound 6 was located at another hydrophobic pocket, surrounded by the residues Pro-383, Leu-386, Ile-387 and Leu-452, forming a stable hydrophobic binding. In addition, cation-π interactions were observed between the kaempferol scaffold of compound 6 and the residues Arg-409, Lys-413 and Arg-484. Importantly, six hydrogen bond interactions were observed between the compound 6 and the residues Thr-448, Arg-409 and Tyr-410 of the BSA.   of the BSA pocket and made a high density of van der Waals contacts, whereas the rhamnopyranosyl group of compound 6 was positioned at the entrance of the pocket and makes only a few contacts. Detailed analysis showed that the acetyl group of compound 6 stretched into the hydrophobic pocket that consisted of Cys-391, Phe-402, Val-432 and Cys-437, while the 4-hydroxylphenyl group of compound 6 was located at another hydrophobic pocket, surrounded by the residues Pro-383, Leu-386, Ile-387 and Leu-452, forming a stable hydrophobic binding. In addition, cation-π interactions were observed between the kaempferol scaffold of compound 6 and the residues Arg-409, Lys-413 and Arg-484. Importantly, six hydrogen bond interactions were observed between the compound 6 and the residues Thr-448, Arg-409 and Tyr-410 of the BSA.   To gain further insight into the binding interaction between compound 6 and the BSA in the molecular level, compound 6 was docked to the Site I of the BSA (Figure 12). The rhamnopyranosyl group of compound 6 fit at the bottom of the BSA pocket and made a high density of van der Waals contacts, whereas the other two sides of compound 6 were positioned near the entrance of the pocket and makes only a few contacts. Detailed analysis showed that the acetyl group of compound 6 stretched into the hydrophobic pocket that consisted of Trp-213, Val-342 and Pro-446, while the kaempferol scaffold of compound 6 was located at the hydrophobic pocket, surrounded by the residues Ile-289, Ala-290 and Val-292, forming a stable hydrophobic binding. Moreover, cation-π interactions were observed between the kaempferol scaffold of compound 6 and the residues Arg-194, Arg-198, Arg-217, Lys-221 and Lys-294. Importantly, six hydrogen bond interactions were observed between the compound 6 and the residues Arg-194, Arg-198, Arg-217, and Lys-294. All of these interactions helped compound 6 to anchor in the binding site of BSA. Molecular docking analysis suggests a better binding interaction for compound 6 with site I, and this is consistent with the results of competitive binding assay. To gain further insight into the binding interaction between compound 6 and the BSA in the molecular level, compound 6 was docked to the Site I of the BSA (Figure 12). The rhamnopyranosyl group of compound 6 fit at the bottom of the BSA pocket and made a high density of van der Waals contacts, whereas the other two sides of compound 6 were positioned near the entrance of the pocket and makes only a few contacts. Detailed analysis showed that the acetyl group of compound 6 stretched into the hydrophobic pocket that consisted of Trp-213, Val-342 and Pro-446, while the kaempferol scaffold of compound 6 was located at the hydrophobic pocket, surrounded by the residues Ile-289, Ala-290 and Val-292, forming a stable hydrophobic binding. Moreover, cation-π interactions were observed between the kaempferol scaffold of compound 6 and the residues Arg-194, Arg-198, Arg-217, Lys-221 and Lys-294. Importantly, six hydrogen bond interactions were observed between the compound 6 and the residues Arg-194, Arg-198, Arg-217, and Lys-294. All of these interactions helped compound 6 to anchor in the binding site of BSA. Molecular docking analysis suggests a better binding interaction for compound 6 with site I, and this is consistent with the results of competitive binding assay. In summary, the above molecular simulations give us a rational explanation of the interactions between compound 6 and the BSA, which will provide a good structural basis to explain the fluorescence quenching of BSA emission in the presence of compound 6.
The present study provided the detailed information about the binding characteristics and of compounds 1-7 to BSA, which provides useful data for future pharmacological studies.

Chemicals and Instrumentation
Fatty acid-free BSA was obtained from Shanghai Shenhang Biotechnology Co., Ltd. (Shanghai, China). The stock solution of compounds 1-7 was prepared in distilled water, respectively. All of the above solutions were kept in the dark at 0-4 °C. Tris-HCl buffer solution (0.05 mol L −1 , pH 7.4) In summary, the above molecular simulations give us a rational explanation of the interactions between compound 6 and the BSA, which will provide a good structural basis to explain the fluorescence quenching of BSA emission in the presence of compound 6.
The present study provided the detailed information about the binding characteristics and of compounds 1-7 to BSA, which provides useful data for future pharmacological studies.

Chemicals and Instrumentation
Fatty acid-free BSA was obtained from Shanghai Shenhang Biotechnology Co., Ltd. (Shanghai, China). The stock solution of compounds 1-7 was prepared in distilled water, respectively. 28.293, and center_z: 36.82 with dimensions size_x: 15, size_y: 15, and size_z: 15, and the IIA site (Site I) was identified as center_x: 95.873, center_y: 20.925, and center_z: 17.631 with dimensions size_x: 15, size_y: 15, and size_z: 15. In order to increase the docking accuracy, the value of exhaustiveness was set to 20. For Vina docking, the default parameters were used if it was not mentioned. The best-scoring pose as judged by the Vina docking score was chosen and visually analyzed using PyMoL software (1.3r1, DeLano Scientific LLC, South San Francisco, USA) [6].

Conclusions
In this work, seven compounds were isolated from the roots of Woodwardia unigemmata. The structures of the compounds were elucidated by spectroscopic analysis, as well as by comparison with literature data. Compound 6 showed comparable MDR reversing effect to verapamil. The interactions between complexes 1-7 and BSA were investigated employing different spectroscopic and molecular docking techniques. The experimental results indicated that these compounds bind to BSA by static quenching mechanisms. The negative ∆H and ∆S values indicated that van der Waals interactions and hydrogen bonds contributed in the binding of compounds 2-6 to BSA. On the other hand, for compounds 1 and 7, the hydrophobic interactions play a major role on BSA binding. There are two binding sites of compound 6 on BSA and the site I is the main one through the molecular docking and competitive binding assay. Meanwhile, there is a slight change in the secondary structure of BSA after the binding of compound 6.
The present study provided the detail information about the binding characteristics and conformation of compounds 1-7 on BSA, which provide useful data for future pharmacological studies.