Bioavailability and Activity of Natural Food Additive Triterpenoids as

Article pubs.acs.org/JAFC

Bioavailability and Activity of Natural Food Additive Triterpenoids as Influenced by Protein Wei Peng,†,‡,§ Fei Ding,*,‡,§,∥ Yu-Ting Jiang,⊥ and Yu-Kui Peng*,† †

College of Food Science & Engineering, Northwest A&F University, Yangling 712100, China Department of Chemistry, China Agricultural University, Beijing 100193, China ∥ Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, United States ⊥ Department of Chemistry, University of Ottawa, 10 Marie Curie, Ottawa, Ontario K1N 6N5, Canada ‡

S Supporting Information *

ABSTRACT: Triterpenoids were thought to be biologically ineffective for a very long time, but aggregating proof on their widely ranging pharmacological activities paired with a dubious toxicity portrait has motivated regenerated attraction for human health and disease. In the current contribution, our central goal was to integratively dissect the biointeraction of two typical triterpenoids, ursolic acid and oleanolic acid, by the most fundamental macromolecule bovine serum albumin (BSA) by employing molecular modeling, steady state and time-resolved fluorescence, and circular dichroism spectra at the molecular scale. Based on molecular modeling, subdomain IIA, which matches Sudlow’s site I, was allocated to retain high affinity for triterpenoids, but the affinity of ursolic acid with subdomain IIA is somewhat inferior compared to that of oleanolic acid, probably because the affinity differentiation arises from the different positions of the methyl group on the E-ring in the two triterpenoids. This sustains the site-specific ligands, and hydrophobic 8-anilino-1-naphthalenesulfonic acid probe results in arranging the triterpenoids at the warfarin−azapropazone site. The data of steady state and time-resolved fluorescence indicated that the recognition of triterpenoids by BSA produced quenching by a static type, in other words, the ground state BSA−triterpenoid complex formation with the affinities of 1.507/1.734, 1.042/1.186, and 0.8395/0.9863 × 104 M−1 at 298, 304, and 310 K for ursolic acid/oleanolic acid, respectively. Thermodynamic analyses show that the basic forces acting between BSA and triterpenoids are hydrogen bonds, van der Waals forces, and hydrophobic interactions; this occurrence provoked the alterations of the BSA spatial structure with a noticeable decline of α-helix evoking perturbation of the protein, as stemmed from circular dichroism, synchronous fluorescence, and three-dimensional fluorescence measurements. We anticipate that the complexation of plant triterpenoids with protein delineated here may be exploited as a biologically relevant model for evaluating the physiologically applicable noncovalent complexes in in vivo examination of triterpenoid properties such as accumulation, bioavailability, and distribution. KEYWORDS: food additive, triterpenoids, bovine serum albumin, molecular modeling, fluorescence, circular dichroism

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INTRODUCTION Triterpenoids are polycyclic chemicals derived from the linear hydrocarbon squalene, and they are secondary plant metabolites pervasively dispersed throughout the plant kingdom and noted for their exceptional structural variety and plentiful biological roles regarding human health and sickness prevention.1 They exist in plants in a free form, but also as esters and glycosidic conjugates named saponins. Among them, the ursolic acid, oleanolic acid, and betulinic acid series of pentacyclic triterpenes originating from α-amyrin, β-amyrin, and lupeol, respectively, are the most extensively spread pentacyclic triterpenes in plants.2 For instance, ursolic acid in apples, basil, bilberries, cranberries, and rosemary; oleanolic acid in olive fruit and leaves, Rosa woodsii, Prosopis glandulosa, Phordendron juniperinum, and Hyptis capitata; and betulinic acid in white birch (Betula pubescens), Ber tree (Ziziphus mauritiana), self-heal (Prunella vulgaris), Triphyophyllum peltatum, and Ancistrocladus heyneanus, where they may consist of as much as 20% of the dry weight of the separate origin.3 Although the biological duty of pentacyclic triterpenes in plant metabolism is not lucidly realized, they are emerge in a vast © 2014 American Chemical Society

scope of plants utilized in traditional remedies. Lately, ursolic acid and oleanolic acid (Figure 1) have been found to exercise significant actions as antioxidant, anti-inflammatory, antimicrobial, antiviral, antiulcer, hepatoprotective, immunomodulatory, hypolipidemic and lowering cholesterol, antiatherosclerotic, wound healing, anticoagulant, anticarcinogenic, and even antitumor agents.4,5 Concretely, ursolic acid could restrain tumorigenesis and tumor promotion and repress angiogenesis in human breast cancer cells, while oleanolic acid may inhibit proliferation and cause apoptosis in osteosarcoma cells.6 Liobikas et al.7 assayed the uncoupling and antioxidant activities of ursolic acid in male Wistar rat heart mitochondria; they found that ursolic acid in a concentration dependent behavior (EC50 of 4.1 ± 1.1 ng mL−1) originated statistically significant uncoupling of oxidative phosphorylation in the heart mitochondria, and a notable reduction in hydrogen peroxide Received: Revised: Accepted: Published: 2271

November 4, 2013 February 11, 2014 February 18, 2014 February 18, 2014 dx.doi.org/10.1021/jf4049512 | J. Agric. Food Chem. 2014, 62, 2271−2283

Journal of Agricultural and Food Chemistry

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μM) and diminished forward scatter, a result arriving statistically significantly at 5.0 μM, revealing cell shrinkage. Also, activation of Ca2+ entry, ceramide shaping, eliciting phospholipid scrambling and to a lesser extent hemolysis in human erythrocytes have been perceived upon addition of ursolic acid. These consequences were ascribed to frank evidence of the ursolic acid influencing erythrocytes from flowing blood and exciting the suicidal demise of erythrocytes, with a consequent generation in anemia. For all of these, the episteme of the existence and biological functions of these natural triterpenoid chemicals, in particular those derived from edible and pharmaceutical herbaceous plants, are of enormous relish. Blood plasma includes many proteins or other substances such as enzymes that are eagerly bound with ligands especially drugs and, consequently, impress the pharmacodynamics and pharmacokinetics of many drugs, but most investigations on the binding of drugs to assorted proteins in blood have evidently displayed that likely the most significant protein is albumin.12 Even though albumin has a net negative charge at the pH of blood, pH = 7.4, it binds both positively and negatively charged ligands, such as tetracyclines, digitoxin, salicylates, sulfonamide, phenylbutazone, barbiturates, acid dyes, vitamins C, uric acid, histamine, bilirubin, etc.13 Pharmacologically, albumin protein recognition is a weighty factor of pharmacokinetics as it decides how much of a drug will be accessible in its free state for therapeutic utility, nontarget effectiveness (side effects), toxicity, and circulation into other human body cabins.14 Drugs that are greatly bound to albumin protein generally have a low proportion of distribution and could be smaller promptly swept from the body as just the free section is usable for renal elimination. Depending on the expected target of a drug and the hopeful period of action, a high extent of albumin protein binding may or may not be an attractive characteristic.15 An in vitro albumin protein recognition forecasts the degree to which an objective chemical will be recognized by albumin protein in the same species in vivo. In arousing pharmacological and toxicological responses, drugs mostly connect either reversibly or irreversibly with target sites on intrastitial biopolymers or organelles, and therefore bring about changes of physicochemical or biochemical procedures in the living organism.16,17 As has been shown, although drugs can bind to different proteins in blood, the chief plasma and tissue protein which is typically chargeable for the broad spectrum binding of most drugs is albumin. Despite a large quantity of literature, there is little consolidated knowledge obtainable on the kinetic and physiological dispersal of albumin−triterpenoids in the human body. It is thereby proper to contemplate the function of albumin in influencing triterpenoid binding and pharmacokinetics. The objective of the present study was to inspect the molecular recognition as well as the solution structure of the complexes fashioned between the triterpenoids ursolic acid and oleanolic acid and the protein bovine serum albumin (abbreviation: BSA, Figure 2), by means of computer-aided molecular modeling, steady state and time-resolved fluorescence, site-specific ligands, and hydrophobic 8-anilino-1-naphthalenesulfonic acid displacement. Furthermore, the conformation of BSA protein upon complexation was monitored by circular dichroism (CD) and synchronous fluorescence along with three-dimensional fluorescence measurements. These clues stated here can probably be useful when exploiting new synthesized drugs, which have analogous configuration with triterpenoids, whether the

Figure 1. Molecular structure of (A) ursolic acid and (B) oleanolic acid.

production in mitochondria upon incubation with 5.0 ng mL−1 ursolic acid occurred. Therefore, they considered that mild mitochondrial uncoupling evoked through pharmacological concentrations (1.6−5.0 ng mL−1) of ursolic acid as discovered in some herbal preparations may be helpful in cardioprotection. In another study, oleanolic acid was found to obstruct lipopolysaccharide-intervened secretion of high-mobility group box 1 protein by endothelial cells as well as high-mobility group box 1 protein-mediated proinflammatory responses, which creates a decline in monocyte adhesion and migration.8 Oleanolic acid also deterred high-mobility group box 1 protein-interfered nuclear factor-κB and tumor necrosis factor-α production, which are the most principal inflammatory mediators. These discoveries could offer that oleanolic acid may be utilized as an anti-inflammatory ligand for vascular inflammatory infirmity medication. Owning to its easily obtainable, low-cost material and various biological roles, humankind in many cultures, as well as animals globally, have been reliably consuming these triterpenoids, e.g., ursolic acid and oleanolic acid, in their victual, or have been utilizing them for curative objects, for numerous centuries without marked disease actions.9,10 Accordingly, an exponential growth in the amount of information about bioactive ursolic acid and oleanolic acid or other triterpenoids over the early decade mirrors the increasing core in their concealed cosmeceutical, nutraceutical, and medicinal applications. Unfavorably, the demerit of applying ursolic acid and oleanolic acid, indeed, is the toxicity related to their hemolytic and cytostatic character.4 In a more recent examination, Jilani et al.11 described that contact of leukocyte depleted erythrocytes for 48 h to Ringer’s solution containing ursolic acid (0.5−10 2272

dx.doi.org/10.1021/jf4049512 | J. Agric. Food Chem. 2014, 62, 2271−2283


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pymol.org) was finally used for visualization of the molecular docking results. Steady State Fluorescence. Fluorescence emission spectra were obtained with a 1.0 cm path length quartz cell using a F-7000 spectrofluorimeter (Hitachi, Japan) equipped with a thermostatic bath. The excitation and emission slits were set at 5.0 nm each, intrinsic fluorescence was carried out by exciting the continuously stirred protein solution at 295 nm to favor tryptophan (Trp) excitation, and the emission spectra were read in the wavelength range of 300−450 nm at a scanning speed of 240 nm min−1. The reference sample consisting of the Tris−HCl buffer of triterpenoid in corresponding concentrations was subtracted from all fluorescence measurements. Time-Resolved Fluorescence. Time-resolved fluorescence was examined with a FLS920 spectrometer (Edinburgh Instruments, U.K.), using the time-correlated single photon counting system with a hydrogen flash lamp excitation source, in air equilibrated solution at ambient temperature. The excitation wavelength was 295 nm, and the number of counts gathered in the channel of maximum intensity was 4,000. The instrument response function (IRF) was gauged exploiting Ludox to scatter light at the excitation wavelength. The data were analyzed with a nonlinear least-squares iterative method utilizing Fluorescence Analysis Software Technology, which is a sophisticated software package designed by Edinburgh Photonics for the analysis of fluorescence and phosphorescence decay kinetics; IRF was deconvoluted from the experimental data, and the resolution limit after deconvolution was 0.2 ns. The value of χ2 (0.9−1.2), the Durbin− Watson parameter (greater than 1.7), and a visual inspection of the residuals were used to assess how well the calculated decay fit the data. Average fluorescence lifetime (τ) for multiexponential function fittings were from the following relation:19

Figure 2. The ribbon model of the BSA derived from X-ray diffraction crystallography (PDB: 4JK4) and the tryptophan (Trp) residues located in domain I and II. This diagram was fabricated using PyMOL on the basis of the atomic coordinates accessible at the Brookhaven Protein Data Bank (http://www.rcsb.org/pdb).

intention is to utilize their depot role or to avoid binding to BSA.

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EXPERIMENTAL SECTION

I(t ) =

Materials. Albumin from bovine serum (A7030, lyophilized powder, fatty acid free, globulin free, ≥98%), ursolic acid (U6753, ≥90%), oleanolic acid (O5504, ≥97%), and 8-anilino-1-naphthalenesulfonic acid (A1028, ≥97%) were received from Sigma-Aldrich (St. Louis, MO) and used without further purification, and deionized water was obtained from a Milli-Q Ultrapure Water Purification System from Millipore (Billerica, MA). Tris (0.2 M)−HCl (0.1 M) buffer of pH = 7.4, with ionic strength 0.1 in the presence of NaCl, was used, and the pH was measured with an Orion Star A211 pH Benchtop Meter (Thermo Scientific, Waltham, MA). Dilutions of the BSA stock solution (10 μM) in Tris−HCl buffer were prepared immediately before use, and the concentration of BSA was checked spectrophoto18 All other reagents employed were of metrically using E1% 1cm = 6.6. analytical grade and purchased from Sigma-Aldrich. Molecular Modeling. Molecular modeling of the BSA− triterpenoid complexes was operated on an SGI Fuel Visual Workstation. The crystal structure of BSA (entry codes 4JK4), determined at a resolution 2.65 Å, was retrieved from the Brookhaven Protein Data Bank (http://www.rcsb.org/pdb). After being imported in the program Sybyl Version 7.3 (http://tripos.com), BSA structure was carefully checked for atom and bond type correctness assignment. Hydrogen atoms were computationally added using the Sybyl Biopolymer and Build/Edit menus. To avoid negative acid/acid interactions and repulsive steric clashes, added hydrogen atoms were energy minimized with the Powell algorithm with 0.05 kcal mol−1 energy gradient convergence criteria for 1500 cycles; this procedure does not change positions to heavy atoms, and the potential of the three-dimensional structure of BSA was assigned according to the AMBER force field with Kollman all-atom charges. The twodimensional structure of triterpenoid was downloaded from PubChem (http://pubchem.ncbi.nlm.nih.gov), and the initial structure of the molecule was produced by Sybyl 7.3. The geometry of triterpenoid was subsequently optimized to minimal energy (tolerance of 0.5 kcal mol−1) using the Tripos force field with Gasteiger−Hückel charges, and the lowest energy conformer was utilized for the docking analysis. The Surflex-Dock program which employs an automatic flexible docking algorithm was applied to analyze the possible conformation of the ligand that binds to BSA, and the program PyMOL (http://www.

∑ Ai e−t/ τi i

(1)

where τi are fluorescence lifetimes and Ai are their relative amplitudes, with i variable from 1 to 2. Site-Specific Ligand. Binding location studies between BSA and triterpenoid in the presence of four typical site markers (phenylbutazone, flufenamic acid, digitoxin, and hemin) were executed using the fluorescence titration approach. The concentrations of BSA and site markers were held equimolar (1.0 μM), and then triterpenoid was added to the BSA-site marker mixtures. An excitation wavelength of 295 nm was chosen, and the fluorescence emission wavelength was acquired from 300 to 450 nm. Hydrophobic ANS Displacement. In the first series of experiments, BSA concentration was kept fixed at 1.0 μM, triterpenoid/ANS concentration was varied from 5.0 to 45 μM, and BSA fluorescence was gained (λex = 295 nm, λem = 341 nm). In the second series of experiments, triterpenoid was added to solutions of BSA and ANS held in equimolar concentration (1.0 μM), the concentration of triterpenoid was also varied from 5.0 to 45 μM, and the fluorescence of ANS was recorded (λex = 370 nm, λem = 465 nm). CD Spectra. Far-UV CD spectra were collected with a Jasco-815 spectropolarimeter (Jasco, Japan) equipped with a microcomputer; the apparatus was sufficiently purged with 99.9% dry nitrogen gas before starting the instrument, and then it was calibrated with d10camphorsulfonic acid. All the CD spectra were obtained at 298 K with a PFD-425S Peltier temperature controller attached to a water bath with an accuracy of ±0.1 °C. Each spectrum was performed with use of a precision quartz cuvette of 1.0 cm path length and taken at wavelengths between 200 and 260 nm, which provides a signal extremely sensitive to small secondary conformational distortions. Every determination was the average of five successive scans encoded with 0.1 nm step resolution and recorded at a speed of 50 nm min−1 and response time of 1 s. All observed CD data were baseline subtracted for buffer, and the estimation of the secondary structure elements was obtained by exploiting Jasco Spectra Manager II, which computes the different designations of secondary structures by comparison with CD spectra, determined from distinct proteins for which high-quality X-ray diffraction data are available.20 2273



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Figure 3. Molecular modeling of triterpenoids docked to BSA. Panel A shows docked triterpenoids in BSA at subdomain IIA, BSA displayed in surface colored in red, to triterpenoids, colored as per the atoms and endowed with opaque surface of electron spin density. Panels B and C indicate the amino acid residues involved in binding of triterpenoids. The ball-and-stick model represents triterpenoids, colored as per the atoms, and the pivotal amino acid residues around triterpenoids have been described in stick model; salmon stick model portrays hydrogen bonds between Arg-194, Ser-201 (B) and Arg-194, Ser-201, Arg-435 (C) residues and ursolic acid and oleanolic acid, respectively; white stick model narrates hydrophobic interactions between Leu-210, Ala-212, Trp-213, Leu-218, Val-342, Leu-454 (B) and Leu-210, Ala-212, Trp-213, Leu-218, Val-342, Leu-454 (C) residues and ursolic acid and oleanolic acid, respectively. Panel D interprets the conformational superposition of ursolic acid (magenta) and oleanolic acid (cyan) in the subdomain IIA of BSA. In this equation, F0 and F are the fluorescence intensities in the absence and presence of quencher, respectively, K and n are the association constant and the number of binding sites, respectively, and [Q] is the concentration of quencher. Thus, a plot of log(F0 − F)/F against log [Q] can be used to calculate K and n. Moreover, the fluorescence intensities were corrected for absorption of the exciting light and reabsorption of the emitted light to decrease the inner filter effect by using the following relationship:21

Three-Dimensional Fluorescence. The emission wavelength was reaped between 200 and 500 nm, the initial excitation wavelength was set to 200 nm with increment of 10 nm, the number of scanning curves was 16, and the other scanning parameters were identical to those for the steady state fluorescence above. Principle of Fluorescence Quenching. Fluorescence quenching refers to any process that decreases the fluorescence intensity of a sample. A variety of molecular interactions can result in quenching, such as excited state reactions, molecular rearrangements, energy transfer, ground state complex formation, and collisional quenching. Fluorescence quenching is described by the well-known Stern−Volmer equation:21

F0 = 1 + kqτ0[Q] = 1 + KSV[Q] F

Fcor = Fobs × e(Aex + Aem)/2

where Fcor and Fobs are the fluorescence intensities corrected and observed, respectively, and Aex and Aem are the absorption of the systems at the excitation and the emission wavelength, respectively. The fluorescence intensity utilized in this work is the corrected intensity. Statistical Analysis. All assays were executed in triplicate, and the mean values, standard deviations, and statistical differences were estimated using analysis of variance (ANOVA). The mean values were compared using Student’s t-test, and all statistic data were treated using the OriginPro Software (OriginLab Corporation, Northampton, MA, USA).

(2)

In this equation, F0 and F are the fluorescence intensities in the absence and presence of quencher, respectively, kq is the bimolecular quenching constant, τ0 is the lifetime of the fluorophore in the absence of quencher, [Q] is the concentration of quencher, and KSV is obtained by linear regression of a plot of F0/F versus [Q]. Calculation of Recognition Ability. When ligand molecules bind independently to a set of equivalent sites on a macromolecule, the equilibrium between free and bound ligand molecules is given by the following relation:22

log

F0 − F = log K + n log[Q] F

(4)

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RESULTS AND DISCUSSION Molecular Modeling. The purpose of ligand−receptor docking is the ability to prefigure the preponderant recognition patterns of a ligand with an acceptor such as protein or nucleic acid of established three-dimensional crystal structure. We first

(3) 2274



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although hydrogen bond energies may seem small, on the order of 4.0−25.0 kJ mol−1, it should be remembered that they commonly occur as a system of interconnections; thus they are additive and donate considerably to the comprehensive stability of systems such as the polypeptide polymer and protein or nucleic acid.28,29 It was evident at first glance that the hydrogen bonds between BSA and triterpenoids look very alike to one another, for example, the carbonyl group in triterpenoids could generate a hydrogen bond network with the Arg-194 residue in BSA, but they do have at least two differences between the two systems. The first is the number of hydrogen bonds. Careful examination of Figure 3B and Figure 3C shows that oleanolic acid may form two strong hydrogen bonds with Ser-201 residue, whereas only one hydrogen bond can be yielded for ursolic acid. Furthermore, another hydrogen bond could be formed between oleanolic acid and the Arg-435 residue. A second theme is the differentiation between these hydrogen bonds. Intuitively, the lengths of hydrogen bonds between ursolic acid and the Arg-194 residue are larger than the oleanolic acid; this means that oleanolic acid has greater affinity than the ursolic acid. These distinctions mentioned above macroscopically behave in the diverse affinities between triterpenoids and BSA, i.e., K = 1.419 × 104 M−1 (ursolic acid−BSA) and K = 1.811 × 104 M−1 (oleanolic acid−BSA), respectively. It is well testified that the biological activities of ursolic acid and oleanolic acid may be related to the methyl group on the Ering. Ovesná et al.30 conducted an in vitro study to scrutinize the protective effects of ursolic acid and oleanolic acid in leukemic cells. They lucidly revealed that in murine leukemia cells L1210 and K562 cells, the antioxidant activity of oleanolic acid measured by single cell gel electrophoresis was notably better in contrast with the antioxidant activity of ursolic acid. But for HL-60 cells, the percentage of ursolic acid protective outcome was 63.8%−61.6% at the concentration range of 2.5− 10 μM, and the antioxidant effect of oleanolic acid was reasonably inferiorly collated with the activity of ursolic acid; however, the inconsistency between the protective activity of ursolic acid and oleanolic acid was not remarkable. In a recent study, ursolic acid and oleanolic acid were found to be powerful inducers of apoptosis in four human liver cancer cell lines involving mitochondrial dysfunctions.31 Interestingly, the anticancer activities of the two triterpenoids were not uniform; ursolic acid at tantamount volume was larger than oleanolic acid in suppressing Na+-K+-ATPase activity in HepG2 cell; ursolic acid at 8.0 μM induced more DNA destruction than oleanolic acid in HepG2 and Hep3B cells, yet oleanolic acid at 8.0 μM stimulated more mitochondrial membrane latent decrease than ursolic acid in Huh7 and HA22T cells. The computational analyses quoted above, as a result, corresponded absolutely closely with these depictions found in the reference, i.e., the position of substituting group (−CH3) on the E-ring may quite likely have an effect on the noncovalent interactions between triterpenoids and the amino acid residues in BSA, and result in different affinities between these triterpenoids. Moreover, the significant driving forces in the BSA− triterpenoid conjugation are hydrogen bonds and hydrophobic interactions; we also presume that the three-dimensional spatial structure of BSA may be perturbed due to the triterpenoids operating on the amino acid residues in a polypeptide chain by noncovalent bonds. Consequently, the information manifested in solution experiments expatiated below, as deduced from steady state and time-resolved fluorescence, competitive ligand

used this computational modeling technique to visually look over the binding modes between the BSA and the triterpenoids. Majorek et al.23 and Bujacz et al.24,25 in the first place declared a structure of albumin from bovine serum to 2.70 Å and 2.47 Å, severally, derived from tetragonal crystals obtained from defatted commercial BSA. The tertiary structure of BSA received from these X-ray diffraction measurements is a heart-shaped molecule, and it includes 583 amino acid residues and has 9 loops formed by 17 disulfides. The three-dimensional configuration is composed of three homologous domains (I, II, and III), each domain in turn is the spawn of two subdomains (A and B), which are largely helical and widely cross-linked through several disulfide bridges. Besides, the 6 subdomains partake of a joint helical motif, and the motif mainly corresponds to the amino acids surrounded within the double disulfide bonds 1, 3, 4, 6, 7, and 9. Two tryptophans (Trp), namely, Trp-134 and Trp-213, situated in domain I and domain II, respectively, and Trp-134 residue are lying in a hydrophilic circumstance, close to the protein surface, while Trp-213 residue is encircled by a hydrophobic surrounding within a protein patch.12,26,27 The best docked energy conformation is shown in Figure 3; it was clear that both ursolic acid and oleanolic acid binding sites are located within subdomain IIA formed by six-helices (Figure 3A), but the recognition ability of subdomain IIA with the latter is higher, compared with the former. For the BSA−ursolic acid mixture, the oxygen atoms of the carbonyl group and the oxygen atom of the hydroxyl group in ursolic acid can make hydrogen bonds (Figure 3B) with the hydrogen atoms of the amino group in Arg-194 and the hydrogen atom of the hydroxyl group in Ser-201 residues; the bond lengths are 2.42 Å, 2.71 Å, 2.81 Å, 3.03 Å, and 2.43 Å, respectively. For BSA−oleanolic acid complex, the oxygen atoms of the carbonyl group, the oxygen atom of the hydroxyl group, the hydrogen atom of the hydroxyl group, and the oxygen atom of the hydroxyl group can form hydrogen bonds with the hydrogen atoms of the amino group in Arg-194, the hydrogen atom of the hydroxyl group in Ser-201, the oxygen atom of the hydroxyl group in Ser-201, and the hydrogen atom of the amino group in Arg-435 residues; the bond lengths, respectively, are 2.18 Å, 2.54 Å, 2.59 Å, 2.80 Å, 2.44 Å, 2.78 Å, and 3.10 Å (Figure 3C). On the basis of surface modification of the amino acid residues composed of subdomain IIA, we may ascertain a hydrophobic region of subdomain IIA composed of Leu-210, Ala-212, Trp-213, Leu-218, Val-342, and Leu-454 residues, the skeleton of 22 carbon atoms organized into five rings toward the domain, which implied that hydrophobic interaction was exerting a heavy effect between them. Apparently, ursolic acid and oleanolic acid have greatly close molecular structures but have only disparate sites of methyl group on the E-ring; if the methyl group at C19 of ursolic acid is shifted to C20, it turns to oleanolic acid. To elaborate the divergence of binding mode between ursolic acid and oleanolic acid, the optimized conformations of ursolic acid and oleanolic acid have been overlapped in the subdomain IIA (Figure 3D). It was effortlessly seen that the dissimilarities of both locality and conformation between ursolic acid and oleanolic acid were tiny. Therefore, we could speculate that the different position of one methyl group on the E-ring might give rise to moderately disparate affinities of triterpenoids. In comparison with these noncovalent interactions, particularly hydrogen bonds residing in the BSA−triterpenoid conjugation, we may obtain some beneficial information regarding this distinction. Chemically, 2275



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displacement, CD spectra, synchronous fluorescence, and three-dimensional fluorescence techniques, was applied to affirm upper molecular modeling corollaries. Photochemical Properties of BSA−Triterpenoids. The molecular recognition of triterpenoids by BSA was evaluated by the assessment of intrinsic fluorescence of Trp residues, and Figure S1 (Supporting Information) conveys the raw data for quenching of BSA fluorescence at pH = 7.4 by addition of triterpenoids. It will be noticed that BSA arose to have a fluorescence peak at 341 nm, following an excitation at 295 nm. In BSA, the three aromatic amino acid residues Phe, Tyr, and Trp are all fluorescent, but emission of BSA is controlled by Trp residues, which absorbs at the longest wavelength and exhibits the largest extinction coefficient.12,26,27 Clearly, a regular shortening of the Trp residues’ fluorescence intensity was perceived from Figure 4 accompanied by growth in the

triterpenoids, the data were dissected according to the Stern− Volmer eq 2, and the corresponding results fitted from Stern− Volmer plots (Figure 5) are collected in Table 1. Usually, a

Figure 4. Trp residue fluorescence quenching of BSA (1.0 μM) at pH = 7.4 and T = 298 K, plotted as expunction of fluorescence intensity (F/F0) against triterpenoid concentrations: 0, 5.0, 10, 15, 20, 25, 30, 35, 40, and 45 μM. The fluorescence intensity was read at λex = 295 nm and the λem maximum obtained at 341 nm. All data were corrected for quencher fluorescence, and each point was the mean of three independent observations ± SD ranging from 0.38% to 4.44%.

Figure 5. Stern−Volmer plot depicting Trp residue quenching of BSA (1.0 μM) at pH = 7.4 in the presence of 5.0, 10, 15, 20, 25, 30, 35, 40, and 45 μM ursolic acid (A) and oleanolic acid (B). Each data was the average of three separate determinations ± SD ranging from 0.62% to 4.64%.

concentration of triterpenoids. Under the present conditions triterpenoids displayed no fluorescence emission in the range 300−450 nm, which did not affect BSA intrinsic Trp residues’ fluorescence. These phenomenon explained plainly that there were association reactions between BSA and triterpenoids and the ligand was localized in the region where Trp residues located within or near the fluorophores.32 Further, the fluorescence quenching effect of oleanolic acid was better than that of ursolic acid, which denotes that oleanolic acid has a good affinity to complex with BSA, and this result is almost totally in agreement with the affinity imparity that branched from molecular modeling simulations. A similar finding has also been illustrated by Bourassa et al.33 for the molecular recognition of antioxidant polyphenols resveratrol, genistein, and curcumin by milk α- and β-caseins. Normally, quenching mechanism research can be employed to expose the localization of fluorophores in proteins and may also signify the dispersal of quenchers over middling large distances comparable to the dimension of proteins.21 In order to expound the essence of fluorescence quenching of BSA by

linear Stern−Volmer plot is exhibitive of a single kind of fluorophore, all equivalently attainable to quencher. The Stern−Volmer plots for triterpenoids are apparently linear, Table 1. Stern−Volmer (KSV) and Bimolecular (kq) Quenching Constants for the Molecular Recognition of BSA with Triterpenoids system BSA + ursolic acid

BSA + oleanolic acid

a

2276

T (K)

KSV (×104 M−1)

kq (×1012 M−1 s−1)

Ra

298 304 310 298 304 310

1.568 1.449 1.374 1.894 1.784 1.704

2.732 2.524 2.394 3.300 3.108 2.969

0.9996 0.9994 0.9992 0.9994 0.9995 0.9996

R is the correlation coefficient. dx.doi.org/10.1021/jf4049512 | J. Agric. Food Chem. 2014, 62, 2271−2283


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different ursolic acid or oleanolic acid concentrations, indicating evidently that fluorescence quenching is truly a static mechanism. The results match our previous discussion based on molecular modeling and steady state fluorescence that quenching is static in nature, triterpenoids binds in the vicinity of Trp residues in subdomain IIA, and BSA−triterpenoid complexes are formed between fluorophore and the triterpenoids, which is nonfluorescent. Recent research by Abou-Zied et al.22 also found similar phenomena when BSA from human serum recognizes with anticancer agents 6-hydroxyquinoline, 7hydroxyquinoline, and 8-hydroxyquinoline. Frequently, drugs possess some specificity for the binding site on the acceptor like target protein; if the specificity is lower, it alludes binding affinity is lower, and a bigger potion of drug will be needed, which is related with some side effects.34,35 Therefore the efficacy of a drug depends on its binding strength for the binding site as well as its binding affinity to generate the desired effects. In order to reckon the binding strength of triterpenoids to BSA, eq 3 was applied to estimate K and n by linear regression of a plot of the log(F0−F)/F versus log [Q] (Figure S3 in the Supporting Information), and the results are also displayed in Table 3. The affinity K reduced with rising temperature, showing the destabilization of the BSA− triterpenoid complex; likewise, the affinity of BSA with oleanolic acid is greater than that of ursolic acid, and this corroborates perfectly our preceding results found on molecular modeling that the strength of ursolic acid with BSA is lower than that of oleanolic acid. According to Dangles and Dufour36 and united with the recent reports on the subject of protein− ligand recognition, such as piperine, bile acids, hydroxyquinoline derivatives, retinol, retinoic acid, and folic acid,37−41 it is fairly plain that the recognition of triterpenoids by BSA belongs to intermediate affinity. In addition, the value of n is properly 1, which may infer the presence of just one sort of binding site in BSA for triterpenoids. This fact also overlaps the foregoing molecular modeling and further agrees with the following sitespecific ligands and hydrophobic 8-anilino-1-naphthalenesulfonic acid displacement experiments. It is should be mentioned that the binding intensity of circulating triterpenoids for albumin is moderately small, but the physiological concentration of albumin is in all probability roomy enough (∼640 μM) to allow triterpenoids vast binding. Albumin binding may effectually prolong the in vivo half-life of triterpenoids, and the concentration of triterpenoids ordinarily accumulated in some organs such as brain, heart, kidney, liver, and plasma, which is in logical harmony with the in vivo experiments in healthy volunteers and different animals specified somewhere. For example, Song et al.42 and Xia et al.43 administrated healthy volunteers with single doses of oleanolic acid and ursolic acid and found that the peak plasma

which hints that just one type of quenching reaction exists. Simultaneously, the Stern−Volmer quenching constant KSV is contrarily connected with temperature and the value of kq is 100-fold greater than the biggest feasible value of bimolecular quenching constant in aqueous solution (1.0 × 1010 M−1 s−1), demonstrating that the probable quenching mechanism for BSA fluorescence emission by triterpenoids is a static type, because higher temperature will classically result in the dissociation of weakly bound complexes, and thus lesser quantities of static quenching. One consideration to bear in mind is that static and dynamic quenching can be discerned by their differing reliance on temperature or viscosity, but preferably by fluorescence lifetime measurements, which can discriminate between static and dynamic processes straightly.21 To further acquire direct proof of the nature of the BSA−triterpenoid recognition, the representative fluorescence decay figures of BSA at different molar ratios of triterpenoids in Tris−HCl buffer, pH = 7.4, are displayed in Figure S2 in the Supporting Information, and the fluorescence lifetime and their amplitudes are summarized in Table 2. Obviously, the decay curves fitted well to a Table 2. Fluorescence Lifetime Data of BSA as a Function of Concentrations of Triterpenoids samples free BSA BSA + ursolic acid (1:1) BSA + ursolic acid (1:2) BSA + ursolic acid (1:4) BSA + oleanolic acid (1:1) BSA + oleanolic acid (1:2) BSA + oleanolic acid (1:4)

τ1 (ns)

τ2 (ns)

A1

A2

τ (ns)

χ2

3.68 3.45 3.21 2.89 3.29

6.95 6.31 6.12 6.07 6.42

0.37 0.29 0.19 0.17 0.31

0.63 0.71 0.81 0.83 0.69

5.74 5.48 5.57 5.53 5.45

0.99 1.02 1.05 0.98 1.07

3.54

6.19

0.22

0.78

5.61

0.99

3.03

5.99

0.19

0.81

5.43

1.03

biexponential function, and the relatively fluorescence lifetimes being exhibited are τ1 = 3.68 ns and τ2 = 6.95 ns of BSA, whereas in the maximum concentration of ursolic acid or oleanolic acid, the lifetimes are τ1 = 2.89/3.03 ns and τ2 = 6.07/ 5.99 ns, respectively. Considering Trp residues are known to show multiexponential decays, a transparent allotment of the observed individual fluorescence lifetime constituents to the dissimilar fluorescent Trp residues is tough for a multiple Trp residue protein such as BSA. Therefore we have not sought to assign the separate components; on the contrary, the average fluorescence lifetime has been employed in order to capture a qualitative analysis. The mean fluorescence lifetime of BSA did not change obviously, just from 5.74 ns to 5.53/5.43 ns, at

Table 3. Affinity Constants (K) and Thermodynamic Parameters for the Molecular Recognition of Triterpenoids by BSA at Different Temperatures system BSA + ursolic acid

BSA + oleanolic acid

a

T (K)

K (×104 M−1)

n

Ra

ΔH (kJ mol−1)

ΔG (kJ mol−1)

ΔS (J mol−1 K−1)

298 304 310 298 304 310

1.507 1.042 0.8395 1.734 1.186 0.9863

0.99 0.97 0.96 0.99 0.96 0.96

0.9995 0.9996 0.9995 0.9993 0.9995 0.9995

−37.50

−23.84 −23.38 −23.29 −24.18 −23.71 −23.70

−46.07

−40.55

−36.19

R is the correlation coefficient. 2277



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concentration (Cmax) and time to peak concentration (Tmax), respectively, are 12.12 ng mL−1 and 5.2 h for oleanolic acid and 3404.6 ng mL−1 and 4.0 h for ursolic acid. These results distinctly proved that oleanolic acid and ursolic acid were distributed to an immense degree out of the blood chamber or accumulated in some tissues. In a study with Sprague−Dawley rats, the Cmax and Tmax were found to be 1.10 μg mL−1 and 0.42 h, and the dispersion order of ursolic acid in various organs was detected as follows: lung > spleen > liver > cerebrum > heart > kidney.44 This in vivo study hence concluded that ursolic acid was mostly distributed in ample blood furnishing tissues such as lung, spleen, and liver, and the spreading depended chiefly upon the blood flow and perfusion rate of the organ. In another very recent pharmacokinetic study, Yin et al.45 announced that triterpenoids were assimilated, metabolized, and stored in tissues which in reverse exerted their regional and systemic activities. In this experiment, male C57BL/6 mice were fed triterpenoids for approximately 8 weeks, and it can unmistakably be heeded that the liver not only had the utmost bioavailability for triterpenoids paralleled with other tissues but also was the outstanding organ for triterpenoid reposition and metabolism. In light of the above experimental observations, we could obviously view that the bioavailability and deposit of triterpenoids such as ursolic acid and oleanolic acid in the human body relied mainly upon the blood circulation, or rather plasma protein binding. Consequently, more attention should be paid to this issue, because albumin can usually act as an important vector for nearly all endogenous and exogenous ligands in human plasma to deliver the triterpenoids to the target organs, where it brings forth its pharmacological and toxicological effect. Thermodynamic Analysis. As noted earlier, molecular recognition commonly show a striking level of specificity and high-affinity. Basically, binding transpires only when it is linked with a negative Gibbs free energy of binding (ΔG°), which may have differing thermodynamic symbols, changing from enthalpy to entropy driven. Therefore, the apprehension of the acting forces running the recognition and interaction request a detailed recountal of the binding thermodynamics, and a relationship of the thermodynamic functions with the structures of interacting mates.46 To attest the noncovalent interactions between BSA and triterpenoids, supposing the enthalpy ΔH° do not vary clearly over the temperature range studied, the three thermodynamic functions are related by the following equations: ln K =

−ΔH ° ΔS° + RT R

ΔG° = ΔH ° − T ΔS°

Figure 6. van’t Hoff plot for the molecular recognition of ursolic acid (■) and oleanolic acid (●) by BSA in Tris-HCl buffer, pH = 7.4.

defined by a positive value of ΔS° and a negative ΔH° (almost zero). A negative ΔH° is observed whenever there is a hydrogen bond in the process. In the current instance of BSA− triterpenoid system, ursolic acid and oleanolic acid involve several hydrophobic cyclic systems (6-membered ring) and these cyclic systems dominantly function with amino acid residues of BSA to generate hydrophobic interactions; therefore, one could reasonably proclaim that hydrogen bonds, van der Waals forces, and hydrophobic interactions all weigh heavily in the molecular recognition of triterpenoids by BSA. Moreover, the BSA−triterpenoid recognition process is an unprompted exothermic reaction owing to the negative value of ΔG°; the experimental data ΔG° (298 K) is also greatly close to the theoretical data ΔG° = −23.69 kJ mol−1 and ΔG° = −24.29 kJ mol−1 for ursolic acid and oleanolic acid, respectively, confirming the reliability of the results of steady state fluorescence. Identification of Binding Domain. As we have seen, both ursolic acid and oleanolic acid were located in subdomain IIA (Sudlow’s site I) by building on the results of molecular modeling and eq 3. To further clarify this aspect, site-specific ligand experiments between the triterpenoids and the other ligands that specifically bind to a known site or domain were executed. The curious feature of albumin to bind various substances is predominantly dependent on the existence of two foremost binding sites, that is, Sudlow’s site I and site II, which are situated within particular regions in subdomains IIA and IIIA,48,49 respectively. Site I is known as the warfarin− azapropazone site and formed as a cave in subdomain IIA, the residue Trp-213 of BSA in this patch. The internal wall of the pocket is formed by hydrophobic side chains, whereas the entry to the cavity is enveloped by positively charged residues. The unusual quality of this site is the binding of the ligand which is a bulky heterocyclic anion with a negative charge localized in the middle of the molecule, such as warfarin, phenylbutazone, 3,5-diiodosalicylic acid, and azapropazone.25,27 Site II corresponds to the hole of subdomain IIIA and is renowned as the indole−benzodiazepine site, which is almost equal in size to site I; the interior of the domain is composed of hydrophobic amino acid residues, and the external pocket existed two basic amino acid residues (Arg-410 and Tyr411).23−25 Ligands binding to site II are aromatic carboxylic

(5) (6)

where K is the affinity constant for a given association reaction under a definite set of experimental conditions, R is the gas constant, and T is the absolute temperature. A linear plot (Figure 6) of ln K versus 1/T produces ΔH° and ΔS°, and the data fitted from Figure 6 also shown in Table 3. Ross and Subramanian47 generalized the signs and magnitudes of the thermodynamic parameters associated with different solitary kinds of interactions that might settle in protein association processes, as recounted below. For classic hydrophobic interactions, both ΔH° and ΔS° are positive, while they are negative for van der Waals forces and hydrogen bonds shaping in low dielectric medium. Furthermore, concrete electrostatic interactions between ionic species in aqueous solution were 2278



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acids with a negatively charged acidic group at the end of the molecule, e.g., ibuprofen, flufenamic acid, diflunisal, and diazepam. Subsequently Brodersen et al.50 proposed that digitoxin binding in albumin is autonomous from Sudlow’s site I and site II, and perch on what was nominated as site III. In the present work, the competitor used involved phenylbutazone, a typical marker for site I, flufenamic acid for site II, digitoxin for site III, and hemin for domain I. According to eq 3, the affinity constants were fitted from raw fluorescence data and found to be 0.1967 × 104/0.1538 × 104 M−1, 1.459 × 104/ 1.522 × 104 M−1, 1.461 × 104/1.653 × 104 M−1, and 1.384 × 104/1.619 × 104 M−1 for ursolic acid and oleanolic acid, respectively, in the presence of phenylbutazone, flufenamic acid, digitoxin, and hemin. The outcomes advocate triterpenoids having the same site with phenylbutazone in BSA, and authorize the simulation of molecular modeling putting the triterpenoids at subdomain IIA and is also in agreement with 8anilino-1-naphthalenesulfonic acid displacement below. Fluorescence dye 8-anilino-1-naphthalenesulfonic acid (ANS) is accepted to bind to hydrophobic patches of proteins and can serve as a perceptive announcer of conjugation in the vicinity of protein Trp residues; thereby it has been employed to specify all the hydrophobic sites of proteins.51 For the purpose of further ensuring the property of the molecular recognition of triterpenoids by BSA, binding studies were carried out in the presence of ANS under unanimous conditions. Based on the protocol designed above, the relative fluorescence intensity (F/F0) against ligand concentration ([Ligand]) plots is indicated in Figure 7. Vividly, at a ligand concentration of 45 μM, both triterpenoids and ANS dribble Trp-213 residue fluorescence, but the pitch of quenching by triterpenoids was to some degree lower than ANS; ANS could extinguish about 69.05%, whereas ursolic acid and oleanolic acid, respectively, could just slake 46.16% and 54.01% of Trp213 residue fluorescence. In a much earlier report, Stryer52 imprimis asserted that the quantum yield of ANS is approximately 0.002 in aqueous buffer, but near 0.4 when bound to protein, with almost no contribution from the unbound probe. When triterpenoids are added to the ANS− BSA mixture, it can compete with ANS for its hydrophobic domain, and the fluorescence would trim. The valid explanation for this is that ANS is virtually nonfluorescent when in aqueous solution, but it will become highly fluorescent in nonpolar solvents or when it is bound to proteins. As can be seen in Figure 7, ANS−BSA fluorescence faded about 30.87% and 35.87% in the presence of ursolic acid and oleanolic acid, respectively, pronouncing that triterpenoids may contend against ANS moderately for its binding site. Although still partially debatable, consensus prevailed today that there are five discrepant binding sites for ANS associated with BSA, but preferentially at a site close to the Trp-213 residue.53 In this context, ANS powerfully stifles the fluorescence of BSA, which justifies that the binding position for ANS in this high-affinity site comes near to Trp-213; about 30.87% and 35.87% displacement of ANS fluorescence illustrates that triterpenoids and ANS likely participate at a conjunct site in BSA, viz., the site is located next to the residue Trp-213. An analogous consequence has also been transmitted by Sen et al.54 for the binding of gold nanoparticle to albumin from human serum. Structure and Stability of BSA. Conformational changes in acceptors upon binding ligands are possibly key in a number of biomedical systems, and frequently a conformation change in the receptor would noticeably be a mechanism for coopera-

Figure 7. Fluorescence quenching silhouettes of BSA and BSA−ANS mixture; binding isotherm of (A) ursolic acid (■) and (B) oleanolic acid (■) and ANS (●) provoked quenching of BSA fluorescence and quenching of BSA−ANS complex fluorescence by (A) ursolic acid (▲) and (B) oleanolic acid (▲); pH = 7.4, T = 298 K. Each data was the mean of three individual measurements ± SD ranging from 0.23% to 4.82%.

tivity.55 In terms of molecular modeling, we deduced that the conformation of BSA has changed after the triterpenoid conjugation. To verify this point, the circular dichroism (CD) spectra of BSA in the absence and presence of triterpenoids are displayed in Figure S4 in the Supporting Information, and the secondary structure components calculated based on raw CD data are pooled in Table 4. The CD curves of BSA exhibited Table 4. Secondary Structure Constituents of BSA Recognition with Triterpenoids at pH = 7.4 Appraised by Jasco Spectra Manager II Software secondary structure components (%)

2279

samples

α-helix

β-sheet

turn

random

free BSA BSA + ursolic acid (1:2) BSA + ursolic acid (1:4) BSA + oleanolic acid (1:2) BSA + oleanolic acid (1:4)

61.3 57.8 52.1 56.4 49.2

8.1 8.7 9.9 8.9 10.1

11.9 12.6 14.2 12.8 14.4

18.7 20.9 23.8 21.9 26.3



Article

two negative peaks in the far-UV CD region at 208 and 222 nm, characteristic of α-helical structure of BSA. The rational elucidation is that the negative bands between 208 and 209 nm and 222 and 223 nm are both contributed by the π → π* and the n → π* transition for the peptide bond of α-helix.56 Free BSA has 61.3% α-helix, 8.1% β-sheet, 11.9% turn, and 18.7% random coil; upon complexing with ursolic acid and oleanolic acid, a decline of α-helix was detected from 61.3% free BSA to 52.1%/49.2% BSA−triterpenoid complexes, while elevation was detected in β-sheet, turn, and random coil from 8.1%, 11.9%, and 18.7% free BSA to 9.9%, 14.2%, and 23.8% BSA−ursolic acid and 10.1%, 14.4%, and 26.3% BSA−oleanolic acid at a molar ratio of BSA to triterpenoids of 1:4. The decrease of αhelix with an increment in the β-sheet, turn, and random coil explained triterpenoids bound with amino acid residues of the polypeptide chain and giving rise to the destabilization of the BSA spatial structure,57 i.e., some degree of BSA destabilization upon triterpenoid complexation. Another testimony of structural changes of BSA after complexing with triterpenoids was represented by synchronous fluorescence spectra. It involves simultaneously scanning of the excitation and emission monochromators while retaining a stable wavelength interval (Δλ) or fixed increment of energy (Δv) between them.58 Fuller and Miller59 suggested that if the D-value (Δλ) between excitation and emission wavelengths was riveted at 60 nm, the synchronous fluorescence states the idiosyncratic clue of Trp residues. Figure 8 displays the synchronous fluorescence of BSA in Tris−HCl buffer in the presence of different amounts of triterpenoids. A faint red shift can be noted from Figure 8, which sustained that the hydrophobicity around the Trp residue was abated and the hydrophilicity expanded.60 Moreover, it has been frankly shown in Figure 9 that the slope was higher for the BSA−oleanolic acid mixture, interpreting that the quenching effect of Trp-213 residue fluorescence by ursolic acid was definitely frailer than that of oleanolic acid. To procure more exhaustive information on the conformational alterations of triterpenoid complexes with BSA, threedimensional fluorescence of BSA and BSA−triterpenoid complex is scanned in Figure S5 in the Supporting Information, and the corresponding parameters are also filed in Table 5. Peak 1 (λex = 280.0 nm, λem = 360.0 nm) chiefly unravels the spectral trait of Tyr and Trp residues, because when BSA is excited at 280.0 nm, it principally shows the intrinsic fluorescence of Tyr and Trp residues, and the Phe residue fluorescence can be trifling.21 Besides peak 1, peak 2 mainly exposits the fluorescence spectral action of polypeptide chain backbone structure CO. The fluorescence intensity of peak 2 alleviated a lot after the addition of triterpenoids, which delivered that the peptide chain structure of BSA was changed and this espouses far-UV CD spectra. Analyzing from the fluorescence intensity variations of the two peaks which decreased tangibly, but to an unequal tune: the intensity of peak 1 has been receded of 40.29% and 50.32%, while peak 2 of 18.4% and 21.16% for BSA−ursolic acid and BSA−oleanolic acid, respectively, evincing that the molecular recognition of triterpenoids with BSA begot the conspicuous destabilizing of the polypeptide chain of protein, which augmented the unwrapping of some hydrophobic patches that had been entombed before.61 All of the above inspections and analyses verified that the conjugation of triterpenoids with BSA evoked spatial structure changes in protein, which may be associated with its physiological function. This phenomenon is also in

Figure 8. Synchronous fluorescence spectra (Δλ = 60 nm) of BSA (1.0 μM) in the presence of 0, 5.0, 10, 15, 20, 25, 30, 35, and 40 μM ursolic acid (A) and oleanolic acid (B); pH = 7.4 and T = 298 K.

Figure 9. Synchronous fluorescence quenching (Δλ = 60 nm) efficiency of BSA (1.0 μM) at pH = 7.4 and T = 298 K, plotted as elimination of fluorescence intensity (F/F0) versus triterpenoid concentration. Each point was the average of three independent experiments ± SD ranging from 0.87% to 2.49%. 2280



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Table 5. Three-Dimensional Fluorescence Spectral Distinctive Parameters of BSA and BSA−Triterpenoid Complex system

peak 1 (λex/λem)

Δλ (nm)

intensity F

peak 2 (λex/λem)

Δλ (nm)

intensity F

BSA BSA + ursolic acid BSA + oleanolic acid

280.0/360.0 280.0/364.0 280.0/367.0

80.0 84.0 87.0

788.5 470.8 391.7

230.0/362.0 230.0/363.0 230.0/366.0

132.0 133.0 136.0

326.1 266.1 257.1

Author Contributions

accordance with the inference based on UV/vis absorption spectra introduced by Rada et al.,62 and then, the extent of αhelical content changes induced by ursolic acid was smaller than that by oleanolic acid, which gave indirect proof to the affinity of human serum protein (mainly albumin) with oleanolic acid being larger than that with ursolic acid. In a word, we have decoded the molecular recognition of two naturally bioactive triterpenoids, ursolic acid and oleanolic acid, by protein BSA under physiological conditions, which were dissected by blending with molecular modeling, steady state and time-resolved fluorescence, and CD spectra. The results explained that both ursolic acid and oleanolic acid are fastened in subdomain IIA, Sudlow’s site I of albumin, substantially by the noncovalent interactions such as hydrogen bonds and van der Waals and hydrophobic interactions, but the strength of oleanolic acid with BSA is better, compared with ursolic acid, which are related to the position of methyl substituting group in the molecular structure of triterpenoids. Further assays will be performed to earn integrative information concerning the stereochemistry and different position of methyl group substitutions on the E-ring meddled with their affinity toward protein albumin by using nuclear magnetic resonance and molecular dynamics simulation, and the work is now in process. Steady state and time-resolved fluorescence endorse that the quenching of BSA Trp residue fluorescence resulted from a static mechanism, or rather the nonfluorescent BSA− triterpenoid complex formation with a comparatively modest intensity of 104 M−1. Furthermore, the conformation of BSA was proved to be moderately disturbed upon the addition of triterpenoids with a shrinkage of α-helix accompanied by a rise in β-sheet, turn, and random coil, counseling a partial disruption of protein. All the data gathered herein fit well with the fact that the molecular recognition of triterpenoids by albumin may also be coherent with the pharmacological or toxicological style of the hugely utilized natural pentacyclic triterpenoids ursolic acid and oleanolic acid, as the biological effect of natural products is, assumedly, regulated through cellular diffusion bound to the most copious plasma protein.

■

§

These authors contributed equally to this work.

Notes

The authors declare no competing financial interest.

■

ACKNOWLEDGMENTS We are greatly indebted to Professor Ulrich Kragh-Hansen of Department of Medical Biochemistry, University of Aarhus, for the priceless gift of his doctoral dissertation. We are much obliged to Professor John W. Finley of Department of Food Science, Louisiana State University, for his warm support during the manuscript processing. Thanks also go to the reviewers of this manuscript for their valuable and constructive suggestions.

■

ABBREVIATIONS USED Ala, alanine; ANOVA, analysis of variance; ANS, 8-anilino-1naphthalenesulfonic acid; Arg, arginine; BSA, bovine serum albumin; CD, circular dichroism; DNA, deoxyribonucleic acid; IRF, instrument response function; Leu, leucine; Phe, phenylalanine; R, correlation coefficient; SD, standard deviation; Ser, serine; Tris, tris(hydroxymethyl)aminomethane; Trp, tryptophan; Tyr, tyrosine; UV/vis, ultraviolet−visible spectroscopy; Val, valine

■

ASSOCIATED CONTENT

* Supporting Information S

Figures depicting steady state fluorescence of BSA, timeresolved fluorescence decays of BSA−ursolic acid and BSA− oleanolic acid, association constant plot illustrating Trp residue quenching of BSA, far-UV CD spectra of the BSA−ursolic acid and BSA−oleanolic acid system, and three-dimensional fluorescence spectra of BSA, BSA−ursolic acid, and BSA− oleanolic acid mixture. This material is available free of charge via the Internet at http://pubs.acs.org.

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REFERENCES

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