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Cytotoxicity and comparative binding mechanism ofpiperine with human serum albumin and α-1-acidglycoproteinDaniel Pushparaju Yeggonia, Aparna Rachamallub, Monika Kallubaia & RajagopalSubramanyama

a Department of Plant Sciences, School of Life Sciences, University of Hyderabad,Hyderabad 500046, Indiab Department of Animal Sciences, School of Life Sciences, University of Hyderabad,Hyderabad 500046, IndiaAccepted author version posted online: 23 Jul 2014.Published online: 20 Aug 2014.

To cite this article: Daniel Pushparaju Yeggoni, Aparna Rachamallu, Monika Kallubai & Rajagopal Subramanyam (2014):Cytotoxicity and comparative binding mechanism of piperine with human serum albumin and α-1-acid glycoprotein, Journalof Biomolecular Structure and Dynamics, DOI: 10.1080/07391102.2014.947326

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Cytotoxicity and comparative binding mechanism of piperine with human serum albumin andα-1-acid glycoprotein

Daniel Pushparaju Yeggonia, Aparna Rachamallub, Monika Kallubaia and Rajagopal Subramanyama*aDepartment of Plant Sciences, School of Life Sciences, University of Hyderabad, Hyderabad 500046, India; bDepartment of AnimalSciences, School of Life Sciences, University of Hyderabad, Hyderabad 500046, India

Communicated by Ramaswamy H. Sarma

(Received 18 June 2014; accepted 18 July 2014)

Human serum albumin (HSA) and α-1-acid glycoprotein (AGP) (acute phase protein) are the plasma proteins in bloodsystem which transports many drugs. To understand the pharmacological importance of piperine molecule, here, we stud-ied the anti-inflammatory activity of piperine on mouse macrophages (RAW 264.7) cell lines, which reveals that piperinecaused an increase in inhibition growth of inflammated macrophages. Further, the fluorescence maximum quenching ofproteins were observed upon binding of piperine to HSA and AGP through a static quenching mechanism. The bindingconstants obtained from fluorescence emission were found to be Kpiperine = 5.7 ± .2 × 105 M−1 and Kpiperine = 9.3± .25 ×104 M−1 which correspond to the free energy of −7.8 and −6.71 kcal M−1at 25 °C for HSA and AGP, respectively. Fur-ther, circular dichrosim studies revealed that there is a marginal change in the secondary structural content of HSA dueto partial destabilization of HSA–piperine complexes. Consequently, inference drawn from the site-specific markers (phe-nylbutazone, site I marker) studies to identify the binding site of HSA noticed that piperine binds at site I (IIA), whichwas further authenticated by molecular docking and molecular dynamic (MD) studies. The binding constants and freeenergy corresponding to experimental and computational analysis suggest that there are hydrophobic and hydrophilicinteractions when piperine binds to HSA. Additionally, the MD studies have showed that HSA–piperine complex reachesequilibration state at around 3 ns, which prove that the HSA–piperine complex is stable in nature.

Keywords: cytotoxicity; drug binding; fluorescence quenching; α-1-acid glycoprotein; human serum albumin; moleculardynamics simulation; piperine

Introduction

Piperine is a main alkaloid phytochemical found inplants from the family of Piperaceae. This compoundcan be obtained from the fruits of Piper nigrum, alsoboth in black and long pepper grains which areP. nigrum and Piper longum L. Further, piperine isone of the major constituent of Sitopaladi churna.Molecular formula of this compound is C17H19NO3

having molecular mass of 285 Da (Figure 1, insert).In animal studies, piperine plays an important role indrug metabolisms (Pandey, Saraf, & Saraf, 2010). Thebiological properties of piperine have been extensivelystudied (Atal, Dubey, & Singh, 1985; Atal, Zutshi, &Rao, 1981). It was liable and found to possess centralnervous system, depressant properties (Lee, Shin, &Woo, 1984; Pei, 1983). It may also interact with theprocess of activation/deactivation of certain metabolicpathways, slowing down the metabolism and biodegra-dation of the drugs. This action of piperine results inhigher plasma levels of drugs, rendering them moreavailable for pharmacological action. It could impro-vise the bioavailability of various compounds and alter

the effectiveness of certain medication and also inhibitcertain enzyme metabolism (Atal et al., 1985). Interest-ingly it acts on digestive enzyme of pancreas stimu-lated by dietary piperine which significantly reducesthe gastrointestinal food transit time (Platel & Sriniva-san, 2004). In general, piperine is found to act onanti-mutagenic and anti-tumor properties (Srinivasan,2007). Also, it has been demonstrated in in vitro stud-ies to protect against oxidative damage by inhibitingor quenching free radicals and reactive oxygen species.Further, piperine is used successfully to thwart mor-phine-induced respiratory depression in experimentalanimal models (Singh, Kulshrestha, Srivastava, &Kohli, 1973). Altogether, piperine is helpful in reduc-ing inflammation, improving digestion, and revealingpain and asthma. Also, it has been found to haveimmune-modulatory, anti-oxidant, anti-asthematic, anti-carcinogenic, anti-inflammatory, and antiulcer properties(Bang, 2009).

Of all the plasma proteins that are present in blood,human serum albumin (HSA) is an abundant protein inhuman blood circulation (Carter et al., 1989). In plasma,

*Corresponding author. Email: srgsl@uohyd.ernet.in

© 2014 Taylor & Francis

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HSA and α-1-acid glycoprotein (AGP) are the major drugcarriers proteins (Kragh-Hansen, Chuang, & Otagiri,2002; Kremer, Wilting, & Janssen, 1988). HSA is anextracellular characterized protein which has an ability ofbinding and transporting a wide variety of endogenousand exogenous ligands to the target sites (Mandeville,Froehlich, & Tajmir-Riahi, 2009; N’soukpoé-Kossi,Sedaghat-Herati, Ragi, Hotchandani, & Tajmir-Riahi,2007). It is a monomeric polypeptide that contains helicalprotein, consisting of 585 natural amino acids having amolecular mass of 67 kDa. Apparently, the whole proteinis stabilized by 17 disulfide bridges which help in main-taining heart-like shape (He & Carter, 1992; Peters,1995). Subsequently, it is a single polypeptide chain com-posed of structurally similar domains that are domains I(Residues 1–195), domain II (196–383), and domain III(384–585), linked by inter domain helices. Each domainis further divided into subdomains A and B containing sixand four α-helices, respectively and all are connected byrandom coils (Bhattacharya, Curry, & Franks, 2000;Curry, Mandelkow, Brick, & Franks, 1998; Ghumanet al., 2005; Sugio, Kashima, Mochizuki, Noda, &Kobayashi, 1999). However, out of all the subdomains,most of the drugs bind within the hydrophobic pocket, i.e.subdomains IIA and IIIA, which are known as Sudlowsite I and site II, respectively (Sudlow, Birkett, & Wade,1976; Varshney et al., 2010). Among these subdomains,in site I subdomain (IIA) and site II (IIIA) have importantphysiological effects in binding to a large number of smallmolecules. Majority binding studies of HSAwith differentdrugs play a role in transport, distribution, elimination,and free concentration of variety of drugs that can influ-ence the function of their binding constants of HSA.There are reports on the limited number of bindingconstants for various ligands that typically binds revers-

ibly to HSA with binding constants of 104–108 M (He &Carter, 1992). It has been reported that there are fewdrugs that bind equally to HSA and AGP and furthermorefrom literature, it is known that there is only one bindingsite on AGP for acidic drugs (Kalra, 2003). The trypto-phan residue (Trp-214) in HSA is located in the subdo-main IIA of site I. The main emission of this protein isdue to the presence of the residue Trp-214; therefore, it isoften used as a probe to investigate the interaction ofligands with HSA. The presence of multiple binding sitesin HSA is an unique ability to bind with organic and inor-ganic molecule to make this protein an important regula-tor, as well as the pharmacokinetic nature of many drugs(Curry, Brick, & Franks, 1999; Curry et al., 1998; Peters,1995; Petitpas, Grüne, Bhattacharya, & Curry, 2001).Thus, our group has recently showed that the natural andsynthesized products, such as coumarin derivatives, chi-tooligomers, asiatic acid and trimethoxy flavone, couma-rintyramine, β-sitosterol, and 7-hydroxy coumarinderivative possess robust binding to HSA, which lead tothe HSA ligand complextion results in the struc-tural changes in the HSA (Garg et al., 2013; Gokara,Kimavath, Podile, & Subramanyam, in press; Gokara,Malavath, Kalangi, Reddana, & Subramanyam, 2014;Gokara, Sudhamalla, Amooru, & Subramanyam, 2010;Neelam, Gokara, Sudhamalla, Amooru, & Subramanyam,2010; Sudhamalla, Gokara, Ahalawat, Amooru, &Subramanyam, 2010; Yeggoni et al., 2014).

Another plasma protein, AGP is an acute phase pro-tein and it is also called as orosomucoid. AGP is havingmolecular mass of ~44 kDa, is comprised of 183 aminoacid residues, and 45% of carbohydrate was presented(Fournier, Medjoubi-N, & Porquet, 2000). HoweverAGP biosynthesis is done in the liver and secreted intothe blood stream (Athineos, Kukral, & Winzler, 1964),and other organs that include the stomach. Also, lungsand heart are been reported to synthesize and secreteAGP as well (Fournier et al., 2000). HSA and AGP arethe major plasma proteins, where in some drugs showedequivalent binding (Kumar, Walle, Bhalla, & Walle,1993). However it’s known that AGP accounts for 3%,whereas HSA is 60% of the plasma proteins (Chuang &Otagiri, 2006). Nevertheless, in chronic diseases such ashepatic, inflammation, infection, renal, and pregnancythe levels of AGP synthesis increase up to 10 folds andhence the drugs binds to AGP rather than HSA(Cheresh, Haynes, & Distasio, 1984; Fey & Fuller, 1987;Stekleneva, Shevtsova, Brazaluk, & Kulinich, 2010).Thus, the pharmacokinetics or dynamics of naturallyoccurred piperine with plasma protein has not been stud-ied. Since HSA is a negative acute-phase protein andAGP is a positive acute-phase protein; therefore, drugbinding with HSA and AGP are important in terms ofcorrect understanding of pharmacokinetics of the drugs.Thus, binding of the phytochemical piperine with HSA

Figure 1. Piperine is showing anti-inflammatory propertiesagainst LPS-induced mouse macrophages (RAW 264.7) in adose-dependent manner. Cell growth was measured by theMTT assay. Insert shows the structure of piperine andthe molecular formula (C17H19NO3) and mass, 284 Da,respectively.

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and AGP may give a platform to understand the pharma-cokinetics role. Hence, for our study, we have used bothbiophysical and computational approach to understandthe binding mechanism of piperine with HSA and AGP.Also, we carried out the cytotoxic studies to understandthe pharmacological role of piperine.

Materials and methods

Preparation of stock solutions

Pure fat-free HSA purchased from Sigma Aldrich wasdissolved in physiological aqueous solution of .1 Mphosphate buffer at a pH 7.4, with a concentration of1.5 mM. Piperine was purchased from Natural remediesPvt., Ltd, Bangalore, India, with purity of 99.2%. Itsstock solution (2 mM) was prepared in 20:80 ethanol–water mixture. From our previous reports, a solutioncontaining 20% ethanol has showed no effect on HSAsecondary structure (Subramanyam, Gollapudi, Bonigala,Chinnaboina, & Amooru, 2009; Subramanyam, Goudet al., 2009). All other chemicals are purchased fromSigma Aldrich.

Here, we also optimized the incubation time ofpiperine binding to HSA and AGP by fluorescenceemission and the maximum binding time was foundto be 5 min. Thus, for all parameters, we have fixedthe incubation of piperine with HSA and AGP as5 min. There is no protein precipitation upon titrationof piperine with HSA which indicates that the mixtureis transparent.

Cell response assay (MTT assay)

Cell response was carried out by the MTT stainingmethod (Mosmann, 1983). The mouse macrophages cells(RAW 264.7) were subcultured and were seeded in 96-well plates at a density of 5 × 103 cells/well. After 16 h,cells were pretreated with lipopolysaccharide (LPS) at1 μg/mL for 4 h to induce inflammation. Later, mousemacrophages were treated with piperine (isolated fromPiperin nigrum, purchased from Natural remedies,Bangalore, India) in increasing concentration of 10, 20,40, 60, 80, and 100 μM for 48 h in a volume of 100 μLof medium. In control experiment, the cells were grownin the same media without piperine. At the end, 20 μLof MTT (5 mg/mL in PBS) was added and the cells areincubated for 3 h. About 100 μL of DMSO is added toeach well and mixed with repeated pipetting to dissolvethe crystals. Cell response was measured at an absor-bance of 570 nm on a micro plate reader (μ QuantBiotek Instrument, Inc.). Three independent experimentswere carried out in triplicate. Cell response was calcu-lated using a control as the 100% reference. The

mean ± SE was calculated and reported as the cellresponse (%) vs. concentration (μM).

Fluorescence emission measurements

The potential interactions between piperine and HSA orAGP were recorded on Perkin-Elmer LS55 fluorescencespectrometer and analyzed quantitatively. A fixed HSAand AGP concentration (.001 mM) and different concen-tration of piperine from .001 to .009 mM in .1 M PBSsolution were used at physiological pH of 7.4. BothHSA and AGP were excited at 285 nm, and the slitwidth (band width) was fixed to 5 nm for excitation andemission. The emission spectra were collected from 300to 500 nm at room temperature. Binding constants werecalculated using fluorescence intensity value at the emis-sion maximum of 340 nm for AGP and 360 nm forHSA. Here, three independent experiments were per-formed and each time identical results were obtained.

Molecular displacement experiment with sub domainIB (lidocaine), site I (phenyl butazone), and II(ibuprofen) markers

The competitors of lidocaine (IB specific marker), phenylbutazone (site I specific marker), and ibuprofen (site IIspecific marker) were used to identify the target bindingsites in HSA while binding piperine. Of all the site mark-ers, lidocaine binds to a particular site formed by resi-dues from subdomain IB facing the central, inter-domaincrevice (Hein et al., 2010). Phenylbutazone (crystallinesubstance) and ibuprofen, both are non-steroidal anti-inflammatory agent, have been considered as stereotypi-cal ligands explicitly, which binds to site I in subdomainIIA and ibuprofen that primarily binds to site II locatedin subdomain IIIA, respectively. Here, phenylbutazoneand other heterocyclic anions binds to site I located insubdomain IIA, whereas other aromatic carboxylates likeibuprofen with extended conformation prefer to bindwith site II located in subdomain IIIA of HSA. The con-centration of HSA and the site probe were maintained atconstant concentration of 1 μM, whereas piperine wastitrated with increase in concentration from 0 to 9 μM.The excitation wavelength for site-specific makers lido-caine, phenyl butazone, and ibuprofen with HSA was285 nm. The modified Stern–Volmer equation is used tomeasure the fluorescence quenching data (Hussein, 2011;Liang, Tajmir-Riahi, & Subirade, 2007; Min et al., 2004;Zsila, Bikádi, & Simonyi, 2003).

Measurement of circular dichroism spectra

Circular dichroism (CD) spectra of HSA and HSA–piper-ine were recorded with a Jasco J-810 spectropolarimeterusing a quartz cell with a path length of .02 cm. Three

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scans were accumulated with scan speed of 100 nm min−1

and data were collected from 190 to 300 nm at roomtemperature. The final concentration of HSA was.001 mM and the concentrations of piperine were .001,.005, and .009 mM. Temperature-dependent circular di-chrosim (TdCD) was performed for HSA–piperine com-plexes with increasing temperature from 25 to 85 °C.Temperature-dependent experiment was controlled byJasco J-715 peltier. An ellipticity of CD spectra isexpressed in millidegrees. Three repeated experimentswere conducted and protein secondary structure was cal-culated using CDNN 2.1 web-based software.

Molecular docking studies

Piperine docking to HSA and AGP was performed withAuto Dock 4.2.3 program using Lamarckian geneticalgorithm (Morris et al., 1998, 2009). The crystal struc-ture of HSA (PDB Id:1A06) and AGP (PDB Id:3KQ0)was obtained from the Brookhaven Protein Data Bank.Three-dimensional structure of piperine was built from2D structure and geometry was optimized using Discov-ery studio 3.5 software. In Auto Dock (4.2.2) program,the water molecules and ions were removed and hydro-gen atoms were added at appropriate geometry withinprotein. To recognize the binding site in HSA and AGP,blind docking was carried out, the grid size set to 126,126, and 126 along X-, Y-, and Z-axis with .586 Å gridspacing. The docking parameters used were: maximumnumber of energy evolutions: 250,000; GA populationsize: 150; and the number of GA runs; 30. During dock-ing, 30 conformations were obtained out of which thelowest free energy conformer which is matched to theexperimental data (fluorescence emission) was used forfurther analysis as reported earlier (Neelam et al., 2010;Sudhamalla et al., 2010).

Ligplot measurement

LIGPLOT, is a program for automatically plotting pro-tein–ligand interactions, was used to analyze hydropho-bic interactions and hydrogen bonds between HSA andpiperine (Wallace, Laskowski, & Thornton, 1995).

Molecular dynamics simulations

A 10,000 ps MD simulations of the complex (HSA–piperine) were carried out with the GROMACS 4.0(Berendsen, van der Spoel, & van Drunen, 1995; Li, Ji,& Sun, 2009) and GROMOS96 43a l force field wasused (van Gunsteren et al., 1996; van Gunsteren, Daura,& Mark, 1998). For MD simulations, the conformationwith binding energy closest to experimental bindingenergy and binding constant was taken. The topologyparameters of HSA were created using GROMACS pro-gram. Dundee PRODRG2.5 server (beta) was used to

build the topology parameter of piperine (Schüttelkopf &van Aalten, 2004). Then, the complex was immersed ina cubic box (7.335 × 6.135 × 8.119 nm) of extendedsimple point charge water molecules (van Gunsterenet al., 1996). The solvated system was neutralized byadding sodium ions in the simulation box. The entiresystem was composed of 5843 atoms of HSA, one piper-ine, 15 Na+ counter ions, and 69,491 solvent atoms. Fur-ther, to release conflicting contacts, energy minimizationwas performed using the steepest descent method of1000 steps followed by the conjugate gradient methodfor 1000 steps. MD simulation studies consist of equili-bration and production phases. In the first stage of equili-bration, the solute (protein, piperine and counter ion)was fixed and was subjected to the position-restraineddynamics simulation of the system, in which the atompositions of HSA were restrained at 300 K for 30 ps.Finally, the full system was subjected to 10,000 ps MDat 300 K temperature and 1 bar pressure. The periodicboundary condition was used and the motion equationswere integrated by applying the leaf-frog algorithm witha time step of 2 fs. At .5 ps, the atomic coordinates wererecorded during the simulation for latter analysis. TheMD simulation and results analysis were performed onOSCAR Linux cluster with 12 nodes (dual xeon proces-sor) at BIF facility, University of Hyderabad.

Results and discussion

Cell response assay data analysis

In order to understand the pharmacological importance,we have examined the anti-inflammatory effect of piper-ine isolated from P. nigrum, on inflammated mouse mac-rophages (RAW 264.7) using the MTT assay. In order toinduce the inflammation, the LPS (1 μg/mL) was used inmouse macrophages cell lines. The results showed that asthe concentration of piperine increases, the percent growthof inflammated macrophages was reduced in a dose-dependent manner with an IC50 of 63.5 μM (Figure 1).Earlier reports show that synthetic compound of piperinehas anti-inflammatory properties and also found that itinhibited the production of prostaglandin E2 and nitricoxide induced by LPS. Furthermore, we have tested anti-cancer properties of piperine with other cell lines likeHepG2 (hepatic carcinoma) and HT-29 (human colonadenocarcinoma cell line); however, this phytochemical isspecific to inflamed mouse macrophages, which indicatesthat piperine can act categorically against inflammatorydiseases. Thus, the phytochemical (piperine) which wasused could be a potential therapeutic agent.

Analysis of fluorescence emission data

The drug–protein interaction binding affinity was doneusing fluorescence emission spectroscopy. The emission

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fluorescence of HSA and AGP comes from tryptophan,tyrosine, and phenylalanine. However, in general, thetyrosine and phenylalanine have low quantum yield;thus, the fluorescence of HSA and AGP is dominated bythe tryptophan emission. The fluorescence emission spec-trum of HSA is due to the presence of single residueTrp-214, which is located in subdomain IIA. When smallmolecules bound to HSA, there is a change in intrinsicfluorescence intensity due to the tryptophan residue(Sułkowska, 2002). Titration of different concentrationsof piperine (.001–.009 mM) to HSA resulted in decreasein fluorescence maximum emission at 360 nm(Figure 2(A)). Hence, the fluorescence quenching ofHSA is concentration dependent of piperine. Thisdecrease in fluorescence intensity may be due to theinteraction of excited state of the fluorophore with itsenvironment within the protein (Lakowicz, 2009). Here,piperine acts as quencher and binds in the vicinity ofTrp 214 which leads to decrease in the fluorescenceemission. These results indicate that, upon binding ofpiperine to the HSA, the piperine–HSA complex wasformed. There are numerous reports which showed thequenching of intrinsic fluorescence of HSA upon inter-acting with various drug molecules (Agudelo et al.,2012; Bourassa et al., 2011; Garg et al., 2013; Gokaraet al., 2010, 2014; Kandagal, Kalanur, Manjunatha, &Seetharamappa, 2008). The marginal blue shift observed

in both HSA and AGP binding to piperine attributes thatthe hydrophobic regions of piperine binding are exposedto hydrophobic cavities of HSA and AGP. We concludethat the fluorescence changes ascribed that piperine bind-ing to HSA or AGP is mainly by hydrophobic interac-tions apart from hydrogen bonds.

Similarly, when different concentrations of piperinetitrated with AGP, maximum AGP fluorescence emissionwas quenched (Figure 3). This indicates that piperinebinds to plasma protein of HSA and also to AGP, thoseare the major carrier proteins for wide range of drugs inthe human blood circulatory system. AGP may abundantin various anti-inflammation or immunomodulatoryevents for the following reasons. The expression of AGPis regulated by both cytokines (interleukin-1, interleukin-6 and tumor necrosis factor-α) and glucocorticoids, unlikeother acute-phase proteins including fibrinogen, cerulo-plasmin, and α2-microglobulin, which only by interleu-kin-6 (Baumann, Prowse, Marinković, Won, & Jahreis,1989; Kulkarni, Reinke, & Feigelson, 1985; Stadnyk &Gauldie, 1991). In fact, various studies propose that theligand binding site of AGP consists of at least three par-tially overlapping subunits, acidic-, basic-, and steroid-binding sites (Matsumoto et al., 2002), Since HSA isnegative acute-phase protein and AGP is a positive acute-phase protein, binding studies with both HSA and AGPplay a crucial role in deciding the pharmacokinetic

Figure 2. Fluorescence emission spectra of HSA-piperine in .1 M phosphate buffer with pH 7.4, λex = 285 nm, andtemperature = 25 ± 1 °C. (A) Free HSA (.001 mM) and free HSA with different concentrations of piperine .001, .002, .003, .004,.005, .006, .007, .008, and .009 mM. (B) Plot of log (dF/F) against log [Q]. (C) Stern-Volmer plots of HSA–piperine complexesshowing fluorescence quenching constant (Kq) and plot of F0/F against [Q] for piperine. λex = 285 nm and λem = 360 nm.

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behavior of the piperine in various diseased and inflam-matory conditions. Thus, binding studies of piperine withAGP along with HSA is utmost important in terms ofunderstanding of pharmacokinetics properties, especiallyduring normal and in pathological conditions.

Moreover, to evade inner filter effect, increasing con-centration of piperine with HSA and AGP, increasingabsorbance of excitation or emission radiation introducesinner filter effect that may decrease the fluorescenceintensity and results in a nonlinear relationship betweenthe observed fluorescence intensity and the concentration[Q] of the piperine. Such effect can be corrected usingthe following equation:

Fcor ¼ Fobs10 Aexc þ Aemið Þ=2 (1)

where Fobs is the observed fluorescence and Fcor is thecorrected fluorescence intensity, and Aexc and Aemi repre-sent the absorbance at the fluorescence excitation(285 nm) and emission wavelengths (360 nm) for HSAand (340 nm) AGP, respectively.

The quenching mechanism is classified as either sta-tic or dynamic quenching. These quenching can be emi-nent by difference in the fluorescence life time,temperature, and viscosity (Lakowicz, 2009). Staticquenching is a process in which the quencher has tophysically interact by making a chemical bond withexcited molecule. However, dynamic quenching is a pro-cess where the quencher will not have a direct contactwith molecule. Quencher and molecule will move in sol-vent and they can have collision, hence quencher canmeet the excited molecule and bring the excited

molecule to ground state. Here, static quenching refers tobe fluorophore of HSA along with quencher of piperinewhich will form HSA–piperine complex formation. Toverify whether the quenching mechanism is static ordynamic in HSA–piperine complex, here, we have plot-ted F0/F against Q, the plot is linear for piperine–HSAcomplexes indicating that the quenching is mainly staticin these protein–drug complexes (Agudelo et al., 2012;Mansouri, Pirouzi, Saberi, Ghaderabad, & Chamani,2013; Zhang, Que, Pan, & Guo, 2008). The Kq was esti-mated according to the Stern–Volmer equation:

F0=F ¼ 1þ kqt0 Q½ � ¼ 1þ KD Q½ � (2)

where F and F0 are the fluorescence intensities in thepresence and absence of quencher, [Q] is the quencherconcentration, and KD is the Stern–Volmer quenchingconstant (Kq), which can be written as KD = kqt0 wherekq is the bimolecular quenching rate constant and t0 isthe lifetime of the fluorophore in the absence ofquencher, lifetime of fluorophore for HSA is 5.6 ns(Tayeh, Rungassamy, & Albani, 2009). The quenchingconstant (Kq) for piperine is calculated (Figure 2(C)) tobe 6.9 × 1013 M−1 s−1. As these value is much greaterthan the maximum collisional quenching constant2.0 × 1010 M−1 s−1 (Agudelo et al., 2012; Zhang, Xu,Ge, Jiang, & Liu, 2012); hence, the static quenchingmechanism is predominant in these HSA–piperinecomplex.

When small ligand molecules bind independently toa set of equivalent sites on a macromolecule, the equilib-rium between bound and free molecule could be repre-sented by a modified Stern–Volmer equation (Feroz,Mohamad, Bujang, Malek, & Tayyab, 2012; Kragh-Han-sen, 1981; Zsila et al., 2003).

log F0 � Fð Þ=F½ � ¼ logKsþ n� log ½Q� (3)

where Q, n, and Ks are the quencher concentration, num-ber of binding molecules, and binding constant, respec-tively. From this equation, the plotted results indicated agood linear relationship. The number of piperine mole-cule binding to HSA was calculated to be 1.2 suggestingthat HSA interacts with piperine in a close relationshipof one-to-one ratio (Figure 2(B)). The binding constantsof piperine were calculated from the intercept as5.7 ± .2 × 105 M−1 which indicates strong binding ofpiperine to HSA. There is a good correlation with thecomputational calculated binding constant as3.3 × 105 M−1 obtained as lowest free energy.

Interestingly, the binding of piperine with AGP wasalso stronger and the binding constant was found to be9.3 ± .25 × 104 M−1 which is in the range of FDA (Fed-eral Drug Administration). It is known that in most ofthe cases, HSA is a vehicle to carry the drug molecules;however, in chronic disease conditions, the AGP alsoplays a major role in transporting the drug molecules.

Figure 3. Fluorescence emission spectra were measured forAGP along with piperine in .1 M phosphate buffer with pH7.4, λex = 285 nm, and temperature = 25 ± 1 °C. Free AGP(.001 mM) and free AGP with different concentrations ofpiperine .001, .002, .003, .004, .005, .006, .007, .008, and.009 mM. Insert: Plot of log (dF/F) against log [Q].λex = 285 nm and λem = 340 nm.

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Acute-phase proteins are proteins whose plasma concen-trations increase in response to inflammation like AGP.The abundance of plasma protein levels follows theimpact on drug-binding extent, and causes modificationsin the mode of drug action, distribution, disposition, andelimination. Thus, AGP is one of the most importantacute-phase proteins that influence the principal bindingproteins for basic drugs. Although plasma concentrationof AGP is much lower than that of HSA, AGP can playthe major drug-binding macromolecule in plasma withsignificant clinical implications.

Additionally, AGP is also involved in drug–druginteractions, particularly in the displacement of drugsand endogenous substances from their binding sites,which implements pharmacokinetic and clinical conse-quences. The binding of several drugs has been shownto increase following surgical interventions, inflammationand stress, and this increased binding is due to anincrease in the plasma concentration of AGP (Otagiri,2009). It is generally assumed that in plasma, acidicdrugs are mainly bound to HSA. However, binding toAGP will contribute significantly to the total plasmabinding of these drugs, especially in diseases in whichthe concentration of AGP increases and/or of HSAdecreases (Urien, Albengres, Pinquier, & Tillement,1986). Recently, we have also reported that chitosanoligomers were strongly bounded to AGP, whereas othermolecules such as 7-hydroxycoumarin derivativesshowed weak binding to AGP (Yeggoni et al., 2014).Thus, piperine is a potent phytochemical which can bindto both HSA and AGP that are pharmacologically andclinically important proteins.

Free energy calculations

In broad spectrum, there are four types of non-covalentinteractions when small molecules bind to proteins.These are electrostatic, hydrogen bonds, van der Waalsforces, and hydrophobic interactions. Thermodynamicparameters are the main evidence for affirming the forcesinvolved in drug-binding studies. The standard freeenergy is calculated according to the following equation:

DG� ¼ �RT lnK (4)

where ΔG is a free energy, K is a binding constant at thecorresponding temperature, which can be obtained fromfluorescence data, and R is the gas constant. Thus, thecalculated free energy change is −7.8 kcal M−1 at 25 °C.We have also calculated the free energy (−6.8 kcal M−1)from computational modeling and results are in agree-ment with the experimental data. Here, the lower freeenergy value is mainly due to hydrophobic interaction ofpiperine binding to HSA. In case of AGP, the freeenergy calculated from experimental and computationalis −6.7 and −6.71 kcal M−1, respectively. Hence, it is

corroboration of both the experimental and computation-ally calculated free energies. In addition, similar types ofinteractions such as hydrophobic and hydrogen bondingwere observed with our recent studies on natural as wellas synthesized compounds, such as trimethoxy flavone,coumaroyltyramine, asiatic acid, chitosan oligomers, and7-hydroxy coumarin derivatives with HSA (Garg et al.,2013; Gokara et al., 2010, in press, 2014; Neelam et al.,2010; Yeggoni et al., 2014).

Site-specific marker analysis of HSA with piperine

In molecular displacement experiments, the concentrationof HSA and site-specific markers lidocaine (subdomain IBmarker), phenylbutazone (site I marker), and ibuprofen(site II marker) concentrations were constant (1 μM). Site-specific marker (1 μM) was added to the HSA (1 μM) andthe fluorescence emission spectrum of the site marker phe-nylbutazone–HSAwas recorded. To this complex (phenyl-butazone–HSA), increased concentration of piperine wastitrated (Figure 4). Hence, phenylbutazone and ibuprofenwere used as site marker fluorescence probes for monitor-ing sites I and II of HSA, respectively. (Sudlow, Birkett, &Wade, 1975; Sudlow et al., 1976; Wanwimolruk, Birkett,& Brooks, 1983). The titration of the different concentra-tion of piperine to HSA–phenylbutazone showed thedecrease in the fluorescence emission as the piperinereplaces the phenylbutazone. Further, the binding constantvalues obtained for this displaced fluorescence quenching

Figure 4. Displacement of phenylbutazone from HSA–phenyl-butazone complex by piperine. Fluorescence emission spectrawere recorded on Perkin Elmer LS55 fluorescence spectrometerfor piperine with HSA in presence of site probe markers. Phe-nylbutazone is referred as site-I probe. The concentrations ofHSA and phenylbutazone were maintained constant at a con-centration of 1 μM whereas, the piperine was varied from 0 to9 μM. Inserts: Modified Stern–Volmer plot. Plot of log (dF/F)against log [Q] λex = 285 nm, λem = 360 nm.

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of piperine + HSA–phenylbutazone found to be1.8 ± .1 × 105 M−1 which is closely correlated withthe HSA–piperine complexes 5.7 ± .2 × 105 M−1 (SeeFigure 2(A)). Thus, the observed emission of HSA– phe-nylbutazone was decreased upon increasing the concentra-tion of piperine which reveals that there is competitionbetween the piperine and phenylbutazone for site I subdo-main of HSA. We have also performed the experimentswith other site-specific marker such as lidocaine, ibupro-fen for subdomain IB and site II probe. However, the bind-ing constants are not close to the value which wasobtained from HSA–piperine complex, and it indicate thatthere is no competition between the piperine with lido-caine and ibuprofen (S. Figures 1 and 2). Thus, from theexperimental evidence, it indicates that the piperine specif-ically binds to the site I domain.

Circular dichroism data analysis for secondarystructure of HSA

CD spectroscopy study is one of the best methods forthe identification of secondary structural changes whosesecondary structural elements may vary according totheir bound state. CD is an important spectroscopicmethod for studying macromolecules that differentiallyabsorb clockwise or counter-clockwise circularly polar-ized light as the result of structural asymmetry. In gen-eral, CD spectroscopy has been used to monitor proteinstability (thermal, pH, ionic strength, or solvent), confor-mational differences in protein from various expressionsystems or species, observe changes in structure uponprotein–protein or protein–ligand interactions, and toconfirm proper folding during purification processes(Gokara et al., 2010; Sreerama & Woody, 2004). CD sig-nal is observed for chromophores that are chiral eitherintrinsically due to its structure through covalent bondingto chiral centers or when in asymmetric environmentsdue to a three-dimensional structure such as proteins andoligonucleotides (Sreerama & Woody, 2004). For exam-ple, protein typically absorbs UV-light due to n-П* andП-П* electronic transitions associated with peptide amidebonds and aromatic residues, respectively. In particular,the amide n-П* transitions observed between 190 and240 nm are highly sensitive to their local symmetry andthus, are useful for a protein secondary structure calcula-tions. CD spectra is read to evaluate α-helix, β-sheets,β-turn, and random coil of the HSA and it exhibit twonegative bands in the UV region at 208 and 218 nm(Gokara et al., 2010; 2014; Kiselev, IuA, Dobretsov, &Komarova, 2001; Subramanyam, Gollapudi, et al., 2009;Subramanyam, Goud et al., 2009; Sudhamalla et al.,2010). Various concentrations of piperine were added tofat-free HSA and incubated for 5 min, the intensity at208 and 218 nm were decreased in concentration-depen-dent manner. This predominately exhibited considerable

changes in the secondary structure of protein. Conse-quently, the loss of helical stability may be the result offormation of complex between HSA and piperine. Here,the secondary structural changes are calculated using theCDNN 2.1 program. Secondary structure of free HSAconsists of ∼58% α-helix, ∼23% β-sheets, and ∼19%random coils, which is in agreement with previousreports (Gokara et al., 2010; Kanakis, Tarantilis,Tajmir-Riahi, & Polissiou, 2007; Neelam et al., 2010;Subramanyam, Gollapudi et al., 2009; Subramanyam,Goud et al., 2009; Sudhamalla et al., 2010; Yeggoniet al., 2014). From this method, it was found that uponcomplexation of HSA with different concentrations ofpiperine (.001, .005, and .009 mM), the α-helical contentof the protein decreased from 58 ± 2.3 to 54 ± 1.6%with an increase in β-sheets from 23 ± .7 to 25 ± .2%and random coils from 19 ± 1.2 to 20 ± .7%, respec-tively (S. Table 1). It is important that the binding ofpiperine to HSA induces alterations in the structure andfunction of the protein. The components of the second-ary structure could become altered due to the formationof hydrophobic interaction and hydrogen bondingbetween the piperine and HSA. This caused the proteinmolecule to become relaxed and certain groups insidethe structure to become exposed. The conformationaltransition probably resulted in the exposure of thehydrophobic cavities and a perturbation of the microen-vironments around the aromatic amino acid residues.Therefore, the results suggest that changes in the second-ary structural elements arise from the marginal unfoldingof HSA upon binding of piperine (Figure 5(A) and (B)).Thus, the addition of piperine induced a partial unfoldingof the secondary structure of HSA. Further, the partialunfolding may be due to the microenvironment changeswithin the surrounding tryptophan residue while bindingto piperine, which was affirmed by the decrease in fluo-rescence emission. The decrease in α-helical content withan increase in the β-strand and random coils may be dueto the formation of HSA-piperine complex upon titrationof different concentrations of piperine to HSA withhydrophobic interactions and formation of hydrogenbond (Iranfar, Rajabi, Salari, & Chamani, 2012; Sattaret al., 2012). Earlier reports also indicate that conforma-tional changes occur in HSA upon complexion withligand (Beauchemin et al., 2007; Jiang, 2008; Kanakiset al., 2007; Subramanyam, Gollapudi et al., 2009;Subramanyam, Goud et al., 2009; Zsila et al.,2003).Thus, the protein conformation in our experimentarise due to change in the local structural mobility in thesecondary structural elements and decrease in the α-helixis changed due to the partial unfolding of the proteinwhich is further supported by the root means squaredeviation (RMSD) through molecular dynamic (MD)simulation of HSA and HSA-piperine complexes,which showed a consistent stability. Hence, the change

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in secondary structure upon binding of piperine withHSA is not certainly due to the destabilization of theHSA.

Thermal stability of HSA and HSA–piperine complexes

In order to understand the stability of HSA–piperinecomplexes, temperature-dependent CD was carried outfor HSA and HSA with .009 mM of piperine, from 25 to85 °C. The secondary structural conformation of proteinis not significantly changed up to 60 °C in HSA–piper-ine complexes (S. Figure 3). Above 65 °C, the α-helicalcontent decreased dramatically, while the β-sheet andrandom coil content increased in both HSA–piperinecomplexes. Earlier report showed that the Tm of theHSA alone was around 65 °C, which shows that theunfolding of protein occurs only after this point (Gokaraet al., 2010). The secondary structural conformation wasnoticed in piperine plus HSA complexation (.009 mM)and the α-helical contents were 54 ± 1.6 and 49.6 ± .7%,β-sheets were 25 ± .7 and 28.4 ± 1.1%, and random

coils were 20 ± 1.2 and 22 ± .9%, respectively, whichindicate that there is no release of piperine from its com-plexation. Thus this result indicated that even at 60 °C,the HSA–piperine complex is stable, thus the proteincomplex is thermodynamically and conformationally sta-ble. Later on, due to thermal denaturation, the conforma-tion of the protein decreases significantly which indicatesuncoupling of piperine from HSA. Inference drawn fromthis result is that HSA–piperine complex is functionallystabilized by non-covalent interactions which requireshigh temperature to breakdown these complexes.

Molecular docking studies

Protein–ligand interactions are important for all processesoccurring in living organisms. Ligand-mediated signaltransmission through molecular complementary is essen-tial to all life processes; these chemical interactions com-prise biological recognition at molecular level. Thebeauty of the evolution of the protein functions dependson the development of specific sites (active sites) whichare designed to bind ligand molecules. Ligand (drugs)-binding nature is essential for the regulation of biologicalfunctions. Protein–ligand interactions occur through themolecular mechanics involving the conformationalchanges among low and high affinity states. Drug bind-ing interactions change the protein state and proteinfunction. Here, HSA is a monomeric single polypeptideprotein comprising 585 amino acids, with three α-helicaldomains (I, II, and III), each containing two subdomains(A and B). Due to presence of multiple binding sites,HSA has exceptional ability to bind various small mole-cules (Carter et al., 1989). Based on the site-specificmarkers used in our experiment, we determined that pip-erine binds specifically to the site I (subdomain IIA). Tofurther, we have defined the binding site using moleculedocking technique to determine the primary binding siteof piperine on HSA. Using Auto Dock software 4.2.2,about 30 conformers were been generated from dockingsimulation (S. Table 2) in which we have chosen onlyone conformer on the basis of least free energy of bind-ing and score ranking which also matches to the freeenergy obtained from fluorescence emission (Jones,Willett, Glen, Leach, & Taylor, 1997). The piperine com-plex is stabilized by a hydrogen bond between piperineand LYS212 of the protein with length of 2.98 Å. Theminimum binding energy conformer was observed as−6.8 kcal M−1 that is very close to the experimentallydetermined values (−7.8 kcal M−1) having inhibitionconstant of 3.46 μM and binding constant was 3.3 ×105 M−1 (Figure 6(A)). Docking results deciphers thatpiperine is binding to HSA at subdomain IIA which isSudlow’s site I with most stable docking conformer(Figure 6(C) and (D)). It is also observed that piperinebinding site in HSA was fully covered by hydrophobic

Figure 5. (A) CD spectra of free HSA and its piperinecomplexes in aqueous solution with a protein concentration of.001 mM and piperine concentrations were .001, .005, and.009 mM. Spectra were recorded with a JASCO J-815 CDspectropolorimeter. A quartz cell with a path length of .02 cmwas used. (B) The secondary structural changes of HSA andHSA–piperine, the plot represent the concentration-dependentsecondary structural changes of free HSA and HSA–piperine.

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Figure 6. Schematic representation of least binding energy docked conformation obtained from docking simulation (A) Piperine boundto IIA domain on HSA (protein and ligand colored blue, red, yellow, and green, respectively). (B) The hydrophobic and hydrophilicamino acid residues surrounding the probe piperine. (C) A graphical representation of Ligplot data to show the hydrophobic interactionsof HSA with piperine and (D) Pymol Stereo view of piperine binding site to HSA IIA domain in which piperine is rendered as cappedsticks and surrounding residues as lines (LYS351, GLU354, GLY328, ALA350, LEU347, ALA213, VAL216, VAL235, LYS212, andASP324).

Figure 7. Docking conformation of AGP–piperine complex obtained from Auto dock v 4.2. (A) Piperine bound to AGP (Proteinrepresented in ribbon model and ligand represented in stick model). (B) The binding pocket showing hydrophobic and hydrophilicamino acid residues surrounding the probe piperine. (C) Ligplot analysis of AGP with piperine to show the hydrophobic interactionsand (D) Pymol Stereo view of piperine bound to AGP in which piperine is rendered as capped sticks and surrounding residues aslines (Lys5, Ile2, Arg149, Lys147, Arg33, Leu8, Pro10, Val9, Leu4, and Asp115).

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interactions (Figure 6(D)). Thus, piperine is mostlysurrounded by hydrophobic and hydrophilic amino acidssuch as LYS351, GLU354, GLY328, ALA350, LEU347,ALA213, VAL216, VAL235, and ASP324. Thus, piper-ine binds with HSA mainly by hydrophobic interactionswith above-mentioned amino acids involved in the inter-actions, along with few hydrophilic interactions that areshown in (Figure 6(C)). These results obtained are inaccordance with the free energy calculations obtainedfrom binding constant which was derived from fluores-cence quenching data.

Interestingly, docking studies revealed that piperineinteracts with AGP with binding constant of1.2 × 104 M−1, and free energy was found to be−6.71 kcal M−1 which is nearer to the experimental9.3 ± .25 × 104 M−1 and computational −6.71 kcal M−1

values (Figure 7). The piperine–AGP complex is stabi-lized by hydrophobic amino acids and with one hydrogenbond between the compound and Arg 33 with bond lengthof 2.89 Å. However, piperine–HSA complex is stabilizedby hydrophobic interactions along with one hydrogenbond between the piperine and Lys212 with bond lengthof 2.98 Å. All the interactions of piperine are variedbetween HSA and AGP, but the binding constants andfree energy of experimental and computational are com-paratively less than HSA, suggesting that HSA–piperinecomplex is more stable than AGP–piperine complex.However, the pharmacological behavior could change dif-ferently in diseased conditions and thus, piperine bindingto plasma protein of AGP would certainly be an importantprocess. Particularly, AGP is an acute-phase protein;hence, there were differential interactions that wereobserved for the piperine molecule with HSA and AGP.

Analysis of the dynamics trajectories

Molecular docking/simulation provided an accurate anduseful picture on HSA–piperine interaction at molecularlevel because MD simulation resembles certain keyaspects such as physiological conditions pH, solvation,and temperature. The least docking energy which isobtained from Auto Dock software based on the experi-mental derived free energies and binding constants wasselected for 10,000 ps MD simulation. The trajectorieswere used to analyze the RMSDs, radius of gyration(Rg), and Root mean square fluctuations (RMSF).

RMSDs data analysis

In this study, to conform the stability of HSA and HSA–piperine, the RMSDs were plotted from 0 to 10000 ps(Figure 8(A)). The RMSD values steadily increased from0 to 1000 ps and then were stabilized at around 3000 psfor piperine-free HSA and HSA–piperine complexes.These results indicate that there was no increment of

RMSD and piperine-free HSA and HSA–piperine com-plexes reached equilibration and oscillate at around3000 ps. The RMSD values of atoms in unligand HSAand ligand HSA were calculated from a 0 to 10000 pstrajectory, where the data points fluctuated by.42 ± .036 nm for HSA alone and .5 ± .69 nm for HSA-piperine complexes. During the MD simulations, thesecomplexes remain in a stable binding position with lowRMSD fluctuations, confirming the credibility of dockedconformer which is predicted by Auto Dock 4.2.2. Inter-estingly, our data is in agreement with the earlier reportsfrom our group (Gokara et al., 2014; Malleda, Ahalawat,Gokara, & Subramanyam, 2012; Sudhamalla et al.,2010; Yeggoni et al., 2014). Also, the computationalsimulation values are in agreement with the fluorescenceemission and CD data; stable complexes were formed(HSA–piperine) due to microenvironmental changearound the tryptophan residue of HSA,

Radius of gyration (Rg) data analysis

The protein integrity is analyzed by plotting Rg valuesagainst function of time. Rg was used to assess the

Figure 8. (A) The root mean square deviation (nm) ofunligand HSA and ligand HSA (HSA–pipeirne). (B) The timedependence of the radius of gyration (Rg) for the backboneatoms of unligand HSA and ligand HSA (HSA–piperine).

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stability of the back bone atoms of HSA and HSA-piper-ine complexes (Figure 8(B)). In HSA alone and HSA–piperine, the Rg value is stabilized at about 1500 ps,which implies that the MD have achieved equilibrium at3000 ps. Firstly, the Rg value of both free HSA andHSA–piperine complex was 2.64 nm. The unligand HSAand HSA-piperine complexes were stabilized at2.57 ± .03 nm (Figure 8(B)). The variation of Rg atequilibration point around 3000 ps from HSA to HSA–piperine over the course of simulation time indicatesconformational rearrangements and stabilization of sec-ondary structure of HSA. These results are perfectlymatching to the conformational changes and stability ofthe complexes observed from CD data (Figure 5 and SFigure 3). The Rg value of HSA, which is shown experi-mentally by neutron scattering in aqueous solution, was2.74 ± .35 nm (Kiselev et al., 2001) which showed thatit is synchronizing with the experimental data. The previ-ous reports showed that the Rg of HSA determinedexperimentally from neutron scattering in aqueous solu-tion 2.74 ± .035 nm, which indicates that the simulationsperformed are identical to the experimental values(Kiselev et al., 2001). Also, the present result is closelymatching with our previous reports that β-sitosterol andasiatic acid stabilize from 2.59 ± .03 to 2.40 ± .031 nm;2.42 ± .03 to 2.45 ± .01 nm for free HSA and HSA–withcomplexes, respectively (Gokara et al., 2014; Sudhamallaet al., 2010). As seen in the (Figure 8(B)), the Rg ofpiperine-HSA decreases upon binding, this implies acompact structure of HSA. However, the protein micro-environment of HSA is changed upon binding leading tothe structural changes of HSA. These results are con-comitant with the CD data that upon binding of piperinewith HSA, there is a considerable conformational changeof secondary structure of protein (S. Table 1) asdiscussed earlier (Figure 5(A)).

Root mean square fluctuations

In order to examine the local protein mobility of HSAwe have calculated RMSF value of HSA alone andHSA-piperine complexes. The RMSF were plottedagainst residue numbers based on the 10000 ps trajectory(Figure 9(A)). The profile atomic fluctuations were foundto be very similar to those of HSA and HSA–piperinecomplex. Here, the results show that except subdomainIIA, rest all domains IA, IB, IIB, and IIIA of HSA areshowing high fluctuation which indicate that piperine ismore rigid at site I, particularly IIA (Figure 9(A)). Thisrigidity was located to individual residues of LYS351,GLU354, ALA213, VAL216, and ASP324 of HSA–piperine (Figure 9(B)). The fluctuations of subdomainIIA and fluctuation of residues at the binding site clearlysuggests that the piperine specifically interacts with themajor ligand-binding site I which is subdomain IIA of

HSA. Also, the rigidity of the residues of IIA domainmay lead the conformational changes of the protein uponbinding of piperine to HSA (Figure 5). Additionally, thespecific binding also confirmed from the site-specificmarkers showed that piperine binds to IIA domain(Figure 4).

In order to know the mobility, stability, and flexibil-ity of least energy conformer, we investigated the dockedconformer at different intervals of 1–10 ns (Figure 10).From these results, it clearly implies that during the per-iod of 4 ns, the flexibility of the piperine in the bindingpocket of subdomain IIA of HSA is not much fluctuated.Further, slight structural rearrangement of amino residueswas obseved till 6 ns and then it got stabilized at 10 nsupon binding of piperine with HSA. The rigidity ofbinding site, which is not varied much even in the caseof RMSD as whole, indicate that piperine binding is sta-ble enough, which is very well supported by the rigidityof subdomain IIA (Figure 9(A) and (B)). Here, the struc-tural arrangement and the variation are firmly supportedby Rg (Figure 8(B)). This study gives that there are

Figure 9. (A) The RMSF values against residue numbers. TheRMSF values of unligand HSA and HSA–piperine complexwere plotted against residue numbers. (B) The profile of atomicfluctuations. Atomic fluctuations of unliganded HSA andHSA–piperine complex to the active site amino acid residuespresent in the IIB subdomain of HSA which is Site I.

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conformational rearrangement of protein structure whichimplies that the piperine binds with site I (subdomainIIA) of HSA. The above results are very much supportedby our pervious experimental and simulation reports onchitosan oligomers and asiatic acid (Gokara et al., 2014,in press).

Conclusions

This study illustrates the concentration-dependent mannerof piperine which showed a clear decrease in the per-centage growth with increase in inhibition of inflammat-ed mouse macrophages (RAW 264.7 cell line). Thus, itindicates that piperine is a potent anti-inflammatoryagent. Further, the fluorescence studies revealed thatquenching of piperine interaction is by static mechanismthus, piperine binds with HSA within the subdomain IIAwith hydrophobic and hydrophilic interactions. Its bind-ing constants and free energy were found to be Kpiperine =5.7± .2 × 10 5 M−1 and −7.8 kcal M−1 at 25 °C, respec-tively. This value is closely synchronized to the compu-tationally calculated free energy −6.8 kcal/mol.Interestingly, piperine is also binding with AGP (anacute phase protein) having a binding constant of9.3 ± .25 × 104 M−1, free energy −6.7 kcal M−1 at25 °C, this value is very closely agreement with thecomputationally calculated free energy −6.71 kcal M−1.Further, CD studies reveal that there are marginalchanges in the secondary structure of the HSA upon

binding of piperine, which showed slight decrease in theα-helices and increase in the β-sheets and random coil.These changes in the secondary structure of protein arewell supported by the partial fluctuation in the RMSDand Rg values of MD. Molecular displacement experi-ments and docking studies showed that piperine bindsspecifically to subdomain IIA, mostly by hydrophobicand hydrophilic interactions. Additionally, rigidity andthe changes in the local protein mobility with piperineand surrounding residues in HSA of 10000 ps is doneusing RMSF, which gives the integrity of piperine andHSA–piperine complex. MD data showed that HSA pluspiperine reaches equilibration state at around 3000 ps,which indicate that stable HSA and piperine complexwere generated. Our work provided a quantitative dataon the binding affinity of piperine with drug carrier pro-tein of HSA and AGP. Thus, HSA and AGP play a spe-cific major role in the pharmacokinetic of piperine andits vital role in the development of piperine inspireddrugs.

Supplementary material

The supplementary material for this paper is availableonline at http://dx.doi.10.1080/07391102.2014.947326.

AcknowledgmentsMK acknowledges UGC-New Delhi for providing financialsupport under the scheme of Dr. D. S. Kothari Postdoctoral

Figure 10. Showing hydrophobic interaction with subdomain IIA at different nanoseconds, using Ligplot for HSA–piperinecomplex.

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Fellowship. We thank CIL and BIF, University of Hyderabad,for CD and Bioinformatics facilities.

FundingThis work was supported by Department of Science and Tech-nology [grant number SR/SO/BB-0123/2010 and DST-FIST],DBT-CREBB, India and UPE-2, University of Hyderabad.

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