Quantum Chemical and Docking Insights into Bioavailability

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Quantum Chemical and Docking Insights into Bioavailability Enhancement of Curcumin by Piperine in Pepper Vaishali M Patil, Sukanya Das, and Krishnan Balasubramanian J. Phys. Chem. A, Just Accepted Manuscript • DOI: 10.1021/acs.jpca.6b01434 • Publication Date (Web): 25 Apr 2016 Downloaded from http://pubs.acs.org on April 28, 2016

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The Journal of Physical Chemistry

Quantum Chemical and Docking Insights into Bioavailability Enhancement of Curcumin by Piperine in Pepper Vaishali M. Patila, Sukanya Dasb and Krishnan Balasubramanian*c a

School of Pharmacy, Bharat Institute of Technology, Partapur, Meerut 250 103, Uttar Pradesh, India b

Discipline of Pharmacology, School of Medicine, The University of Adelaide, Adelaide, South Australia 5005, Australia

c

School of Molecular Sciences, Arizona State University, Tempe AZ 85287-1604

Abstract

We combine quantum chemical and molecular docking techniques to provide new insights into how piperine molecule in various forms of pepper enhances bioavailability of a number of drugs including curcumin in turmeric for which it increases its bioavailability by a 20-fold. We have carried out docking studies of quantum chemically optimized piperine structure binding to curcumin, CYP3A4 in cytochrome P450, UDP-glucose dehydrogenase (UDP-GDH), pGlycoprotein

and

UDP-glucuronosyltransferase

(UGT),

the

enzyme

responsible

for

glucuronosylation, which increases the solubility of curcumin. All of these studies establish that piperine binds to multiple sites on the enzymes and also intercalates with curcumin forming a hydrogen bonded complex with curcumin. The conjugated network of double bonds and the presence of multiple charge centers of piperine offer optimal binding sites for piperine to bind to enzymes such as UDP-GDH, UGT, and CYP3A4. Piperine competes for curcumin’s intermolecular hydrogen bonding and its stacking propensity by hydrogen bonding with enolic proton of curcumin. This facilitates its metabolic transport, thereby increasing its bioavailability both through intercalation into curcumin layers through intermolecular hydrogen bonding, and by inhibiting enzymes that cause glucuronosylation of curcumin.

*

Address Correspondence to [email protected] P: (480)965-3461

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1. Introduction Piperine is a naturally occurring heterocyclic compound found in black pepper, white pepper and green pepper, and this molecule is attributed to the pungency and heat of all forms of pepper.1 Furthermore the medicinal enhancing and sedative properties of piperine are well-known.2-18 Piperine shares with many alkaloids a common nitrogen-containing heterocycle; in this case derived from a saturated six-membered ring containing N or piperidine. The presence of a conjugated aliphatic chain that acts as a bridge between the piperidine heterocycle and a 5-(3,4-Methylenedioxyphenyl) group makes piperine a structurally interesting candidate for providing optimal electronic features for its propensity to bind to cytochrome P450 enzymes, for example, CYP3A4 and UGT enzymes involved in drug metabolism,6-18 as well as a transporter of partially hydrophobic molecules like curcumin through metabolic pathways. These enzymes are major phase I and phase II drug-metabolizing enzymes.6-18 Curcumin, a yellow compound that occurs in nature in turmeric rhizome or curcuma longa, exhibits a number of medicinal properties such as anti-cancer, anti-Alzheimer’s, anti-bacterial, and antiinflammatory.5,19-42 However curcumin is highly insoluble in water at pH 7 and 18 °C, that is, a solubility of less than 0.1 mg/ml, although it is more readily soluble in ethanol (10 mg/ml). Curcumin’s insolubility in water arises primarily from its hydrophobic aliphatic conjugated bridge, even though it contains an enolic group and phenolic OH groups which are partially acidic in nature. The presence of these groups, in fact, makes it much more soluble under alkaline pH yielding a dark red solution.38-39 It has been recently observed by Jagnnathan et al.5 that curcumin’s intermolecular hydrogen bonding, responsible for stacking of layers of curcumin molecules, can be broken down by heating an aqueous solution of curcumin, thus making it more soluble under higher temperatures. Although Piperine molecule in also not much soluble in water, that is, 1 g/25 L at 18 °C and neutral pH, in this study we show that the electronic features of piperine are such that it can compete for intramolecular hydrogen bonding of curcumin that leads to the breaking of stacking of curcumin layers. Consequently, when curcumin and piperine are heated together in an aqueous solution, piperine may effectively enhance transport of curcumin through intercalation mechanism that we describe in this work. Moreover, we show here that the electronic features of piperine provide optimal orbitals, electrostatic potentials, and charges for piperine to form a stronger intermolecular bonding between curcumin and piperine, thus competing with curcumincurcumin molecular stacking.

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It is well known that piperine enhances bioavailability of many drugs such as digoxin, cyclosporine and bioactive curcumin14 but the exact mechanism has not been well understood. A proposed mechanism of bioavailability enhancement of piperine is primarily through inhibiting first-pass metabolism enzymes that increases the metabolic rate of excretion of such drugs through oxidation and glucuronosylation, a process that converts hydrophobic molecules such as curcumin into more water soluble molecules by a transfer of glucuronic acid to bioactives by linking through a glycosidic bond in the intestine and liver.6-14 This makes curcumin less bioavailable as once glucuronosylated, it becomes more water soluble and excreted.15 As shown by Shoba et al.8 piperine is known to increase curcumin’s bioavailability by a factor of 20. Although the mechanism of how piperine enhances the bioavailability of curcumin is not well understood, it has been attributed to piperine’s inhibition of critical enzymes such as UDP-GDH, UGT, and CYP3A4 in cytochrome P450 family of monoamine oxidase enzymes.16 These enzymes play important roles in metabolizing many drugs primarily by increasing the solubility of drugs by adding glucuronic acid, thereby speeding up their excretion out of the body. Piperine appears to bind to these enzymes thereby inhibiting their action thus facilitating greater availability of drugs or therapeutic bioactives such as curcumin in the body allowing for greater time for their absorption in the intestine. However exact mechanisms are far from understood and there is much to be desired by way of molecular insights into piperine’s 20-fold increase of bioavailability of curcumin. In this study we have carried out high level quantum chemical calculations to derive new insights into the structure and binding of piperine. We have obtained the optimized geometry of piperine which is then used for docking piperine to a number of biologically relevant metabolic proteins. We have employed high-level correlation consistent aug-cc-pVTZ basis sets that included 1403 basis functions as well as 6311G+(d,p) basis sets to obtain the electronic properties and spectra of piperine. We have also carried out docking studies of piperine-C3PYA4, piperine-UGT, curcumin-C3PYA4, curcumin-UGT, piperine-p-glycoprotein and piperine-curcumin complexes. Our computations provide significant new insights into explaining piperine’s bioavailability enhancing features through the docked complexes, binding energies and inhibition constants. Laplacians of charge densities provide optimal features for piperine to compete with intramolecular hydrogen bonding and π-stacking as well as its ability to bind to critical enzymes in cytochrome P450, causing their inhibition in metabolizing curcumin, thus allowing more time for the body to absorb it. On the basis of our computed piperine-curcumin complex, we also predict that heating a solution of piperine and curcumin in water should facilitate thermal breaking of stacking between curcumin layers, and remaking of bonding between curcumin and piperine. The present study provides valuable molecular insights for future drug developments with molecular similarity methods43,44 and in general, properties of related polycyclic aromatics45 and alkaloid compounds. 3 ACS Paragon Plus Environment


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2. Method of Computations. A. Quantum Chemical Computations. We have employed Dunning’s46 both aug-CC-pVTZ and CC-pVTZ basis sets for all atoms of piperine with 5D and 7F pure Gaussian functions options for the 3d and 4f polarization functions. This resulted in 1403 Gaussian basis functions for the piperine molecule. We have also employed a less expensive 6-311G+(d,p) basis sets. Geometry optimizations were performed for both the basis sets to compare the equilibrium geometries. The equilibrium geometries came out quite similar but there were differences in the energetics. We have used the Becke-Lee-Yang-Parr47,48 density functional method abbreviated with wellknown B3LYP acronym for the computation of equilibrium geometries and energies. We have also compared the B3LYP method47,48 with the second-order Møller-Plesset level of theory((MP2)49 computations with the 6-311G+(d,p) basis sets. As we did not find significant differences in the optimized geometries, we uniformly chose the B3LYP method for optimization of the structure of piperine. We have carried out full geometry optimization of the ground states of piperine. We have obtained the Laplacians of the charge densities in order to provide further insights into the binding sites and binding propensity of piperine with cytochrome P450 enzyme CYP3A4, UGT, and curcumin molecule. All of the quantum chemical computations were carried out using Gaussian ’09 package of codes,54 electrostatic potentials and Laplacians were obtained using Molden package while structural properties, and structures were presented by visualization software Chemcraft. B. Molecular Docking Studies In order to gain further insights into enzyme-piperine, enzyme-curcumin and curcumin-piperine interactions we carried out sets of docking studies of each of these molecules with the two key enzymes: CYP3A4 and UGT using the optimized geometry of curcumin and piperine at aug-cc-pvTZ level. The molecular level interactions of curcumin and piperine towards CYP3A4 in cytochrome P450, UDP-glucuronosyltransferase (UGT) and P-glycoprotein (P-GP) were carried out using AutoDock4.0.50 All computational simulations were performed on a Hewlett-Packard 110-216ix workstation with Intel Core i3 processor, 2GB RAM and Red Hat Linux as the operating system. The major steps in docking study are as follows: Protein preparation: Previously reported docking protocol has been followed with X-ray crystal structure of CYP3A4 in cytochrome P450 (PDB ID: 4D78), UDP-glucuronosyltransferase (PDB ID: 1IIR) and P-GP (PDB ID: 3G60) downloaded from Protein Data Bank.51,52,9 Protein preparation was carried out using the AutoDock Protein Preparation Wizard. In the protein preparation process in order to satisfy the valency, we have add the polar hydrogens, merged the lone pairs and added the Merz-Kollman charges to the selected macromolecule/protein. AutoDock uses the united atom model to represent the molecules and the AD4 4 ACS Paragon Plus Environment

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scoring function needs to be calibrated using the charges present on both protein as well as ligand. The water molecules were removed from the protein surface to mask the surface and the protein file was saved in a PDBqt file format. Ligands preparation: Ligands were prepared using Chemaxon software (Chemaxon Ltd., Budapest, Hungary). Ligands were imported in to the AutoDock workspace and torsion information was added i.e. number of torsions, which torsions are to be treated as rotatable during docking. The output for ligand was saved in PDBqt file. Calculation of Grid maps AutoGrid, a part of ADT calculates the energy of non-covalent interactions between the protein and probe atoms that are located in the different grid points of a lattice that defines the area of the macromolecule where the possibility of ligand binding is studied. Receptor grids were generated using 60 × 60 × 60 Grid points in xyz with grid spacing of 0.375 Å and Map types were generated using AutoGrid-4.0. The grid parameter file (.gpf) was written using MGL Tools-1.4.6.50 Docking parameters and running docking simulation For docking, the docking parameter files (.dpf) were written using MGLTools-1.4.650 and docking was carried out with following parameters: number of runs: 50, population size: 150, number of evaluations: 2,500,000 and number of generations: 27,000, using AutoDock4.0. The results of AutoDock contain an output file (.dlg) and the generated conformers were scored and ranked as per the interaction energy. Analysis of docking results obtained through Lamarckian genetic algorithm was done using MGLTools-1.4.6 and the top scoring molecule in the largest cluster was analyzed for its interaction with the protein. Validation The molecular docking method was internally validated by redocking the co-crystal ligands as well as reported inhibitors51,52,53 and the validated protocols were applied to study the binding mode of curcumin and piperine in the CYP3A4, UDP-glucuronosyltransferase (UGT) and P-GP active sites. As pointed out by Plewczynski et al.,72 care should be exercised in validating docking results.

3. Results and Discussion

We start with a depiction of a 2D sketch of piperine molecule in Fig. 1. Piperine molecule possesses some of the typical alkaloid features such as a nitrogen-containing heterocycle and a ring with oxygen atoms. The presence of the C=O group would lend some polarity to the molecule but the conjugated bridge and the connecting benzene ring would make this part of the molecule somewhat non-polar or hydrophobic. The conjugated network of six alternating double bonds starting with the C=O groups and ending with the 5 ACS Paragon Plus Environment


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benzene ring should provide a framework of π-orbitals perpendicular to that part of the plane of the molecule. Thus layers of piperine molecule could arrange themselves with π−π interaction favoring a layered structure in the solid state similar to the one proposed by Jagannathan et al.5 for curcumin. The optimized three-dimensional geometry of piperine is shown in Fig. 2 at the aug-cc-pvTZ level with Cartesian coordinates of the optimized structure deposited as supplementary information. It is evident from Fig. 2 that the saturated 6-membered heterocyclic ring is puckered and projects out-of-plane of the conjugated network part of piperine. The conjugated set of C-C bonds clearly exhibit bond alternation. These structural features and our computed spectra which will be the subject of a future publication are consistent with the observed spectra of piperine.55-59 The primary portion of piperine that would be involved in any binding to another molecule or to an enzyme would be the part that starts with C=O, N and ends with the five-membered ring. This part of the network, C=O group, and the basic Nitrogen atom would be the ones that would participate in such intermolecular bonding or binding to an enzyme. Next we consider the electronic features and properties of the piperine molecule in order to understand its bioavailability enhancing properties. The HOMO is delocalized over the entire conjugated aliphatic and aromatic network consisting primarily of the p-orbitals of the unsaturated network, although the N-atom of the heterocyclic ring also furnishes its 2p orbital to the HOMO. Likewise the LUMO of piperine involves the conjugated network of bonds. The delocalization stabilizes the LUMO facilitating ready acceptance of electrons from the multiple donating sites of P450 (CYP3A4) and UGT enzymes that are involved in the metabolism of molecules such as curcumin by glucuronosylation.8,14,15 As piperine provides a framework of extended HOMO and LUMO that extends all the way from nitrogen atom to the aromatic carbons, piperine can readily accept and also donate electrons to the enzyme at multiple binding sites. This is consistent with the previous experimental studies11-12 of various enzymes such as CYP3A4 and UGT that suggest the existence of multiple binding sites that can facilitate binding of multiple molecules and also multiple sites of a molecule like piperine. We can obtain further insights through the Laplacian of the charge density, the second derivative of charge density, shown in Figure 3. The Laplacian of the charge density exhibits peaks on carbon attached to the nitrogen and a few other centers, suggesting electron depletion on these sites. A nucleophile such as OHwould primarily cleave the C-N bond of piperine. This is consistent with the observed reaction of piperine with OH- which cleaves the molecule at the C-N bond, producing piperic acid upon attachment of OH- to the C attached to the heterocyclic 6-memberd ring of piperine. Thus piperine exhibits multi-faceted electronic features, providing multiple sites as it has delocalized sites of accumulation and depletion of charge densities. Fig. 4 shows different views of the computed molecular electrostatic potentials (MEP) of the piperine molecule. It is evident from Fig. 4 that the primary red region of the MEP is at the oxygen of C=O 6 ACS Paragon Plus Environment

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group which is consistent with the charges. This is the region of strongest affinity for a proton while the blue region (cold region) is the region of affinity for an electron. As seen from Fig. 4, the blue and cyan regions are delocalized over the entire molecule due to extended conjugation including the aromatic ring which is primarily cyan. The yellow centers correspond to the oxygen atoms of the five membered ring which are somewhat unreactive. Thus the extended cyan and blue regions in piperine offer multiple binding sites to accept electronic density in the piperine molecule with the C=O oxygen, rich in electronic density, would act as an electron donor or a proton acceptor. The above analysis reveals multifaceted nature of piperine in its ability to bind to multiple sites both through the operation of electron acceptance and donation and through excitonic transfer of electron density over the conjugated network. Thus piperine consists of appropriate electronic features extended over the molecule to intercalate with curcumin by competing with curcumin-curcumin intermolecular hydrogen bonding forces. We note that the crystal structure of piperine exhibits intermolecular C-H..O hydrogen bonding and π−π Intermolecular interactions as shown by Karunakar et al.

73

The solid piperine structure is

comprised of packing of the tetramer formed by C-H…O and C-H…π-π Intermolecular interactions between the adjacent layers of piperine. Jagannathan et al.5 have carried out spectroscopic studies on curcumin as a function of temperature, as curcumin’s solubility in water varies with temperature strongly. Thus Jagannathan et al.5 have suggested that the variation of solubility of curcumin at different temperatures could be due to the breaking of intermolecular hydrogen bonding between the layers formed primarily through the acidic enolic proton exchange. The enolic proton in curcumin appears to play a critical role in binding layers of curcumin together with intermolecular sharing of proton with adjacent layers through hydrogen bonding. Heating up an aqueous solution of curcumin appears to break the H-Bonding thus exposing the polar groups of different layers of curcumin and hence making it more soluble in water. This aggregation−disaggregation of curcumin observed by Jagannathan et al.5 at various temperatures is not only consistent with the observed changes in temperature-dependent electronic transition but also suggests the possibility that the C=O group of piperine can induce the breaking of the hydrogen bonding between two layers of curcumin by a competitive mechanism, where the ionic C=O group of piperine competes for the enolic proton of curcumin. Consequently, we suggest that when an aqueous solution of piperine with curcumin is heated, the supplied thermal energy not only would assist in the breaking of hydrogen bonding between layers of curcumin but we suggest that it would also aid in intercalating piperine between adjacent layers of curcumin though reformation of hydrogen bonding and π−π Intermolecular interactions between alternating layers of curcumin and piperine. The primary mechanism of such insertion of piperine would be through the exchange of a proton from curcumin to the C=O group of piperine. This is strongly supported by both the molecular 7 ACS Paragon Plus Environment


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electrostatic potentials and charges on curcumin. Consequently, at higher temperatures, weak interactions between the layers of curcumin can be broken down, and we suggest that piperine molecule can be intercalated in alternating layers as the solution cools. Moreover the conjugated π-electronic networks of both piperine and curcumin can align in a complimentary manner enabling weak dative bond between the layers. This may explain the fact that although piperine is known to enhance the bioavailability of other drugs, it exhibits significant enhancement of the bioavailability of curcumin, that is, a 20-fold enhancement in the case of curcumin owing to our proposed intercalation of piperine with curcumin, aiding in transport of curcumin though the metabolic pathway, in addition to inhibition of P450 (CYP3A4) and UGT enzymes. Thus heating a solution of pepper with turmeric enhances intermolecular interaction between the two species. Our suggestion of intercalation appears to be the first mechanism for piperine’s enhancement in transporting curcumin through the metabolic pathway. The second mechanism of piperine’s role in the bioavailability enhancement of curcumin is through its binding to CYP3A4 enzyme of cytochrome P450 family of enzymes and to UDP-glucose dehydrogenase (UDP-GDH) and UDP-glucuronosyltransferase (UGT), the enzymes responsible for glucuronosylation both in intestine and liver.15 Any such donation or acceptance of charge would induce excitonic stabilizing feature in piperine extending over the entire conjugated network. This is consistent with the HOMO and LUMO which support such excitonic delocalization over the network. Once piperine binds to UGT, it inhibits UGT’s action to glucuronosylate curcumin which would essentially convert hydrophobic curcumin to soluble form through attachment of glucuronic acid (glucose-like) molecule, thus enhancing its water solubility. When curcumin becomes water soluble by glucuronosylation, it would be readily excreted from the body. Consequently, piperine prevents glucuronosylation of curcumin in P450 (CYP3A4) thereby allowing greater time for the guts to absorb curcumin. Consequently, a 20-fold increase in the bioavailability of curcumin by piperine is explained by a dual mechanism: (1) enhanced transportation of curcumin through our proposed intercalation and (2) binding to cytochrome P450 enzyme CYP3A4 and UGT enzyme thereby preventing glucuronosylation of curcumin. In the human body the CYP3A4 enzyme is responsible for the metabolism of drugs through oxidation, hydroxylation, and heteroalkyl removal. Curcumin acts as a substrate by binding to the active site of CYP3A4 having broad selectivity and highest inhibitory activity,61-64 thus exemplifying its importance for curcumin biotransformation. In silico structure-based approaches and availability of the crystal structure of CYP3A4 offers an opportunity to gain further insight into protein-ligand interaction.65-67 Both Piperine and Curcumin can modulate CYP3A4 expression and modify its pharmacokinetic profile in male SpragueDawley rats. Curcumin attenuates the level of CYP3A4 in small intestine.68-71 Piperine inhibits the major 8 ACS Paragon Plus Environment

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drug-metabolizing enzyme CYP3A4 as well as the drug transporter P-glycoprotein and influences the firstpass elimination of many drugs. Thus dietary piperine along with orally administered drugs could affect the plasma concentrations of CYP3A4 and P-glycoprotein in humans.70-71 In silico studies of Piperine-Fe conjugate into CYP450 3A4 (PDB ID 1W0E) have generated unique chemical arrangement in each of the five reported cavities. Piperine has been suggested as an enhancer of iron bioavailability and metabolism. Inhibition of CYP3A4 can help to explain the various drug-drug interactions as many drugs are CYP3A4 substrates and higher elevated plasma concentration of drugs may be attributed to a dual effect on drug transport and metabolism as supported by docking studies of Piperine with CYP3A4, Ferritin and Pglycoprotein.71 In view of the reported data and in order to test our hypothesis of the dual action of piperine’s bioavailability enhancement features, we have performed a molecular docking study to shed further light into an understanding of glucuronosylation inhibition of curcumin by piperine. Docking helps to specify the energetically stable ligand conformation at the active site of receptor. The various docking programs have an accuracy of only ~ 60% in predicting the correct docking conformation.73 Moreover the method could also produce false positive structure-based virtual screening when receptor plasticity is considered.74 In view of this flexible docking has been adopted and the root mean square deviations (RMSDs) of the receptors and ligands were compared (RMSD < 0.971Å) to correlate the crystallographically bound ligands with the test ligands. The chemical properties of curcumin such as the H-bond donating and accepting capacity of the βdicarbonyl moiety, H-bond accepting and donating capacity of the phenyl hydroxyl residues, H-bond accepting capacity of the ether residue in the methoxy groups facilitate intermolecular interactions and association with biomolecular targets. Our results of the six different docking studies are shown in Figures 5A-E, Figure 6, and Figure 7. Table 1 shows the energetic parameters, inhibition constant and the principal amino acid binding sites for curcumin and piperine with the three enzymes of interest, namely, CYP3A4 in cytochrome P450, UGT and P-GP. Binding mode analysis of Curcumin with CYP3A4 in cytochrome P450 has revealed one H-bonding interaction of Phenolic hydroxyl oxygen of Curcumin with the backbone amino proton of TYR347 in the hydrophilic pocket formed by TYR347, THR346, VAL146, PRO147, and ILE148. As the enolic proton of curcumin is held relatively tightly by the β-diketone groups of curcumin, we find that the enolic proton of curcumin does not contribute to any H-bonding interactions with the amino acid binding sites of the two enzymes. The other amino acids forming hydrophobic cavity are LYS143, GLU144, and ARG288 (Fig. 5A). Similarly the Piperine molecule has shown one H-bonding interaction with one of the benzodioxole oxygen with backbone amino proton of GLY438. The hydrophobic interactions were observed with ASN361, ASN426, ILE427, TYR432, PRO434, PHE435, MET445 and LEU449 (Fig. 5B). Kapelyukh et al.12 have 9 ACS Paragon Plus Environment


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reported the same binding site with slightly different binding energies for 7-benzyloxyquinoline (7BQ) molecule with the unbound crystal structures of human microsomal P450 3A4 (PDB ID: 1tqn and 1w0e). The 7BQ molecule exhibits multiple binding without any structural changes in the active site and the predicted binding energies for different sites A to E were found to be broadly similar i.e., isoenergetic interaction. Up to five molecules of 7BQ have been observed to bind simultaneously12 with different active sites of CYP3A4 suggesting both edge-to-face and stacking interactions between the aromatic rings of the 7BQ molecules. Piperine and Curcumin exhibit binding at different sites of the CYP3A4 enzyme with quiet similar docking score. Moreover, the sensitivity of the inhibition mechanism may be reduced if the inhibitors have two different substrate binding sites. The simultaneous binding of at least five substrates with different sites of CYP3A4 suggests that piperine can exhibit a synergistic effect with curcumin by adopting the simultaneous docking profile. Binding mode analysis of both curcumin and piperine with UGT indicates the presence of (i) Hbonding interaction between phenolic hydroxyl hydrogen with backbone oxygen of TYR122, (ii) H-bonding interaction between phenolic hydroxyl hydrogen with backbone oxygen of TRP216 and (iii) π-π interaction between curcumin benzene ring and PHE124. This concludes towards the polar and electronegative nature of pocket formed by PRO16, TYR122, PHE124, HIS125, TYR129, TRP218, ILE219, ALA311, GLY312, and ASP332 (Fig. 5C). Piperine exhibits binding of benzoxazole ring in the hydrophilic pocket formed by TYR122, PHE124, TYR129 and TYR190 with H-bonding interaction of benzoxazole oxygen with TYR122. The piperidine ring is accommodated in the hydrophobic pocket formed by amino acids GLY310, ALA311, ASP332, and GLN333 (Fig. 5D). As can be seen from Table 4 both piperine and curcumin have relative binding energies with cytochrome P450 enzyme CYP3A4 as well as UGT, but piperine has a dramatically larger inhibition constant toward UGT, the enzyme that is responsible for glucuronosylation of curcumin. The reported data suggest a strong correlation between the docked complex of P-GP-piperine with ADP, AMP-PNP and ATP and enhanced bioavailability of P-GP substrates in the presence of piperine.9 We carried out a similar docking study for piperine at the transmembrane domain (TMD, PDB ID: 3G60) and the binding energy that we compute is −6.29 kcal/mol with an inhibitory constant (K i ) 24.31µM. Our computed value is slightly less than the previously reported result9 (Docking score −7.90 kcal/mol at TMD of P-GP). As can be seen from Figure 7, piperine exhibits hydrophobic interactions at the active site formed by the residues TYR303, PHE332, PHE339, PHE724, SER 725, TYR949, SER975, VAL978, and ALA981. This binding pocket is the same for all of the conformers generated for piperine and it is similar to that reported for the cocrystallized ligand in PDB ID: 3G60.9 For other binding sites, competitive docking score was not observed. While the data presented by Singh et al.9 suggest an effective interaction of piperine at the nucleotide binding domain (NBD) (PDB ID: 2HYD; Dockscore = −8.60 and binding score = −9.65 10 ACS Paragon Plus Environment

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kcal/mol). Furthermore piperine shares the same amino acids with ADP, ATP, and AMP-PNP thus confirming that piperine binds in close proximity to NBD. In the present study, the AutoDock binding score of −6.29 kcal/mol with inhibitory constant in micromolar range defines piperine as a base molecule for potent P-GP inhibition binding at the TMD domain and thus facilitating passage for efflux of P-GP substrates. These features are consistent with the peak in the Laplacian plot of the charge density shown in Fig. 3, and the base nature of piperine is consistent with its alkaloid family’s features. Consequently, our docking studies confirm the dual action of piperine both as a P-GP inhibitor by binding at the NBD9 and TMD domains and as an inhibitor of CYP3A4 and UGT. In order to assess the intercalating propensity of piperine with curcumin as a carrier of the curcumin molecule through metabolic pathways, we have considered the nature of the intermolecular complex between curcumin and piperine. Figure 8 shows this complex which has a binding energy of −3.1 kcal/mol, typical for a π−π sandwich interaction. As seen from Fig. 8, the intermolecular complex formed by curcumin with Piperine is facilitated by the sandwich π-π interactions between both the benzene rings of curcumin and the benzene ring Piperine. As we find the distance between the enolic proton of curcumin to piperine to be smaller than the phenolic protons of curcumin and piperine, we suggest that the primary mode of intercalation is through enolic intermolecular hydrogen bonding and the sandwich π-π interactions between the aromatic rings of the two molecules. Consequently, these interactions between piperine and curcumin compete for intercalating between adjacent layers of curcumin thus providing for a channel for piperine to bind to curcumin in aiding its transportation through metabolic pathways. Hence piperine aids both in transporting curcumin and in inhibiting UGT, thus preventing its glucuronosylation of curcumin into a more water-soluble form. Once glucuronosylation of curcumin is inhibited by piperine, curcumin stays longer in the body providing more time for its absorption thus enhancing its bioavailability by 20-fold through this dual mode of action.

4. Conclusion Computational insights derived here are supported by empirical observations and administration of curcumin in traditional Indian medicinal recipes. Typically turmeric and black pepper in powdered forms are heated with milk, and then taken to increase its effectiveness for a variety of medical conditions ranging from cough due to common cold to prevention of Alzheimer’s disease. In the latter case we propose that piperine enhances curcumin’s bioavailability by intercalating with it, transporting it through the blood-brain11 ACS Paragon Plus Environment


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barrier, and finally release of curcumin to bind to amyloid-β protein. Thus curcumin prevents further misfolding of amyloid-β into neurotoxic conformations found in plaques that cause Alzheimer disease by inhibition of neurotransmitter acetyl-choline and neurotoxicity. Our high-level quantum chemical studies combined with docking studies that we have performed between piperine and P-GP, UGT, and CYP3A4 enzymes provide insight into a dual mechanism, consistent with our suggestion of formation of an intercalation complex between piperine and curcumin thus aiding in transport of curcumin, and piperine’s propensity to bind to multiple sites of Cytochrome P450 enzyme CYP3A4 and UGT an enzyme responsible for glucuronosylation of curcumin. We note that the structural flexibility of macromolecules the selected flexible docking protocol helps to minimize the chances of false positive results.74 Thus the dual action of piperine can be considered as the basis for a 20-fold increase of curcumin’s bioavailability in the presence of piperine. Based on this study, we suggest that optimal administration of curcumin should include piperine after heating and cooling in an encapsulated nanoform. The advantage of nanocapsule form is that it would be metabolized directly in liver and intestine, thus avoiding highly acidic environment of stomach, whereas under alkaline pH conditions in the intestine curcumin forms a more soluble red form which together with piperine’s dual action can more readily be absorbed in the gut.

Conflict of Interest The authors do not have any conflict of interests.

Acknowledgement A part of this work was carried out by Balasubramanian and Das, while they were at California State University, East Bay, supported by office of basic energy sciences, chemical, bio and geo sciences division of US Department of Energy.

Supporting Information The quantum chemically optimized geometry of piperine molecule at the CC-pvTZ level employing the B3LYP method is included as supporting information. This file contains the Cartesian (x,y,z) coordinates of each atom with a label for the atom followed by its charge, and then the x,y,z coordinates of the optimized geometry. All distances are in Å units. 12 ACS Paragon Plus Environment

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References (1) Khare, C. P. Encyclopedia of Indian Medicinal Plants; Springer-Verlag: Berlin, Germany, 2004. (2) Anand, P.; Kunnumakkara, A. B.; Newman, R. A; Aggarwal, B. B. Bioavaliability of CurcuminProblems and Promises. Mol. Pharmaceut. 2007, 4, 807-818. (3) Wilken, R; Veena, M. S.; Wang, M. B.; Srivatsan, E. S. Curcumin: A Review of Anti-cancer Properties and Therapeutic Activity in Head and Neck Squamous Cell Carcinoma. Mol. Cancer 2011, 10:12, doi: 10.1186/1476-4598-10-12. (4) Moorthi, C.; Kiran, K.; Manavalan, R.; Kathiresan, K. Preparation of Curcumin-peprine Dual Drug Loaded Nanoparticles. Asian Pac J Trop Biomed. 2012, 2, 841–848. (5) Jagannathan, R; Abraham, P. M.; Poddar, P. Temperature-Dependent Spectroscopic Evidences of Curcumin in Aqueous Medium: A Mechanistic Study of Its Solubility and Stability. J. Phys. Chem. B. 2012, 116, 14533−14540. (6) Atal, C. K.; Dubey, R. K.; Singh, J. Biochemical Basis of Enhanced Drug Bioavailability by Piperine: Evidence that Piperine is a Potent Inhibitor of Drug Metabolism. J Pharmacol Exp Ther. 1985, 232, 258–62. PMID: 3917507. (7) Kumar, A; Khan, I. A.; Koul, S; Koul, J. L.; Taneja, S. C.; Ali, I; et al. Novel Structural Analogues of Piperine as Inhibitors of the NorA Efflux Pump of Staphylococcus Aureus. J Antimicrob Chemother. 2008, 61, 1270–6. PMID: 18334493. (8) Shoba , G.; Joy, D.; Joseph , T.; Majeed, M.; Rajendran, R.; Srinivas, P.S. S. R. Influence of Piperine on the Pharmacokinetics of Curcumin in Animals and Human Volunteers. Planta Med. 1998, 64, 353356 (9) Singh, D. V; Godbole, M. M.; Misra, K. A Plausible Explanation for Enhanced Bioavailability of PGP Substrates in Presence of Piperine: Simulation for Next Generation of P-GP Inhibitors. J Mol Model. 2013, 19, 227–38. PMID: 22864626. (10) Khom, S.; Strommer, B.; Schoffmann , A.; Hintersteiner, J.; Baburin, I.; Erker, T.; Schwarz, T.; Schwarzer, C.; Zaugg , J.; Hamburger, M.; Hering, S. GABAA Receptor Modulation by Piperine and a non-TRPV1 Activating Derivative. Biochemical Pharm. 2013, 85, 1827–1836. (11) Davydov, D. R.; Halpert, J. R. Allosteric Mechanisms in P450: Multiple Binding Sites, Multiple Conformers or Both. Expert Opinion Drug Metabolism AMP Toxicology. 2008, 4, 1523-1535. (12) Kapelyukh, Y.; Paine, M. J. I.; Marechal, J. D.; Sutcliffe, M. J. Multiple Substrate Binding by Cytochrome P450 3A4: Estimation of the Number of Bound Substrate Molecules. Drug Metabolism and Disposition. 2008, 36, 2136–2144. 13 ACS Paragon Plus Environment


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 14 of 26

(13) Sohl, C. D.; Isin, E. M.; Eoff, R. L. Cooperativity in Oxidation Reactions Catalyzed by Cytochrome P450 1A2- Highly cooperative Pyrene Hydroxylation and Multiphasic Kinetics of Ligand Binding. J Bio. Chem. 2008, 283, 7293 -308. (14) Bharadwaj, R. K.; Glaeser, H.; Becquemont, L.; Klotz, U.; Gupta, S. K.; Fromm, M. F. Piperine, a Major Constituent of Black Pepper, Inhibits Human P-glycoprotein and CYP3A4. J. Pharma. and Expt. Therapeutics. 2002, 302, 645-650. (15) Hoehle, S. I.; Pfeiffer, E.; Metzler, M. Glucuronidation of curcuminoids by human microsomal and recombinant UDP-glucuronosyltransferases. Mol. Nutr. Food Res. 2007, 51, 932-8 (16) Reen, R. K.; Jamwal, D. S.; Taneja, S. C.,;Koul, J. L., Debey, R. K., Wiebel, F. J.; Singh, J. Impairment of UDP-glucose Dehydrogenase and Glucuronidation Activities in Liver and Small Intestine of Rat and Guinea Pig in vitro by Piperine. Biochem. Pharm. 1993, 46, 229-238. (17) Volak, L. P.; Ghirmai, S.; Cashman, J. R.; Court, M. H. Curcuminoids Inhibit Multiple Human Cytochromes P450, UDP-Glucuronosyltransferase, and Sulfotransferase Enzymes, whereas Piperine Is a Relatively Selective CYP3A4 Inhibitor. Drug Metabolism and Disp. 2008, 36, 1594-1605. (18) Koul, S.; Koul, J. L.; Taneja, S. C.; Dhar, K. L.; Jamwal, D. S.; Singh, K.; Reen, R. K. ; Singh, J. Structure-Activity Relationship of Piperine and its Synthetic Analogues for Their Inhibitory Potentials of Rat Hepatic Microsomal Constitutive and Inducible Cytochrome P450 Activities. Biorg. Med. Chem. 2000, 8, 251-268. (19) Li, J. W.; Vederas, J. C. Drug Discovery and Natural Products: End of an Era or an Endless Frontier? Science 2009, 325, 161−165. (20) Sneharani, A. H.; Karakkat, J. V.; Singh, S. A.; Rao, A. G. Interaction of Curcumin with – Lactoglobulin-stability, Spectroscopic Analysis, and Molecular Modeling of the Complex. J. Agric. Food Chem. 2010, 58, 11130−11139. (21) Yang, F.; Lim, G. P.; Begum, A. N.; Ubeda, O. J.; Simmons, M. R.; Ambegaokar, S. S.; Chen, P.; Kayed, R.; Glabe, C. G. Frautschy, S. A.; Cole, G. M. Curcumin Inhibits Formation of Amyloid f3 Oligomers and Fibrils, Binds Plaques, and Reduces Amyloid in vivo. J. Biol. Chem. 2004, 280, 58925901. (22) Weggen, S.; Eriksen, J. L.; Das, P.; Sagi, S. A.; Wang, R.; Pietrizik, C. U.; Findlay, K. A.; Smith, T. E.; Murphy, M. P.; Butler, T.; Kang, D. E.; Marquez-Sterling, N.; Golde, T. E.; Koo, E. H. A Subset of NSAIDs Lower Amyloidogenic Af342 Independently of Cyclooxygenase Activity. Nature 2001, 414, 212-216.


Page 15 of 26

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


(23) Gasparini, L.; Rusconi, L.; Xu, H.; delSoldato, P.; Ongini, E. Modulation of β-amyloid Metabolism by Non-steroidal Anti-inflammatory Drugs in Neuronal Cell Cultures. J. Neurochem. 2004, 88, 337348. (24) Lim, G. P.; Chiu, T.; Yang, F.; Beech, W.; Frautschy, S. A.; Cole, G. M. The Curry Spice Curcumin Reduces Oxidative Damage and Amyloid Pathology in an Alzheimer Transgenic Mouse. J. Neurosci. 2001, 21, 8370-8377. (25) Zsila, F.; Bika´di, Z.; Simonyi, M. Molecular Basis of Cotton Effects Induced by the Binding of Curcumin to Human Serum Albumin. Tetrahedron: Asymmetry 2003, 14, 2433-2444. (26) Rovner, S. L. Untangling Alzheimer’s: Cover Story. Chem. Eng. News 2005, 83, 38-45. (27) Masuda, T.; Toi, Y.; Bando, H.; Maekawa, T.; Takeda, Y.; Yamaguchi, H. Structural Identification of New Curcumin Dimers and Their Contribution to the Antioxidant Mechanism of Curcumin. J. Agric. Food Chem. 2002, 50, 2524-2530. (28) Woo, J.; Kim, Y.; Choi, Y.; Kim, D.; Lee, K.; Bae, J. H.; Min, D. S.; Chang, J. S.; Jeong, Y. J.; Lee, Y. H.; Park, J.; Kwon, T. K. Molecular Mechanisms of Curcumin-induced Cyclotoxicity: Induction of Apoptosis through Generation of Reactive Oxygen Species, Down-regulation of Bcl-XL and IAP, the Release of Cytochrome c and Inhibition of Akt. Carcinogenesis 2003, 24, 1199-1208. (29) Moos, P. J.; Edes, K.; Mullally, J.; Fitzpatrick, J. Curcumin Impairs Tumor Suppressor p53 Function in Colon Cancer Cells. Carcinogenesis 2004, 9, 1611-1617. (30) Rashmi, R.; Kumar, S.; Karunagaran, D. Human Colon Cancer Cells Lacking Bax Resisit CurcuminInduced Apoptosis and Bax Requirement is Dispensable with Ectopic Expression of Smacor Downregulation of Bcl-Xl. Carcinogenisis 2005, 26, 713-723. (31) Khafif, A.; Schantz, S. P.; Chou, T. C.; Edelstein, D.; Sacks, P. G. Quantification of Chemopreventive Synergism Between (-)-epigallocatechin-3-gallate and Curcumin in Normal, Premalignant and Malignant Human Oral Epithelial Cells. Carcinogenisis 1998, 19, 419-424. (32) Balasubramanian, K.; Eckert, R. Green Tea Polyphenols and Curcumin Inversely Regulate Human Involucr in Promoter Activity via Opposing Effects on CCAAT/enhancer-binding Protein Function. J. Biol. Chem. 2004, 23, 24007-24014. (33) VanErk, M. J.; Teuling, E.; Staal, Y.C.M.; Huybers, S.; van Bladeren, P. J.; Aarts, J. M. M. J. G.; van Ommen, B. Time- and Dose-dependent Effects of Curcumin on Gene Expression in Human Colon Cancer Cells. J. Carcinogen. 2004, 3, 1-17. (34) Kim, M.-K.; Choi, G.-J.; Lee, H.-S. Fungicidal Property of Curcumalonga L. Rhizome-derived Curcumin against phyto-pathogenic Fungi in a Green House. J. Agric. Food Chem. 2003, 51, 15781581. 15 ACS Paragon Plus Environment


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 16 of 26

(35) Fujisawa, S.; Atsumi, T.; Ishihara, M.; Kadoma, Y. Cyctotoxicity, ROS Generation Activity and Radical-scavenging Activity of Curcumin and Related compounds. Anticancer Res. 2004, 24, 563569. (36) Jovanovic, S. V.; Steenken, S.; Boone, C. W.; Simic, M. G. H-atom Transfer is a Preferred Antioxidant Mechanism of Curcumin. J. Am. Chem. Soc.1999, 121, 9677-9681. (37) Basak, S. C.; Gute, B. D.; Grunwald, G. D. In Advances in Molecular Similarity, Vol. 2; CarboDorca, R.; Mezey, P. G., Eds.; JAI Press: Stamford, CT, 1998, pp 171-185. (38) Balasubramanian, K. Two Colorful Applications of the PPP Method. Int. J. Quantum Chem.1990, 37, 449-464. (39) Balasubramanian, K. Theoretical Calculations on the UV-visible Spectra of the Curcumin Pigment in Turmeric. Indian J. Chem.1991, 30A, 61-65. (40) Wright, J. S. Predicting the Antioxidant Activity of Curcumin and Curcuminoids. J. Mol. Struct. (THEOCHEM) 2002, 591, 207-217. (41) Balasubramanian, K. Molecular Orbital Basis for Yellow Curry Spice Curcumin’s Prevention of Alzheimer’s Disease. J. Agric. Food Chem. 2006, 54, 3512−3520. (42) Sun, Y. M.; Wang, R. X.; Yuan, S. L.; Lin, X. J.; Liu, C. B. Theoretical study of the Antioxidant Activity of Curcumin. Chinese J .Chem. 2004, 22, 827-830. (43) Basak, S. C. Use of Molecular Complexity Indices in Predictive Pharmacology and Toxicology: A QSAR Approach. Med. Sci. Res.1987, 15, 605-640. (44) Basak, S. C.; Mills, D.; Gute, B. D.; Hawkins, D. Predicting Mutagenicity of Congeneric and Diverse Sets of Chemicals Using Computed Molecular Descriptors: A Hierarchical Approach. Quantitative Structure-Activity Relationship (QSAR) Models of Mutagens and Carcinogens; Benigni,R., Ed.; CRC Press: BocaRaton, FL, 2003, Chapter 7, pp 207-234. (45) Dias, J. R. Handbook of Polycyclic Aromatic Hydrocarbons; Elsevier: Amsterdam, The Netherlands, 1987. (46) Dunning, Jr., T. H. Gaussian Basis Sets for Use in Correlated Molecular Calculations. I. The Atoms Boron through Neon and Hydrogen; J. Chem. Phys.1989, 90, 1007-23. (47) Parr, R. G.; Yang, W. Density Functional Theory of Atoms and Molecules; Oxford University Press: New York, 1989. (48) Becke, A. D. Density-functional Thermochemistry. III. The Role of Exact Exchange. J. Chem. Phys. 1993, 98, 5648-5652. (49) Møller, C.; Plesset, M. S. Note on an Approximation Treatment for Many Electron Systems. Phys. Rev. 1934, 46, 618-622. 16 ACS Paragon Plus Environment

Page 17 of 26

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


(50) Morris, G. M.; Huey, R.; Lindstrom, W.; Sanner, M. F.; Belew, R. K.; Goodsell, D. S.; Olson, A. J. AutoDock4 and AutoDockTools4: Automated Docking with Selective Receptor Flexibility. J Comput. Chem. 2009, 30, 2785–2791. (51) Kaur, P.; Chamberlin, R.; Poulos, T.L.; Sevrioukova, I.F. Structure-based Inhibitor Design for Evaluation

of

a

Cyp3A4

Pharmacophore

Model.

J.

Med.

Chem.

2015,

DOI:

10.1021/acs.jmedchem.5b01146 (52) Mulichak, A. M.; Losey, H. C.; Walsh, C. T.; Garavito, R.M. Structure of the UDPGlucosyltransferase GtfB that Modifies the Heptapeptide Aglycone in the Biosynthesis of Vancomycin Group Antibiotics. Structure 2001, 9, 547-557. (53) Laakkonen, L.; Finel, M. A Molecular Model of the Human UDP-Glucuronosyltransferase 1A1, Its Membrane Orientation, and the Interactions between Different Parts of the Enzyme. Molecular Pharm. 2010, 77, 931-939. (54) Gaussian 09, Revision D.01, M. J. Frisch, G. W. Trucks, H. B. Schlegel, G. E. Scuseria, M. A. Robb, J. R. Cheeseman, G. Scalmani, V. Barone, B. Mennucci, G. A. Petersson, et al. Gaussian, Inc., Wallingford CT, 2009. (55) Schulz, H.; Baranska, M. Identification and Quantification of Valuable Plant Substances by IR and Raman Spectroscopy. Vibr. Spectrosc. 2007, 43, 13–25. (56) Schulz, H.; Baranska, M.; Quilitzsch, R.; Schutze, W.; Losing, G. J. Characterization of Peppercorn, Pepper Oil, and Pepper Oleoresin by Vibrational Spectroscopy Methods. J. Agric. Food Chem. 2005, 53, 3358-3363. (57) Shingate, P. N.; Dongre, P. P.; Kannur, D. M. New Method of Development for Extraction and Isolation of Piperine from Black Pepper. Int. J. Phram. Sci.2013, 4, 3165-3170. (58) Adosraku, R. K.; Kyekyeu, J. P.; Attah, I.Y. Characterization and HPLC Quantification of Piperine Isolated from Piper Guinesse (Fam. PIPERACEAE) Int. J. Phram. Res. 2013, 5, 252-256. (59) Namjoyan, F.; Hejazi, H.; Ramezani, Z. Ethanolic Extract by High Performance Liquid Chromatography with Diode Array Detector. Jundishapur J. Nat. Pharm. Prod. 2012, 7, 163-167. (60) Kumar, S. P.; Rahul, B.; Rashmi, A. Phytochemical Investigation of Ethanolic Extract of Piper Nigrum, Zingiber Offcianale, and Alium Sativum and Spectrophotometric Detection of Piperine, Gingerol, and Allicin. Int. Res. J. Phram. 2014, 5, 814-816. (61) Rendic S. Summary of Information on Human CYP Enzymes: Human P450 Metabolism Data, Drug Metab. Rev. 2002, 34, 83-448.



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 18 of 26

(62) Appiah-Opong, R.; Commandeur, J.N.M.; Axson, C.; Vermeulen, N.P.E. Interactions between Cytochromes P450, Glutathione S-transferases and Ghanaian Medicinal Plants, Food Chem. Toxicol. 2008, 46, 3598-3603. (63) Appiah-Opong, R.; Commandeur, J.N.M.; van Vugt-Lussenburg, B.; Vermeulen, N.P.E. Inhibition of Human Recombinant Cytochrome P450s by Curcumin and Curcumin Decomposition Products, Toxicology, 2007, 235, 83-91. (64) Appiah-Opong R.; de Esch I.; Commandeur, J.N.M.; Andarini, M.; Vermeulen, N.P.E. StructureActivity Relationships for the Inhibition of Recombinant Human Cytochromes P450 by Curcumin Analogues, Eur. J. Med. Chem. 2008, 43, 1621-1631. (65) Sevrioukova, I. F.; Poulos, T.L. Structural and Mechanistic Insights into the Interaction of Cytochrome P450 3A4 with Bromoergocryptine, a Type I Ligand. J. Biol. Chem. 2012, 287, 35103517. (66) de Graaf C.; Vermeulen, N.P.E.; Feenstra, K.A. Cytochrome p450 in silico: An Integrative Modeling Approach, J. Med. Chem. 2005, 48, 2725-2755. (67) Watkins, P. B. The Barrier Function of CYP3A4 and P-glycoprotein in the Small Bowel. Adv Drug Deliv Rev. 1997, 27, 161-170. (68) Zhang, W.; Lim, L.Y. Effects of Spice Constituents on P-glycoprotein-mediated Transport and CYP3A4-mediated Metabolism in vitro. Drug Metab Dispos. 2008, 36, 1283-1290. (69) Velpandian, T.; Jasuja, R.; Bhardwaj, R. K.; Jaiswal, J.; Gupta, S. K. Piperine in food: Interference in the Pharmacokinetics of Phenytoin. Eur J Drug Metab Pharmacokinet. 2001, 26, 241-247. (70) Bhardwaj, R. K.; Glaeser, H.; Becquemont, L.; Klotz, U.; Gupta, S. K.; Fromm, M. F. Piperine, a Major Constituent of Black Pepper, Inhibits Human P-glycoprotein and CYP3A4. J Pharmacol Exp Ther. 2002, 302, 645-650. (71) Alugolu, V.; Rentala, S.; Komarraju, A. L.; Parimi, U. D. Docking Studies of Piperine-iron Conjugate with Human CYP450 3A4. Bioinform. 2013, 9, 334-338. (72) Plewczynski, D; Łaźniewski,M; Augustyniak, R.; Ginalski, K. Can We Trust Docking Results? J. Comput. Chem. 2011, 32, 742–755. (73) Karunakar,P.; Krishnamurthy,V.; Girija,C. R.; Krishna,V.; Vasundhara,D.E.; Begum, N. S.; Syed, A. A. In silico docking analysis of piperine with cyclooxygenases. J. BioChem. Tech.. 2012, 3, S122127. (74) Awuni, Y.; Mu, Y. Reduction of false positives in structure-based virtual screening when receptor plasticity is considered. Molecules 2015, 20, 5152-5164.


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Figure 1 Two-D Sketch of Piperine.

Figure 2 Optimized Structure of Piperine with various important bond distances (cc-pVTZ)

Figure 3 Laplacian plot of piperine shows highest peak at Carbon attached to Nitrogen indicating nucleophilic site; OH- attacks this C to cleave the C-N bond and produce piperic acid. 19 ACS Paragon Plus Environment


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Figure 4 Molecular Electrostatic Potentials of piperine: E(max)=0.1858 E(min)=-0.11246, Blue >0.1 Red

Quantum Chemical and Docking Insights into Bioavailability

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