(Pseudo-)Halogenating Activity of Lactoperoxidase - ACS Publications

Article pubs.acs.org/jnp

Tannins and Tannin-Related Derivatives Enhance the (Pseudo-)Halogenating Activity of Lactoperoxidase Jana Gau,*,† Martine Prévost,‡ Pierre Van Antwerpen,§ Menyhárt-Botond Sarosi,⊥ Steffen Rodewald,∥ Jürgen Arnhold,† and Jörg Flemmig† †

Institute for Medical Physics and Biophysics, Medical Faculty, University of Leipzig, Härtelstraße 16-18, 04107 Leipzig, Germany Laboratory of Structure and Function of Biological Membranes and §Laboratory of Pharmaceutical Organic Chemistry, Université Libre de Bruxelles, Boulevard de Triomphe, 1050 Brussels, Belgium ⊥ Institute of Inorganic Chemistry, Faculty of Chemistry and Mineralogy and ∥Institute of Pharmacy, Faculty of Biosciences, Pharmacy and Psychology, University of Leipzig, Johannissallee, 04103 Leipzig, Germany ‡

S Supporting Information *

ABSTRACT: Several hydrolyzable tannins, proanthocyanidins, tannin derivatives, and a tannin-rich plant extract of tormentil rhizome were tested for their potential to regenerate the (pseudo‑)halogenating activity, i.e., the oxidation of SCN− to hypothiocyanite −OSCN, of lactoperoxidase (LPO) after hydrogen peroxide-mediated enzyme inactivation. Measurements were performed using 5-thio-2nitrobenzoic acid in the presence of tannins and related substances in order to determine kinetic parameters and to trace the LPO-mediated −OSCN formation. The results were combined with docking studies and molecular orbital analysis. The −OSCN-regenerating effect of tannin derivatives relates well with their binding properties toward LPO as well as their occupied molecular orbitals. Especially simple compounds like ellagic acid or methyl gallate and the complex plant extract were found as potent enzyme-regenerating compounds. As the (pseudo‑)halogenating activity of LPO contributes to the maintenance of oral bacterial homeostasis, the results provide new insights into the antibacterial mode of action of tannins and related compounds. Furthermore, chemical properties of the tested compounds that are important for efficient enzyme−substrate interaction and regeneration of the −OSCN formation by LPO were identified.

T

(reaction 3, Scheme 1). Other reactions coupled with oneelectron substrate oxidation are the interconversion of Compound I* to Compound II (see reaction 6, Scheme 1) and Compound II to ferric enzyme (reaction 4, Scheme 1). Reactions 1, 3, and 4, or alternatively reactions 1, 5, 6, and 4, comprise the −OSCN-independent peroxidase cycle, with reaction 4 being the rate-limiting reaction. Furthermore, excess H2O2 can lead to the formation of Compound III (i.e., oxyperoxidase, P-Por-FeIII·O2•−/P-Por-FeII·O2) followed either by a slow conversion to the ferric enzyme accompanied by the release of superoxide (not shown in Scheme 1) or by the spontaneous return of LPO to its ferrous state accompanied by the release of dioxygen (not shown in Scheme 1).10 Compound III is also unable to oxidize SCN−. The −OSCN formation by LPO is known to be impaired under pathological conditions.11,12 Oxidative destruction or enzyme inactivation contributes to a diminished antimicrobial activity in the oral cavity associated with recurrent inflammatory diseases like gingivitis, stomatitis, or caries.7,13,14 Thus, the

he heme-containing protein lactoperoxidase (LPO) is released from secretory epithelial cells and contributes to innate immune responses in human body fluids like saliva, milk, or tears by catalyzing the oxidation of thiocyanate, SCN−, to antimicrobial hypothiocyanite, −OSCN.1−3 After entering bacterial biofilms, −OSCN reacts with free thiol groups inside the microbes and thus inhibits bacterial growth.4−6 The hydrogen peroxide (H2O2)-dependent activity of LPO further prevents mucous surfaces from oxidative damage and diminishes cytotoxic effects of H2O2, which is secreted, for example, by oral pathogens like Lactobacilli and Streptococci.5,7 The formation of −OSCN is initiated in the presence of H2O2 that oxidizes the ferric heme of LPO to Compound I, an oxoiron(IV) porphyryl radical species (P-+•Por-FeIV) (reaction 1, Scheme 1).2 Compound I is able to oxidize SCN− to −OSCN under recovery of the ferric enzyme (reaction 2, Scheme 1).8 Both reactions comprise the (pseudo-)halogenation cycle. Compound I can also be converted into Compound I* (+•P-PorFeIV−OH) by spontaneous transfer of the radical moiety to the apoprotein3 (reaction 5, Scheme 1) or, in the presence of suitable substrates, into Compound II, a nonradical oxoiron(IV) species (P-Por-FeIV-OH) ,9 by one-electron donation © 2017 American Chemical Society and American Society of Pharmacognosy

Received: October 7, 2016 Published: April 3, 2017 1328

DOI: 10.1021/acs.jnatprod.6b00915 J. Nat. Prod. 2017, 80, 1328−1338

Journal of Natural Products

Article

Scheme 1. Schematic Presentation of the Catalytic Cycle of LPOa

a In the presence of H2O2 the ferric enzyme is oxidized to Compound I (reaction 1), which is able to oxidize SCN− to −OSCN. Thereby Compound I is reduced to the ferric enzyme (reaction 2). Both reactions comprise the (pseudo‑)halogenating cycle (or −OSCN-active cycle). The native enzyme can also be regenerated from Compound I by two consecutive one-electron reactions (reaction 3) via formation of Compound II. In addition, a spontaneous transition of Compound I to Compound I* (reaction 4 and 5) can occur. Compound I* is also able to oxidize small substrates (reaction 6). Both Compound I* and Compound II are unable to oxidize SCN−. The reactions 1, 3, and 4 (or alternatively 1, 5, 6, and 4) comprise the −OSCN-independent peroxidase cycle. AH and A• indicate an oxidizable substrate and the substrate radical, respectively.

used in all experiments. Although the applied Cl− concentration of 140 mM does not reflect the oral situation,3 Cl− has no effect on the −OSCN formation rate of LPO, as this enzyme is not able to oxidize chloride.8 Hydrogen peroxide was used at a final concentration of 80 μM to simulate a pro-inflammatory situation. At this concentration a considerable decrease of the LPO-mediated −OSCN formation rate was found as compared to that found at 20 μM H2O2 (Figure 1). The described in vitro system was subsequently applied to test the effect of proanthocyanidins and hydrolyzable tannins on the (pseudo‑)halogenating LPO activity. Effect of Proanthocyanidins. Proanthocyanidins are flavan-3-ol oligomers,22 which were shown to be good promoters of the (pseudo‑)halogenating activity of LPO in a

regeneration of the (pseudo‑)halogenating activity of LPO is an important tool for the treatment and prophylaxis of oral inflammatory processes. Several studies described the peroxidase-mediated antimicrobial effect against several periodontal pathogens,15−17 thus emphasizing the relevance of a functional peroxidase system. In preliminary studies phenolic substances were systematically tested for their potential to regenerate the (pseudo‑)halogenating activity of LPO after H2O2-mediated Compound II accumulation.9,18−20 Thereby 3′,4′-dihydroxylated flavonoids, especially flavones, were identified as highly efficient compounds to enhance this activity under simulated (patho‑)physiological conditions in the oral cavity.19,20 In the present work we extended our studies to the chemical group of tannins and tannin-related substances as well as tannin-rich plant extracts. Tannins can be divided into proanthocyanidins and hydrolyzable tannins.21 While proanthocyanidins comprise dimers, trimers, or polymers of catechin or epicatechin and a variety of other flavan-3-ols,22 hydrolyzable tannins consist of gallic acid or gallic acid depsides esterified to glucose or other polyols.23 Complex tannins are built up of catechins, hydrolyzable tannins, and other minor phenols.24 Several monomeric components of tannins like epicatechin, catechin, or gallic acid have been identified as reactivating substances for the LPO.19,20 Therefore, in this study it was assessed whether tannins are also able to reactivate the (pseudo‑)halogenating activity of LPO. In addition, a pharmaceutically relevant, tannin-rich plant extract of tormentil rhizome that is used, e.g., for the local treatment of minor oral inflammations25 was tested and analyzed. The obtained kinetic data were used together with docking studies and molecular orbital analysis to examine the binding of tannins and tanninrelated derivatives in the substrate channel of LPO.

Figure 1. Dependence of the LPO-mediated −OSCN formation from the H2O2 concentration. Effect of an increasing H2O2 concentration was tested at 37 °C and pH 7.4 in the presence of 5 nM LPO and 2 mM SCN−. A concentration of 80 μM H2O2 led to a significant decrease of the relative −OSCN formation (−OSCN formation rate divided by the applied amount of H2O2). At H2O2 concentrations over 100 μM almost no (pseudo‑)halogenating LPO activity was detectable. Mean and standard deviation of n = 3 experiments are given. p < 0.05 (*), p < 0.01 (**), p < 0.001 (***).

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RESULTS AND DISCUSSION Setup of the Test System. In accordance with the physiological conditions in the buccal cavity, 2 mM SCN−4 was 1329



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Figure 2. Effect of proanthocyanidins on the LPO-mediated −OSCN-formation. (A) Specificity constants (kcat/KM) for the reaction of proanthocyanidin tannins with LPO. (B) Appropriate chemical structures. The highest specificity constant was found for procyanidin B 2, followed by procyanidin A 2. The lowest effect was determined for the trimer procyanidin C 1. Mean and standard deviation of n = 3 experiments are given. p < 0.05 (*), p < 0.01 (**), p < 0.001 (***).

former study.19 Three different types of epicatechin oligomers were tested (Figure 2B): the singly linked B-type procyanidin, procyanidin B 2 (PCB2); the doubly linked A-type procyanidin A 2 (PCA2); and procyanidin C 1 (PCC1). To quantify the regenerating effect of the condensed tannins on the −OSCN formation rate of LPO, the specificity constant (kcat/KM) was used (Figure 2A). As indicated by single kinetic parameters in Table 1, the differences for the three proanthocyanidins resulted from both varied KM and kcat values. PCB2 exhibited the highest effect on the regeneration of the LPO-mediated −OSCN production (kcat/KM = 12.1 ± 5.7 μM−1 s−1), followed by PCA2 (kcat/KM = 4.7 ± 0.0 μM−1 s−1) and PCC1 (kcat/KM = 0.8 ± 0.0 μM−1 s−1). It could be suggested that the number of linkages as well as the type of the interflavanyl bond, i.e., epicatechin-(4β→8)epicatechin for PCB2 and epicatechin-(4β→8,2β→O→7)epicatechin for PCA2, affect the binding to the enzyme (low KM value for PCB2). The additional 2β→O→7 bond in PCA2 may lead to a higher susceptibility toward oxidation and reactivity i.e., a higher kcat value, but apparently decreases the enzymatic binding properties. Indeed, the B-type dimer is a more compact molecule as compared to the A-type procyanidin that has a higher rigidity and extended conformation.26 For PCC1 the kcat value was comparable to that of PCB2, reflecting a similar orientation in the enzyme−substrate pocket. As the KM value of PCC1 is markedly higher than that observed for

Table 1. Determined Kinetic Parameters compound procyanidin B 2 procyanidin A 2 procyanidin C 1

KM, μM

kcat, s−1

Proanthocyanidins 0.37 ± 0.09 4.53 ± 1.07 12.66 ± 0.01 60.03 ± 0.02 8.41 ± 0.24 6.43 ± 0.19

Hydrolyzable Tannin-Related Derivatives punicalagins 10.55 ± 0.61 1,2,3,4,6− pentagalloylglucose epicatechin 0.61 ± 0.02 epigallocatechin 0.81 ± 0.01 chlorogenic acid 1.26 ± 0.00 epigallocatechin gallate 0.32 ± 0.01 ellagic acid 0.30 ± 0.01 methyl gallate 1.24 ± 0.00 1,3,6-trigalloylglucose 1.37 ± 0.04 gallic acid 176.78 ± 0.02

kcat/KM, μM−1 s−1 12.1 ± 5.7 4.74 ± 0.0 0.8 ± 0.0

and Basic Structural Elements 11.76 ± 0.68 1.1 ± 0.1 − − 41.40 43.57 60.59 13.92 29.34 80.32 63.74 30.90

± ± ± ± ± ± ± ±

1.52 0.48 0.16 0.42 0.55 0.06 2.10 0.00

68.4 53.7 48.1 43.8 98.3 64.7 50.3 0.2

± ± ± ± ± ± ± ±

5.0 1.2 0.3 2.7 3.7 0.1 3.1 0.0

PCB2, it can be assumed that the presence of an additional epicatechin moiety in PCC1 affects the binding properties inside the substrate channel because of the increased molecular size. The considerably higher kcat value of PCA2 compared to that of PCC1 indicates again that the presence of the 1330



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Figure 3. Effect of hydrolyzable tannins on the LPO-mediated −OSCN formation. Specificity constant (kcat/KM) for the reaction of different gallotannin-related compounds and basic structural elements with LPO are shown for (A) flavan-3-ol derivatives as well as chlorogenic acid and (B) further galloyl derivatives. (C,D) Chemical structures. The highest specificity constant was found for ellagic acid followed by methyl gallate. The specificity constants determined for epigallocatechin, trigalloylglucose, chlorogenic acid, and epigallocatechin gallate are in the same range. Compared to the other compounds, for gallic acid an extremely low specificity constant was computed. Mean and standard deviation of n = 3 experiments are given. p < 0.05 (*), p < 0.01 (**), p < 0.001 (***).

of small amounts of ellagic acid or side products.27 Accordingly, the docking calculations showed no binding of, e.g., punicalagins in the substrate channel of LPO. Therefore, in further experiments we concentrated on simple galloyl derivatives and constituent units of proanthocyanidin compounds. The structures of these compounds are illustrated in Figure 3C,D. In Figure 3A,B the activities of the compounds are shown, separated into flavan-3-ol derivatives (Figure 3A, including the depside, chlorogenic acid) and further galloyl derivatives (Figure 3B). The kcat/KM values of the former group are in the same range. However, differences were found for the kcat and KM constants. While for all tested flavan-3-ol-related compounds the KM values were smaller than 1 μM, chlorogenic acid exhibited a higher value (1.26 ± 0.0 μM). Regarding the reaction rate, the highest kcat value in this substance group was also found for chlorogenic acid (60.59 ± 0.16 s−1). Although epigallocatechin exhibited the lowest kcat value (13.92 ± 0.42

epicatechin-(2β→O→7)-epicatechin moiety causes a distinctively higher reactivity. The molecule, however, exhibited poor binding properties, probably due to steric hindrance. Effect of Hydrolyzable Tannins and Their Basic Structural Elements. Gallotannins are a quite complex group of natural products. Thus, only selected derivatives and structurally related compounds were tested for their effect to regenerate the (pseudo‑)halogenating activity of LPO. Large ellagi- and gallotannins like punicalagins or pentagalloylglucose (Figure S1, Supporting Information) exhibited either low kcat/ KM ratio, i.e., 1.11 ± 0.13 μM−1 s−1, for punicalagins or almost no effects for pentagalloylglucose regarding the recovery of the (pseudo‑)halogenating activity of LPO. Although the experimental conditions in this study should not contribute to the appreciable cleavage of the ester bond in hydrolyzable tannins,23 the minor effect of the punicalagins may be explained by some degradation processes that could lead to the formation 1331



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Figure 4. Best-scored docked poses of several galloyl derivatives toward the catalytic site of LPO (PDB code 3UBA). The docked ligands, the heme group, and the interacting amino acids are indicated as green, gray, and blue sticks, respectively. The central iron cation is shown as a blue sphere. Amino acids that are involved in the ligand interactions (namely hydrogen bonds, black dashed lines; π−π interactions, blue dashed lines; cation−π interactions, orange dashed lines) are indicated. Images were generated using PyMol, version 0.99rc6.

s−1), it also showed the lowest KM value within this group (0.32 ± 0.01 μM), which led to a specificity constant comparable to the other compounds. In the group of galloyl derivatives, the highest specificity constant was found for ellagic acid (98.3 ± 3.7 μM−1 s−1), followed by methyl gallate (64.7 ± 0.1 μM−1 s−1), trigalloylglucose (50.3 ± 3.1 μM−1 s−1), and gallic acid (0.2 ± 0.0 μM−1 s−1). Compared to ellagic acid, a 2.6- or 2.1-fold higher kcat value was determined for methyl gallate and trigalloylglucose, respectively. Thus, the higher specificity constant for ellagic acid can be attributed to the 4−5 times lower KM value compared to the former ones. Surprisingly, kcat = 30.90 ± 0.00 s−1 was found for gallic acid, which is comparable to that of the other tannin-related derivatives. The activity of this compound results mainly from the low enzyme affinity (KM = 176.78 ± 0.02 μM), most probably caused by the hydrophilic anion (pKa = 4.44 for the carboxylic group).28 Although the kcat/KM values of the other tested gallotannin-related substances are almost in the same range, different binding and oxidizing properties were found by means of computational studies. Computational Studies. Docking calculations predicted that all examined galloyl and flavan-3-ol derivatives form hydrogen bonds with at least one component of the LPO catalytic triad, His109, Arg255, and Gln10529 (Figures 4 and 5). Moreover, stacking of one aromatic moiety of these compounds onto the heme pyrrole D-ring as well as other π−π and/or cation−π interactions were also observed. In contrast, PCB2 exhibits no stacking with the pyrrole moiety and, instead, hydrogen bonds are formed with amino acids located farther from the heme pocket in the LPO channel, i.e., Ser121, Lys126, Lys232, and His426 (Figure 6).30 This could be rationalized by the higher rigidity of this molecule compared to other docked

galloyl derivatives. These results could explain the lower ability of PCB2 to promote the −OSCN regeneration as compared to the hydrolyzable compounds. With epigallocatechin gallate and 1,3,6-trigalloylglucose distinctive interactions were also found with amino acids of the access channel, i.e., Glu116, Lys126, Glu118, Phe254, Glu258, and Arg44030 (Figures 4 and 5) potentially favoring the binding as shown by the free energy of binding value for 1,3,6-trigalloylglucose (ΔG = −12.4 kcal/ mol) and to a lesser extent for epigallocatechin gallate (ΔG = −8.9 kcal/mol). In summary, the predicted values of the binding free energy (ΔG, given in Table 2) show a score ranking as follows: 1,3,6-trigalloylglucose (ΔG = −12.4 kcal/ mol) < epigallocatechin gallate (ΔG = −8.9 kcal/mol) ≈ procyanidin B 2 (ΔG = −8.5 kcal/mol) < epigallocatechin (ΔG = −7.9 kcal/mol) < epicatechin (ΔG = −7.5 kcal/mol) < ellagic acid (ΔG = −6.6 kcal/mol) ≈ gallic acid (ΔG = −6.5 kcal/mol) ≈ methyl gallate (ΔG = −6.1 kcal/mol). The correlation of the predicted ΔG values with the measured KM is limited as the KM values are directly linked with the −OSCN formation rate. Therefore, optimal binding behaviors inside the substrate channel resulting in low ΔG values31 are not necessarily reflected by the KM values as long as the substrate orientation is not ideal for oxidation. Moreover not only thermodynamic aspects regarding the binding of the substrates to LPO have to be taken into account. Especially regarding their oxidizability the electron distribution in the substrates has also to be considered. For this reason the molecular orbitals of the substrates were calculated additionally in order to estimate the position with the highest electron-donating (oxidizing) ability of a molecule. As shown in Figure 7, in most molecules C 4 or parapositioned hydroxyl groups showed the highest participation to 1332



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Figure 5. Best-scored docked poses of several proanthocyanidin flavan-3-ol constituent units toward the catalytic site of LPO (PDB code 3UBA). The ligands used for docking calculations, the heme group as well as interacting amino acids are highlighted by green, gray and blue sticks, respectively. The central heme iron cation is characterized with a blue sphere. The amino acid interactions are marked as black dashed lines (hydrogen bonds), dashed lines (π−π interactions), and orange dashed lines (cation−π interactions). The images were generated using PyMol, version 0.99rc6.

Table 2. Free Binding Energy (ΔG) of Predicted Docked Poses Performed in LPO compound

ΔG, kcal/mol

procyanidin B 2 epicatechin epigallocatechin epigallocatechin gallate ellagic acid methyl gallate 1,3,6-trigalloylglucose gallic acid

−8.5 −7.5 −7.9 −8.9 −6.6 −6.1 −12.4 −6.5

was found in the docked poses. The OH moiety, however, points toward the heme center. The calculated HOMO energies are given in Table 3. The higher the EHOMO, the stronger are the electron-donating properties.32 For epicatechin, methyl gallate, epigallocatechin, and procyanidin B 2, the hydroxy group that has the highest oxidizing capacity indeed interacts with or points toward the active center of LPO. For ellagic acid, the OH group at C 4/C 4′ is predicted to interact with the LPO active center. However, according to a computed study, the most acidic phenolic groups are 3-OH and 3′-OH, with pKa = 5.73 and 5.75.33 The least acidic hydrogen atom belongs to the 4-OH and 4′-OH groups, with pKa = 7.05 and 7.12.33 Furthermore, at the experimental

Figure 6. Best-scored docked pose of procyanidin B 2 toward the catalytic site of LPO (PDB code 3UBA). Docked procyanidin B 2, the heme group and the interacting amino acids are shown as green, gray, and blue sticks, respectively. A blue sphere represents the central heme iron cation. Amino acid interactions are shown as dashed lines (black, hydrogen bonds; blue, π−π interactions; orange, cation−π interactions). The images were generated using PyMol, version 0.99rc6.

the HOMO. Remarkably the previous docking results predicted that this OH-group forms hydrogen bonds with His109, an essential part of the catalytic triad of LPO29 for all molecules except for PCB2. In the latter no hydrogen bond with His109 1333



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Figure 7. Calculated molecular orbitals (visualized in blue and yellow) for selected tannin-related substances and basic structural elements. The hydroxy group with the highest HOMO contribution is marked with black-line circles in the corresponding chemical structure. In addition the hydroxy moieties that were found to interact with the active center with focus on His109 as part of the catalytic triad by docking studies are highlighted with gray-line circles. In the case of procyanidin B 2 the gray circle corresponds to the hydroxy group that is located close to the active center.

in its deprotonated form under the experimental conditions of pH 7.4, the HOMO of this compound is, in contrast to the other tested substances, entirely located on the carboxylate moiety. In the case of epigallocatechin gallate and 1,3,6trigalloylglucose the hydroxy group with the highest HOMO contribution did not correspond to the hydroxy group interacting with the active center in the docked poses. Therefore, for gallic acid, 1,3,6-trigalloylglucose, and epigallocatechin gallate the HOMO energies were also calculated for

and physiological pH 7.4 the mono-deprotonated ellagic acid anion dominates.34 The deprotonated hydroxy moiety in position C 3 or C 3′ contributes largely to the HOMO of the ellagic acid anion. However, a large contribution comes from the 4-OH-group as well. Since the docking simulations for the non-ionized ellagic acid predicted a stabilizing interaction between Glu116 and the C3/C3′- as well as the C4/C4′hydroxy groups, it can be assumed that the phenoxide anion would point toward the iron cationic center of LPO. As gallic acid (pKa = 4.44 for the carboxylic group28) also mainly exists 1334



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and qualify single extract components. Therefore, an HPLC fingerprint (Figure 8A) was recorded. By comparing the retention times with a reference, ellagic acid was identified as a main component of the extract (red marked peak in Figure 8A). As shown in the present work, this compound is an efficient regenerator of the (pseudo‑)halogenating activity of LPO. In Figure 8B the corresponding absorbance spectra are shown indicating two absorbance maxima at 255 and 360 nm. The tannin-related compounds in the ethanol extract of tormentil rhizome could be separated by applying the mobile phase composition that is used to separate (poly-)phenolic compounds.24 As shown in Figure 9, the whole tested plant extract exhibited a pronounced −OSCN regenerating effect on LPO.

Table 3. Frontier Molecular Orbital Energies for the HOMO Distribution of Selected Tannin-Related Compounds compound

EHOMO, eV

procyanidin B 2 epicatechin epigallocatechin epigallocatechin gallate ellagic acid methyl gallate 1,3,6-trigalloylglucose gallic acid

−8.10 −8.42 −8.31 −8.53a −2.42b −8.60 −8.33c −2.66d

a

Additionally calculated HOMO energies for the structures that show differences between the hydroxy groups with the highest HOMO contribution and their predicted interaction with the active center of LPO: EHOMO‑4 = −9.48 eV. bEHOMO‑1 = −5.05 eV. cEHOMO‑3 = −9.34 eV. dEHOMO‑3 = −4.53 eV.

the structural parts predicted to be close to the heme center by docking studies and given as EHOMO‑3 and EHOMO‑4 in Table 3. As 1,3,6-trigalloylglucose and epigallocatechin gallate are bulky structures of similar size, steric conditions could impede the positioning of a substrate for optimal oxidation. The LPO regenerating potential of tannin-related substances is limited by their size, which could be observed for the more bulky compounds punicalagins and pentagalloylglucose. Effect and Characterization of an Ethanol Extract of Tormentil Rhizome. The ethanol extract of tormentil rhizome was tested for its effect on the −OSCN-production by LPO. In 2013, The European Scientific Cooperative on Phytotherapy (ESCOP) published the monograph for Tormentillae rhizoma and proposed its application for the treatment of minor oral infections.25,35 As the ESCOP monographs are based on the evidence and leading expertise about the therapeutic use of herbal products, the tormentil extract was chosen to provide a link between the single tested compounds, a pharmaceutically relevant application, and the pseudohalogenating activity of LPO in the buccal cavity. By applying the hide powder method the tannin content of this extract was determined to be 11.90%, which meets the requirements of the European Pharmacopoeia (Ph. Eur.) of at least 7%.36 As the investigation mainly focuses on the enzymatic activity of LPO and the options and mechanisms to regenerate its −OSCN formation with respect to naturally occurring (poly-)phenolic compounds it was beyond the scope of this study to elucidate

Figure 9. Effect of a tormentil rhizome ethanol extract on the LPOderived −OSCN rate. The concentration-dependent increase of the − OSCN formation rate was fitted to a sigmoidal equation (dashed line) to calculate the EC50 value. Initially in the presence of lower extract concentrations the LPO-derived −OSCN formation significantly increased. After reaching a maximum at 50 μg/mL the effect strongly dropped to the initial value. Mean and standard deviation of n = 3 experiments are given. p < 0.05 (*), p < 0.01 (**), p < 0.001 (***).

The half maximal effective concentration was reached at a quite low concentration of 3.33 ± 0.04 μg/mL leading to a 4fold increase of the −OSCN formation rate. The maximum effect was reached at an extract concentration of 50 μg/mL. At higher concentrations the effect strongly decreases to the initial value that was measured for the −OSCN formation rate in the absence of the plant extract, which could be explained by the accumulation of Compound II (Scheme 1). In former studies

Figure 8. Chromatographic analysis of the ethanol extract of tormentil rhizome. (A) HPLC fingerprint chromatogram of the extract. The marked peak was identified as ellagic acid by comparing the retention time with the reference compound. (B) The corresponding absorbance spectra the marked peak in (A) (continuous line) and the reference ellagic acid (dashed line), indicating two absorbance maxima at 255 and 360 nm. 1335



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concentration of the H2O2 stock solution was determined at 230 and 240 nm (ε230 = 74 M−1 cm−1, ε240 = 43.6 M−1 cm−1).50 H2O2 and DTNB (≥98%) for TNB preparation18 were purchased from SigmaAldrich, Taufkirchen, Germany. Reversed-phase (RP)-HPLC measurements were performed using a C18 column (EC Nucleodur 100-5 C18 ec 250 × 4 mm i.d; 5 μM, Macherey-Nagel, Düren, Germany). The analytes were separated by a Dionex UltiMate 3000 HPLC system (Dionex, California, USA), which consisted of an UltiMate 3000 RS pump and an UltiMate 3000 RS column compartment combined with a Waters 717 plus autosampler (Waters, Eschborn, Germany) and a Waters 996 photodiode array detector operated at 250, 280, 320, and 480 nm. The elution was performed in gradient mode with a mobile phase A consisting of MeOH, H2O, and formic acid (5:95:0.1 v/v/v) and a mobile phase B consisting of MeOH and formic acid (100:0.1 v/v).24 The gradients between the time points were applied as follows: 10 min, 10% B; 15 min, 100% B; 10 min, 100% B; 5 min, 10% B; 5 min, 5% B. The flow rate and the column temperature were set to 1 mL/ min and 40 °C, respectively Chemicals. Chlorogenic acid (≥95%), ellagic acid (≥95%), epigallocatechin (≥90%), epigallocatechin gallate (≥95%), FolinCiocalteu reagent, gallic acid (≥95%), hide powder from bovine, methyl gallate (≥98%), pyrogallol (≥98%), 1,3,6-tri-O-galloyl-β-Dglucose (≥95%), and 1,2,3,4,6-penta-O-galloyl-β-D-glucose (≥96%) were obtained from Sigma-Aldrich, Taufkirchen, Germany. Procyanidin A 2 (≥95%), procyanidin B 2 (≥95%), procyanidin C 1 (≥95%), and punicalagins (≥95%) were purchased from PhytoLab GmbH & Co. KG, Vestenbergsgreuth, Germany. Plant Material. The dried and cut tormentil rhizome (Tormentilla erecta, according to the Ph. Eur) was obtained from Alfred Galke GmbH, Gittelde, Germany (batch number: 26738). Determination of Kinetic Parameters. For all tested single polyphenolic compounds a 5 or 10 mM stock solution was prepared in DMSO. The final substrate concentrations were in the range of 0.01− 500 μM. Control measurements showed no significant effect of DMSO up to 5% on the (pseudo‑)halogenating activity of LPO (data not shown). The tested plant extracts were dissolved in PBS to 5 mg/ mL. The final concentrations ranged between 0.1 and 1000 μg/mL. All kinetic measurements were performed in PBS (pH 7.4) at 37 °C using a multiplate reader (Tecan Infinite 200 Pro, Männerdorf, Switzerland) as described before.19 Based on the TNB-degradation in the presence of −OSCN, kinetic parameters (Michaelis−Menten constant (KM), maximal rate (Vmax), turnover number (kcat), and specificity constant (KM/kcat) were calculated via Lineweaver−Burk plots.19 For the tested plant extract the half-maximal effective concentration (EC50 value) was calculated by using exponential curve fitting. All measurements were performed at least in triplicates. For all kinetic parameters (KM, Vmax, and EC50) the indicated standard deviations correspond to the coefficient of determination for the linear curve fit after Lineweaver−Burk calculation or the corresponding sigmoidal curve fit. 19 Indicated asterisks correspond to the corresponding level of significance (p < 0.05 (*), p < 0.01 (**), p < 0.001 (***)), as determined by Student’s t test. Extraction and Extract Analysis. The crushed tormentil rhizome was extracted with EtOH (70% v/v) at room temperature (24 h, 4×). The extract was evaporated to dryness (the drug extract ratio was calculated to be 9.35:1) and stored at −20 °C. The total tannin content of the extract was quantified with the hide-powder method, which is based on the adsorption of tannins.24 Briefly, the dried extracts were dissolved in EtOH (20% v/v) and divided into two samples. One part of the sample was treated with hide powder. In both samples the polyphenol content was determined by the reduction of phosphomolybdate and phosphotungstate (Folin-Ciocalteu reagent) to blue colored polytungstates51 that were quantified at 760 nm.52 The tannin dependent polyphenol was calculated according to the Ph. Eur.53 For RP-HPLC measurements a stock solution (5 mg/mL) of the dried extract was prepared in MetOH (70%, v/v). The reference compounds gallic acid, chlorogenic acid, ellagic acid, procyanidin A 2, methyl gallate, trigalloylglucose, epigallocatechin, and epigallocatechin

the ability of (poly-)phenolic compounds to provoke the formation of Compound II was shown using stopped-flow measurements.19,20 On the other hand, the protein precipitating effect of higher molecular weight tannins has to be taken into account. Although precipitation is mainly obtained at a pHvalue near the isoelectric point37 (in case of LPO 9.2−9.938) higher concentrations of gallotannins could be able to interact with the protein surface or form complexes between the protein molecules39 that may influence the (pseudo‑)halogenating activity of LPO. Physiological Relevance. In summary, this study indicates a relationship between the −OSCN-regenerating effect of tannins and tannin-related compounds and their binding properties in the substrate channel of LPO as well as with their electron delocalization. These results show that the estimation of potent enzyme regenerators requires the consideration of substrate-specific, e.g., electron-donating ability and molecular size, and enzyme-specific features of the substrate channel properties. Thus, especially small gallotanninrelated structures or basic structural elements efficiently regenerate the LPO-mediated −OSCN production. These results may provide a further explanation for the phyto-pharmaceutical application of tannins and related compounds, which are known for their antibacterial, antiinflammatory, as well as plaque inhibiting effects in the mouth.40−43 The abirritant and antiphlogistic effects of tannins are mainly related to their astringent properties that lead to the formation of coagulant membranes after interaction with proteins located in the upper mucus layers.44 Several studies on the antibacterial effect of tannin derivatives against pathogens like Staphylococcus aureus, Helicobacter pylori, or Escherichia coli have been published.45−47 Comparison of the minimum inhibitory concentration value (MIC) with the kinetic parameters for the LPO reactivation, calculated in this study, shows that much lower compound concentrations are needed to regenerate the (pseudo‑)halogenating activity of LPO. This enzyme could be considered as an additional target for this substance class, which can be activated in the presence of low substance concentrations. The (pseudo‑)halogenating activity of LPO is known to be an essential part for the maintenance of oral microbiological homeostasis. The disturbance of this system is associated with several oral diseases like caries or gingivitis. This justifies the local application of (poly-)phenolic compounds as potent −OSCN-activating substances to support oral health. Additionally, tannin-rich phyto-pharmaceuticals like tormentil rhizome are traditionally used as mouth rinses or teas and are proposed for the treatment of minor oral mucosa inflammation according to the ESCOP monograph,35,48 underlining the pharmaceutical importance of tannins and tannin-rich plant extracts.

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EXPERIMENTAL SECTION

General Experimental Procedures. Lactoperoxidase from bovine milk was obtained from Sigma-Aldrich, Taufkirchen, Germany, as a lyophilized powder (≥200 U/mg; about 80% purity according to the A412/A280 ratio). Stock aliquots of the enzyme (5 μM) were prepared in phosphate buffered saline (PBS, 10 mM, pH 7.4) and stored at −25 °C. The final enzyme concentration for kinetic measurements was 5 nM. The LPO-mediated −OSCN formation was followed by the generation of Ellman’s reagent [5,5′-dithiobis(2nitrobenzoic acid), DTNB] from 5-thio-2-nitrobenzoic acid (TNB) at 412 nm.49 TNB and SCN− were used in final concentrations of 50 μM and 2 mM, respectively. The H2O2 working solution was freshly prepared each day and added at a final concentration of 80 μM. The 1336



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gallate were dissolved in MetOH (70%, v/v) as well to a final concentration of 1 mg/mL. For system control and data collection the program Dionex Chromeleon (Version 6.80) was applied. Docking Calculations. For docking studies of the selected phenolic derivatives gallic acid, methyl gallate, ellagic acid, trigalloylglucose, and procyanidin B 2 the X-ray structures of bovine lactoperoxidase complexed to hydroxycinnamic acid (PDB ID: 3UBA) was used. Preparation of the protein and of ligands as well as the docking procedures were performed as described before.19 Molecular Orbital Calculations. For computation of the electron-donating abilities of selected compounds, starting structures were downloaded from the ChemSpider web service of the Royal Society of Chemistry.54 The molecular volume was calculated with the online tool for calculation of molecular properties and bioactive score offered by Molinspiration.55 All geometry optimizations for the electronic structures were carried out with the Grimme’s 3-corrected Hartree−Fock method (HF-3c).56,57 The HF-3c computations were performed with the quantum chemistry program package ORCA.58 The images of the optimized structures and the highest occupied molecular orbitals (HOMOs) were rendered with the UCSF Chimera program package.59

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(9) Flemmig, J.; Rusch, D.; Czerwinska, M. E.; Rauwald, H. W.; Arnhold, J. Arch. Biochem. Biophys. 2014, 549, 17−25. (10) Jantschko, W.; Furtmuller, P. G.; Zederbauer, M.; Lanz, M.; Jakopitsch, C.; Obinger, C. Biochem. Biophys. Res. Commun. 2003, 312, 292−298. (11) Lorentzen, D.; Durairaj, L.; Pezzulo, A. A.; Nakano, Y.; Launspach, J.; Stoltz, D. A.; Zamba, G.; McCray, P. B., Jr.; Zabner, J.; Welsh, M. J.; Nauseef, W. M.; Banfi, B. Free Radical Biol. Med. 2011, 50, 1144−1150. (12) Tenovuo, J.; Pruitt, K. M. J. Oral Pathol. Med. 1984, 13, 573− 584. (13) Van Steenberghe, D.; Van den Eynde, E.; Jacobs, R.; Quirynen, M. Int. Dent. J. 1994, 44, 133−138. (14) Lagerlof, F.; Oliveby, A. Adv. Dent. Res. 1994, 8, 229−238. (15) Tenovuo, J. Oral Dis. 2002, 8, 23−29. (16) Fabian, T. K.; Hermann, P.; Beck, A.; Fejerdy, P.; Fabian, G. Int. J. Mol. Sci. 2012, 13, 4295−4320. (17) Tenovuo, J.; Lumikari, M.; Soukka, T. Proc. Finn. Dent. Soc. 1991, 87, 197−208. (18) Flemmig, J.; Noetzel, I.; Arnhold, J.; Rauwald, H. W. J. Funct. Foods 2015, 17, 328−339. (19) Gau, J.; Furtmuller, P. G.; Obinger, C.; Prevost, M.; Van Antwerpen, P.; Arnhold, J.; Flemmig, J. Free Radical Biol. Med. 2016, 97, 307−319. (20) Gau, J.; Furtmuller, P. G.; Obinger, C.; Arnhold, J.; Flemmig, J. Biochem. Biophys. Rep. 2015, 4, 257−267. (21) Khanbabaee, K.; Van Ree, T. Nat. Prod. Rep. 2001, 18, 641−649. (22) Ferreira, D.; Slade, D.; Marais, J. P. J. Flavans and Proanthocyanidins. In Flavonoids: Chemistry, Biochemistry and Applications; Andersen, Ø. M., Markham, K. R.,, Eds.; CRC Press, Taylor & Francis Group: Boca Raton, FL, 2006; pp 553−616. (23) Hagerman, A. E. The Tannin Handbook; Department of Chemistry and Biochemistry: Miami, FL, 2002. (24) Moller, C.; Hansen, S. H.; Cornett, C. Phytochem. Anal. 2009, 20, 231−239. (25) ESCOP Scientific Committee. Phytomedicine 2013, 20, 469. (26) Verstraeten, S. V.; Hammerstone, J. F.; Keen, C. L.; Fraga, C. G.; Oteiza, P. I. J. Agric. Food Chem. 2005, 53, 5041−5048. (27) Larrosa, M.; Tomas-Barberan, F. A.; Espin, J. C. J. Nutr. Biochem. 2006, 17, 611−625. (28) Strobel, B. W. Geoderma 2001, 99, 169−198. (29) Sharma, S.; Singh, A. K.; Kaushik, S.; Sinha, M.; Singh, R. P.; Sharma, P.; Sirohi, H.; Kaur, P.; Singh, T. P. Int. J. Biochem. Mol. Biol. 2013, 4, 108−128. (30) Flemmig, J.; Gau, J.; Schlorke, D.; Arnhold, J. Expert Opin. Ther. Targets 2016, 20, 447−461. (31) Van Antwerpen, P.; Prevost, M.; Zouaoui-Boudjeltia, K.; Babar, S.; Legssyer, I.; Moreau, P.; Moguilevsky, N.; Vanhaeverbeek, M.; Ducobu, J.; Neve, J.; Dufrasne, F. Bioorg. Med. Chem. 2008, 16, 1702− 1720. (32) Saqib, M.; Mahmood, A.; Akram, R.; Khalid, B.; Afzal, S.; Kamal, G. M. J. Pharm. Appl. Chem. 2015, 1, 65−71. (33) Markovic, Z.; Milenkovic, D.; Đorovic, J.; Dimitric Markovic, J. M.; Lucic, B.; Amic, D. Monatsh. Chem. 2013, 144, 803−812. (34) Galano, A.; Francisco Marquez, M.; Perez-Gonzalez, A. Chem. Res. Toxicol. 2014, 27, 904−918. (35) Krenn, L. Phytomedicine 2013, 20, 469. (36) Einzelmonographien zu Pflanzlichen Drogen und Zubereitungen. In Europäisches Arzneibuch (Amtliche deutsche Ausgabe); Deutscher Apothekerverlag: Stuttgart, Germany, 2011; p 1917. (37) Adamczyk, B.; Salminen, J. P.; Smolander, A.; Kitunen, V. Int. J. Food Sci. Technol. 2012, 47, 875−878. (38) Fee, C. J.; Chand, A. Sep. Purif. Technol. 2006, 48, 143−149. (39) Hagerman, A. E.; Rice, M. E.; Ritchard, N. T. J. Agric. Food Chem. 1998, 46, 2590−2595. (40) Langmead, L.; Dawson, C.; Hawkins, C.; Banna, N.; Loo, S.; Rampton, D. S. Aliment. Pharmacol. Ther. 2002, 16, 197−205. (41) Tomczyk, M.; Latté, K. P. J. Ethnopharmacol. 2009, 122, 184− 204.

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jnatprod.6b00915. Figure S1, showing the chemical structures of the complex tannin punicalagins and the hydrolyzable tannin pentagalloyl-glucose (PDF)

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Tel: +49 341 9715773. Fax: +49 341 9715709. ORCID

Jana Gau: 0000-0002-5000-7919 Menyhárt-Botond Sarosi: 0000-0003-4222-0717 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS J.G. was supported by a state stipend of the free state of Saxony (grant LAU-R-N-03-2-0714) provided from the Saxon Ministry ̂ of Science and Fine Arts (SMWK). M.P. is “Maitre de Recherche” at the Fonds National de la Recherche Scientif ique (FRS-FNRS, Belgium).

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REFERENCES

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(Pseudo-)Halogenating Activity of Lactoperoxidase - ACS Publications

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