Clovamide and Phenolics from Cocoa Beans (Theobroma Cacao L.) Inhibit Lipid Peroxidation in Liposomal Systems

Food Research International 50 (2013) 129–134

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Food Research International

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Clovamide and phenolics from cocoa beans (Theobroma cacao L.) inhibit lipid peroxidation in liposomal systems

Monica Locatelli ⁎, Fabiano Travaglia, Lorella Giovannelli, Jean Daniel Coïsson, Matteo Bordiga, Franco Pattarino, Marco Arlorio

Dipartimento di Scienze del Farmaco, Università degli Studi del Piemonte Orientale “A. Avogadro”, 28100, Novara, Italy article info abstract

Article history: Neo-synthesized clovamide and a phenolic cocoa extract (fermented cocoa from Ghana) were evaluated for Received 8 August 2012 their capacity to inhibit lipid peroxidation. Their effect was first investigated on phospholipid organic solu- Accepted 10 October 2012 tions, and then on liposomal systems prepared by high pressure homogenization process. Antioxidants Available online 17 October 2012 were added to liposomal system following two different protocols (before and after the homogenization treatment) and their protective action was evaluated monitoring the oxidative status of liposomes (exposed Keywords: to light at room temperature or heated at 40 °C) over three weeks. The results confirmed a significant protec- Clovamide Theobroma cacao tive effect of clovamide on liposomal model systems and, in a minor extent, also of cocoa extract. The capacity Liposomes of phosphatidylcholine liposomes to incorporate clovamide was also evaluated; it was shown that more than Lipid peroxidation 50% of clovamide was englobed in liposomes, although the addition of clovamide solution before the homog- Antioxidant activity enization process led to the isomerization of the molecule from trans to cis form. © 2012 Elsevier Ltd. All rights reserved.

1. Introduction (ranging from 1.36 up to 2.64 mg/kg in unroasted fermented beans, dry weight), and is drastically reduced after roasting process (Arlorio et al., Naturally occurring polyphenolic compounds have been widely 2008). Also cocoa hulls, obtained as by-product after pre-roasting of studied for their beneficial effects on human health (Fraga et al., 2010; whole beans, are rich in phenolic antioxidants and contain interesting Hervert-Hernández & Goñi, 2011; Link, Balaguer, & Goel, 2010). Poly- amounts of clovamide (in the range 1.817±0.059 mg/kg, dry weight) phenols are the main antioxidant-active fraction of cocoa and particu- (Arlorio et al., 2008). larly catechin, epicatechin and procyanidins have been suggested as Such as other N-phenylpropenoyl amino acids naturally occurring in potent bioactive compounds (Heo & Lee, 2005). However, other minor cocoa, clovamide contribute to the astringent taste of unfermented cocoa phenolic cocoa constituents, such as clovamide, should be considered beans as well as roasted cocoa nibs. The sensation induced by these and more investigated for their beneficial properties. amides in the oral cavity was described as mouth-drying and puckering Clovamide is a polyphenolic amide, belonging to the class of astringent (Stark & Hofmann, 2005). Clovamide is structurally related to N-phenylpropenoyl amino acids. It was identified for the first time by some β-adrenergic ligands (dobutamine, denopamine), and clovamide Yoshihara, Yoshikawa, Sakamura, and Sakuma (1974) in the red clover derivatives N-coumaroyldopamine and N-caffeoyldopamine (also found (from which the name “clovamide” was derived) and only later was in cocoa) have been shown to be potent β2-adrenoceptor agonists described in cocoa. Sanbongi et al. (1998) identified for the first time (Park, 2005) and able to suppress platelet–leukocyte interactions via clovamide and its derivative deoxyclovamide in cocoa liquor, which is inhibiting P-selectin expression (Park & Schoene, 2006). the liquid (or paste) produced during chocolate manufacturing, after Clovamide represents an interesting compound for its remarkable cocoa beans are roasted and finely ground. More recently clovamide antioxidant activity (Arlorio et al., 2008; Ley & Bertram, 2003; Sanbongi was identified also in cocoa beans (both fermented unroasted cocoa et al., 1998); it was shown that clovamide has antiradical properties sim- beans and roasted nibs) and hulls. It was shown that the clovamide con- ilar to rosmarinic acid and caffeic acid, resulting more effective than other tent in cocoa beans varies depending on the geographic origin of cocoa well known antioxidant such as epicatechin, trolox, kaempferol and butylated hydroxyanisole (BHA) (Locatelli et al., 2009). Clovamide also Abbreviations: HPH, high pressure homogenization; PL90G, Phospholipon® 90G; showed a remarkable binding affinity for the p56lck SH2 domain, so FAMEs, fatty acid methyl esters; TGA, thermogravimetric analysis; PCS, Photon Corre- highlighting interesting properties for the treatment of a broad range of lation Spectroscopy; ZAve, average diameter; Poly Index, polydispersity index; EE, en- human diseases such as cancer, autoimmune disease, osteoporosis, and capsulation efficiency; PV, peroxide value; OI, oxidation index; DTG, derivative curves chronic inflammatory disease (Park et al., 2007). More recently, of thermogravimetric plots. fl ⁎ Corresponding author. Tel.: +39 0321 375774; fax: +39 0321 375751. neuroprotective effects (Fallarini et al., 2009)andanti-inammatory E-mail address: [email protected] (M. Locatelli). properties (Zeng et al., 2011) were also demonstrated. All these findings

0963-9969/$ – see front matter © 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.foodres.2012.10.008 130 M. Locatelli et al. / Food Research International 50 (2013) 129–134 make clovamide an interesting bioactive compound for pharmaceutical which the most abundant was linoleic acid (C18:2n−6, 62.5%). Other and food research, potentially useful as nutraceutical ingredient. principal fatty acids identified were palmitic acid (C16:0, 14.5%), oleic Liposomes are spherical, self-closed structures formed by one or acid (C18:1n−9, 12.6%), linolenic acid (C18:3n−6, 5.1%) and stearic several concentric lipid bilayers surrounding an aqueous core. They acid (C18:0, 3.7%). can entrap hydrophilic molecules in their internal water compartment and hydrophobic molecules into the membrane (Bangham, Standish, 2.4. Thermal analysis (TGA) & Watkins, 1965; Torchilin, 2005). For their characteristics, liposomes are widely used in pharmaceutical applications (drug delivery, medical The potential protective action of the antioxidants against the diagnostic, gene therapy), in cosmetics and agro-food industry (Lasic, phospholipid degradation was evaluated by thermogravimetric anal- 1995), and can also be used in biomedical field as membrane model ysis (Pyris1 TGA, Perkin Elmer, Italy). For this purpose, methanolic for the study of lipid peroxidation (Chatterjee & Agarwal, 1988). solutions with two different concentrations of clovamide or cocoa phe- The aim of the present work was to evaluate the capacity of nolic extract were mixed with PL90G dissolved in methanol. The final clovamide and of cocoa phenolic extract to inhibit lipid peroxidation: concentrations of the samples were 50 and 500 μg/mL for clovamide; the effect of these antioxidants was investigated on phospholipid 50 and 500 μg/mL for cocoa extract; 15 mg/mL for phospholipids. organic solutions and on liposomal systems prepared by high pres- Each system was submitted to thermal analysis in standard platinum sure homogenization process. The vesicle size and the encapsulation pans from 30 to 600 °C (scan rate 20 °C/min, under 20 mL/min nitrogen efficiency of clovamide in liposomes were also determined. atmosphere). 2. Materials and methods 2.5. Sample preparation 2.1. Clovamide and cocoa extract Liposome dispersions (control) were prepared by treatment of PLG90 Clovamide was obtained by chemical synthesis, as previously in bidistilled water (10 mg/mL) with a high-performance disperser described by Arlorio et al. (2008).Briefly, L-3,4-dihydroxyphenylalanine (Ultra-Turrax®, T 25 basic Ika®-Werke GmbH, Staufen, Germany) at was protected as methyl ester and condensed with caffeic acid to obtain 16,000 rpm for 3 min, followed by high pressure homogenization crude clovamide methyl ester. After hydrolyzing the methyl ester (HPH) — 15,000 psi, single pass — using a M-110L Microfluidizer® (treatment with LiOH), the residue was purified on silica gel column Processor (Microfluidics, Newton, MA, USA). Ethanolic solutions of to obtain clovamide. clovamide and cocoa extract (at the concentration of 2 mg/mL) were Cocoa polyphenolic extract was obtained from fermented beans added to liposomes before (PRE) and after (POST) the homogenization from Ghana. Cocoa beans were finely ground and extracted in auto- treatment (final concentration of clovamide or cocoa extract: 40 ppm), matic Soxhlet (Büchi Labortechnik AG, Flawil, Switzerland) for 12 h, in order to assess the impact of different preparation protocols. As con- using dichloromethane to remove the lipid fraction. Then, the pheno- trol, liposomes were also prepared without antioxidant (EtOH PRE and lic fraction was extracted from dry defatted powder in the same EtOH POST samples). Soxhlet apparatus, using methanol as the solvent (4 h). The solvent was finally evaporated to dryness (vacuum, 40 °C), and the dry extract was stored at −20 °C until use. The complete characterization 2.6. Characterization of liposomes of the cocoa phenol extract is reported in Arlorio et al. (2009). Size and size distribution of liposomes were evaluated by Photon 2.2. Chemicals Correlation Spectroscopy (PCS): the average diameter (ZAve) and polydispersity index (Poly Index) of the vesicles were determined in Phospholipon® 90G (PL90G) was obtained from AVG s.r.l. (Milan, KCl (0.03% w/v) by a Zetasizer 3000 HS (Malvern Instruments, UK). Italy). Methanol, acetonitrile (all HPLC grade) and formic acid (50%, Measurements were made in triplicate. LC–MS grade), were purchased from Sigma-Aldrich (Milan, Italy). Water was obtained by Milli-Q instrument (Millipore S.p.A., Milan, 2.7. Encapsulation efficiency of clovamide in liposomes Italy). All the other chemicals and reagents were of analytical grade and purchased from Sigma-Aldrich (Milan, Italy). Liposomal vesicles were isolated from the aqueous medium by ultrafiltration. Five hundred microlitres of the liposomal suspensions 2.3. Characterization of phospholipidic matrix were transferred in the sample reservoir of 10 kDa Microcon® centrifu- gal filter devices (Millipore S.p.A., Milan, Italy) and centrifuged at Phospholipidic matrix was characterized by GC-analysis of fatty acid 14,000 rpm (17 °C) in a 1754 R Centrifuge (Eppendorf, Hamburg, methyl esters (FAMEs), obtained by phospholipid transesterification. Germany), until the complete separation of liposomes from the aqueous Briefly, 200 mg of PL90G was solved in 500 μL of methanol and sodium media (2 h for the “clovamide POST” sample and 3 h for the “clovamide methylate 0.5 M (500 μL) was added. The reaction was carried out into PRE”). Liposomal fractions were then weighted and dissolved in 1 mL of a closed vial at 80 °C under stirring for 20 min at 350 rpm (Thermomixer ethanol, prior to the chromatographic analyses. Both liposomal and comfort, Eppendorf, Milan, Italy). The content of each vial was then sup- aqueous fractions were analyzed by HPLC to determine the clovamide plied with 250 μL of distilled water and 500 μL of diethyl ether, and concentration. FAMEs were gently extracted by inversion of the vial. Ten microlitres of The encapsulation efficiency (Fang, Hwang, Huang, & Fang, 2006) the organic phase was diluted with 1 mL of CH Cl . 2 2 was calculated as follows: FAMEs were analyzed using a gas chromatograph Shimadzu GC-17A (Shimadzu Italia, Milan, Italy), according to the method previously described by Locatelli et al. (2011). The identification of free EE% ¼ ½ðÞT−C =T 100; fatty acid methyl esters was obtained by comparison with retention times of commercial standards (Supelco 37 Component FAME Mix, Sigma-Aldrich); the quantification was performed on the basis of where T is the total amount of clovamide (i.e. the sum of clovamide the sum of peak areas, and expressed as internal relative percentage. detected in the liposomal and aqueous fractions) and C is the amount It was shown a prevalence of unsaturated fatty acids (80.2%), among of clovamide detected only in the aqueous fraction. M. Locatelli et al. / Food Research International 50 (2013) 129–134 131

2.8. HPLC–DAD–ESI–MS/MS analysis 100 Temperature (ºC) 280 300 320 340 360 380 90 0 The chromatographic separation was performed on a Phenomenex 80 -0,2 Luna C18 (2) (150×2 mm i.d., with particle size of 5 μm, Phenomenex, -0,4 70 Torrance, CA, USA) maintained at 25 °C. The mobile phase consisted of -0,6 water–formic acid 0.1% (eluent A) and acetonitrile–formic acid 0.1% 60 -0,8 -1 (eluent B) using the following program gradient: from 6% to 16.5% B 50 -1,2 (14 min), from 16.5% to 17% B (2 min), from 17% to 17.5% B (2 min), 40 -1,4 isocratic 17.5% B (2 min), from 17.5% to 100% B (10 min), isocratic Derivative Weight % (%/m) -1,6 100% B (5 min), from 100% to 6% B (1 min), isocratic 6% B (5 min). Weight % (%) 30 Total run time was 41 min, at a constant flow-rate of 400 μL/min. Chro- 20 PL90G matograms were recorded at 280, 320 and 365 nm. The eluate was 10 PL-Clovamide 500 injected into the electrospray ion source with a splitting of 40%; the con- PL-Cocoa 500 0 ditions employed for the MS analyses were the same as reported in 30 130 230 330 430 530 Arlorio et al. (2008). MS and MS/MS spectra were acquired and Temperature (ºC) interpreted using the software Xcalibur®. The injection volume was 10 μL. For the quantification, a stock solution of clovamide (2 mg/mL Fig. 1. Thermogravimetric profiles of PL90G (- - -) and PL90G systems containing ethanol) was prepared; this solution was then opportunely diluted in 500 μg/mL of clovamide (−−−) or cocoa extract (――). The corresponding deriva- water or ethanol, in order to quantify clovamide in aqueous and liposo- tive plots (DTG) are reported in the box. mal fractions, respectively. Six different concentration levels were used for each calibration curve. temperatures of the second step shifted to higher values, suggesting a considerable interaction between antioxidant and PL90G. 2.9. Antioxidant activity (inhibition of lipid peroxidation) As expected, differences observed in the onset temperatures between samples with and without antioxidants (ΔT) were higher Protective action of clovamide and cocoa extract against liposome with the highest concentration of antioxidant (Table 1). oxidation was evaluated during: i) storage at room temperature In particular, at the concentration of 500 μg/mL cocoa displayed a (25 °C) and ii) storage in oven at 40 °C (accelerated oxidation). The more evident effect than clovamide against phospholipid decomposition: oxidation degree of liposomal systems was monitored over three this could be explained by taking into account that clovamide is only a weeks, measuring both the peroxide value and the oxidation index. minor component of the cocoa phenolic fraction (Arlorio et al., 2009), The peroxide value (PV) was determined by iodometric titration and the cocoa effect could be principally due to other phenolic com- and expressed as milliequivalents of oxygen per kilogram of sample pounds (i.e., catechins). (International Union of Pure and Chemistry, 1992). The oxidation index (OI) was determined using the method previously described 3.2. Size characterization of liposomes in Gordon and Roedig-Penman (1998), with some modifications. Briefly, 15 μL of liposome suspension was diluted with absolute etha- Liposomes were characterized for their dimensions (ZAve) and nol to a final volume of 1500 μL, then absorbance at 233 and 215 nm polydispersity degree (Poly Index) (Table 2). Compared to control, the was measured. The oxidation index was calculated as the ratio: addition of ethanol before and after the homogenization did not influence the dimension of liposomes (EtOH PRE and EtOH POST samples). fi OI ¼ Abs =Abs : Moreover, the size of lipid vesicles did not signi cantly change also 233 nm 215 nm when clovamide (p=0.663) or cocoa extract (p=0.328) ethanolic solutions were added to the liposomal dispersions after the HPH pro- The inhibition of lipid peroxidation was calculated as the percent- cess (Clovamide POST and Cocoa POST). On the contrary, the addition age with respect to the liposome control. of the antioxidants before the homogenization has generated lipid vesicles of higher dimensions, particularly in the case of clovamide 2.10. Statistical analysis (pb0.001), assuming that an encapsulation of these molecules probably occurred. Results were expressed as mean±standard deviation of three dif- Regarding the polydispersity index, the results showed a homoge- ferent determinations. Statistical significance was determined by analy- neous degree of homogeneity, having observed no significant differ- sis of variance and t test (paired t test in the case of comparison between ences among the samples. The Poly Index values obtained for “PRE” and “POST” samples), using the free statistical software R 2.8.1 Clovamide PRE and Clovamide POST sample were 0.506 and 0.590, version. Differences were considered significant at pb0.05. respectively.

3. Results 3.3. Inhibition of lipid peroxidation in liposomal system

3.1. Thermogravimetric analysis The antioxidant activity of clovamide and cocoa extract was studied as inhibition of lipid peroxidation, monitoring the formation of Thermogravimetric analysis gave some interesting information about the interactions between antioxidants and phospholipids. The Table 1 thermal patterns of phospholipids alone and of the phospholipid sys- Values of ΔT between systems with and without clovamide or cocoa extract. μ tems containing antioxidants (500 g/mL) are depicted in Fig. 1. Methanolic solutions ΔT (°C) The weight loss of phospholipid methanolic solutions occurred in two fi PL90G 0.0 stages: the rst step corresponds to methanol evaporation, the second PL90G+clovamide 50.0 μg/mL 7.2±0.5 c one, in the range 280–380 °C, to the decomposition of lipids. The presence PL90G+clovamide 500.0 μg/mL 9.3±0.7 b of clovamide or cocoa extract influenced the PL90G degradation behavior. PL90G+cocoa 50.0 μg/mL 6.0±0.7 c The analysis of first derivative curves of thermogravimetric plots (DTG) PL90G+cocoa 500.0 μg/mL 18.1±0.8 a indicates that when clovamide or cocoa extract was present, the Mean±SD followed by the same letter are not significantly different (p>0.05). 132 M. Locatelli et al. / Food Research International 50 (2013) 129–134

Table 2 Average diameter (ZAve: nm) and polydispersity index (Poly Index) of liposomes. A

Liposomal systems ZAve (nm) Poly Index

Control 121.9±4.3 c 0.534±0.006 EtOH PRE 126.9±5.0 b,c 0.607±0.077 EtOH POST 121.1±4.8 c 0.505±0.070 Clovamide PRE 211.7±9.4 a 0.506±0.090 Clovamide POST 123.2±1.6 c 0.590±0.014 Cocoa extract PRE 143.6±9.3 b 0.562±0.084 Cocoa extract POST 125.9±4.5 b,c 0.530±0.076

Mean±SD followed by the same letter are not significantly different (p>0.05). No significant differences were observed for the Poly Index values. Oxidation Index peroxides and conjugated dienes (expressed as OI) in liposomal model-systems, during three weeks of storage. Peroxide values obtained after 7, 14 and 21 days of storage in two different conditions (accelerated oxidation at 40 °C and storage at 0.0 0.5 1.0 1.5 2.0 room temperature under light exposure) are reported in Table 3. 0 5 10 15 20 PVs were also measured on PL90G and liposomal dispersions imme- Time (days) diately after their preparation, obtaining values equal to zero. With respect to the light exposure at room temperature, the thermal treat- B ment at 40 °C improved the liposome oxidation, showing PVs about three times higher. Peroxide values obtained for the control sample increased from 15.9 after 7 days of oxidation at 40 °C to 20.4 and 26.0 after 14 and 21 days, respectively. Clovamide and cocoa extract significantly protect liposomes from oxidation (pb0.001); this was only marginally due to the presence of the solvent, since a highly significant protection (pb0.001) was observed also with respect to the samples containing ethanol. The addition of the antioxidant molecules before (PRE) or after (POST) the homogenization process was not influent (paired t test, p>0.05). As expected, liposomes stored Oxidation Index at room temperature were less oxidized than liposomes maintained at 40 °C, showing after 21 days a maximum PV of 8.2. Clovamide and cocoa extract explicated antioxidant properties; however, comparing the results of “PRE” and “POST” experiments, it was observed

that the addition of antioxidant compounds before the homogeniza- 0.0 0.5 1.0 1.5 2.0 tion process increased their protective action toward lipid oxidation 0 5 10 15 b (paired t test, p 0.01). In both these experiments, clovamide showed Time (days) higher antioxidant properties than cocoa extracts, inhibiting lipid peroxidation up to 92.4% and 83.1% in samples stored at 40 °C and Fig. 2. Oxidation degree of liposomal systems measured as oxidation index (OI). Panel A: ex- 25 °C, respectively. periments performed at room temperature, 25 °C, panel B: experiments performed at 40 °C. … … The oxidation index was determined between 1 and 22 days of stor- Legend: Control ( ● );EtOHPRE( ..• ..); EtOH POST ( —♦—); Cocoa PRE (−−■−−); . ● . —▲— . ♦ . age in the conditions previously described (Fig. 2). Considering the Cocoa POST ( ); Clovamide PRE ( ); Clovamide POST ( ). accelerated oxidation (40 °C), OI values obtained for both control and liposomes containing ethanol decreased during the last days of monitoring, probably because of the formation of secondary oxidation prod- containing clovamide reached the maximum values of 0.43 and 0.48 ucts, such as aldehydes, ketones and alcohols, not detectable by this (Clovamide PRE samples) at 40 °C and room temperature, respectively; method; so, in Fig. 2B only values obtained until day 17 are reported. considering the polyphenol cocoa extract, the maximum values were As previously obtained for PVs, the major oxidation degree was 0.99 and 1.23, respectively. The antioxidant activity was mainly observed for liposomes thermally treated, showing values up to 1.73 observed in samples stored at 40 °C. Clovamide revealed major antioxi- for both control and EtOH POST samples (day 17). Considering lipo- dant properties than cocoa, inhibiting lipid peroxidation up to 96.1% somes maintained at room temperature, the maximum OI value was (40 °C, day 8). The maximum inhibition percentage registered for obtained for control (1.06) after 22 days of storage. The OI of liposomes cocoa samples was 78.4 and 73.4%, obtained for Cocoa POST sample

Table 3

Peroxide values (meq O2/kg) of liposomal systems after 7, 14 and 21 days of storage at room temperature (RT, 25 °C) and 40 °C (accelerated oxidation).

Liposomal systems 7 days 14 days 21 days

RT 40 °C RT 40 °C RT 40 °C

Control 4.9±0.4 a 15.9±0.3 a 6.9±0.2 a,b 20.4±0.9 b 8.2±0.2 a 26.0±1.0 a EtOH PRE 4.3±0.4 a 15.4±0.9 a 6.5±0.1 b 22.3±0.5 a 7.6±0.1 a 20.5±0.9 c EtOH POST 4.8±0.2 a 13.7±0.4 b 7.4±0.1 a 18.5±1.1 c 7.8±0.4 a 23.3±0.6 b Clovamide PRE 0.8±0.0 c 1.8±0.1 d 1.6±0.1 f 2.1±0.2 e 1.7±0.1 e 2.0±0.2 f Clovamide POST 1.0±0.1 c 2.3±0.4 d 2.3±0.3 e 3.1±0.1 e 2.7±0.2 d 3.3±0.4 f Cocoa extract PRE 2.1±0.1 b 5.5±0.1 c 3.7±0.1 d 9.5±0.5 d 5.1±0.5 c 11.2±1.0 d Cocoa extract POST 2.7±0.4 b 5.3±0.4 c 4.7±0.2 c 8.6±0.7 d 6.6±0.2 b 7.7±0.3 e

Mean±SD followed by the same letter, within a column, are not significantly different (p>0.05). M. Locatelli et al. / Food Research International 50 (2013) 129–134 133 after 1 and 2 days of storage (40 °C), respectively. The protective effect of liposomes were prepared using the high pressure homogenization cocoa extract drastically decreased during storage; this effect was partic- process, a useful technique to obtain very small and homogeneous ularly evident in liposomes maintained at room temperature, for which liposomes (Brandl, Bachmann, Drechsler, & Bauer, 1993), and apply- the inhibition of lipid oxidation was reduced to 11.8 and 18.6% in cocoa ing gentle operating conditions (single pass, approximately few “POST” and “PRE” samples, respectively. On average, the addition of microseconds long) in order to minimize any degradation of the clovamide to liposomes before or after the homogenization process phospholipid matrix. Clovamide and cocoa polyphenols (phenolic was not influent for the antioxidant activity; on the contrary, Cocoa extract from fermented unroasted cocoa from Ghana) were added POST sample was more effective than Cocoa PRE in experiments to liposomal dispersions before or after the homogenization process, performed at 40 °C, and vice versa for experiments at room temperature. in order to assess the impact of different preparation protocols; then, the oxidative status of liposomes stored in two different condi- 3.4. Clovamide quantification in liposomes and determination of tions (exposed to light at room temperature or heated at 40 °C) was encapsulation efficiency monitored over three weeks. The oxidation degree of the liposomal dispersions was evaluated using the PV and the OI. The peroxide First, the effect of the HPH process on the recovery of clovamide was value is generally employed to describe the oxidative status of lipid determined. A standard solution of clovamide (40 ppm) obtained as pre- samples, particularly measuring the primary products of the oxida- viously described for liposome preparation, but omitting phospholipidic tion (hydroperoxides). Different to other methods (e.g. determina- matrix, was prepared; then, one aliquot was directly analyzed by HPLC tion of p-anisidine value), the PV does not measure the secondary for the quantification and another was previously subjected to the products of oxidation (e.g. aldehydes generated during the decompo- homogenization process. Clovamide was quantified as 40.11± sition of hydroperoxides). Nevertheless, considering that the aim of 0.44 μg/mL in the first aliquot (not homogenized solution), and only this work was not to describe the progressive oxidation of the liposo- 17.72±0.25 μg/mL in the solution passed through the microfluidizer mal systems, and that over three weeks the peroxide value of the processor, obtaining a 56% reduction of the clovamide concentration. In liposomes “control” continuously increased (indicating that the fact, the chromatographic profile of the homogenized solution showed a decomposition of hydroperoxides has not yet occurred), the PV was new peak (tr=12.54 min), missing in the profile of not-homogenized considered a useful and satisfactory parameter to estimate the capac- solution, besides that of clovamide (tr=15.01 min). The new peak ity of clovamide and cocoa extract to inhibit liposome oxidation. In showed the same molecular weight and the same MS/MS fragmentation addition to the PV, we decided to evaluate the oxidation index, pattern as clovamide (prevalent daughter ions: m/z 222, 178, 161), because is a simple and commonly used procedure to determine the suggesting the isomerization of the molecule from trans to cis form, dur- oxidation degree of phosphatidylcholine liposomes (Gordon & ing the homogenization process. Roedig-Penman, 1998). The OI is expressed as the ratio Abs233nm/ The clovamide quantification was then performed in both liposo- Abs215 nm, where Abs233nm reflects the absorption of conjugated mal and aqueous fractions, and then encapsulation efficiency was cal- dienes and the latter wavelength is chosen since there is little contri- culated. The total clovamide content (T) was 41.3±0.8 μg/mL and bution of the fatty acid carbonyl to the absorbance at this wavelength, 15.1±0.9 μg/mL in samples “POST” and “PRE”, respectively. The EE% allowing Beer's Law to be followed (Klein, 1970). On the basis of both calculated was 56.14 and 58.18% for Clovamide POST and Clovamide PV and OI results, clovamide revealed a strong antioxidant activity in PRE, respectively. all the conditions studied, particularly in the case of accelerated oxidation. The addition of clovamide before or after the homogenization 4. Discussion treatment did not significantly influence the inhibition of liposome oxidation. Also the polyphenolic cocoa extract showed interesting Clovamide and, more generally, cocoa polyphenols were widely lipid peroxidation inhibiting properties, but in a minor extent with studied for their antioxidant properties. The chemical structures of fla- respect to clovamide. The higher antioxidant activity values were vonols and procyanidins are important for their antioxidant activity as observed for the accelerate oxidation conditions; as observed for they possess both free radical trapping and chelation of redox-active clovamide, the antioxidant activity was more effective when severe metal properties (Verstraeten, Oteiza, & Fraga, 2004). Moreover, cocoa oxidation conditions were employed (heating). Considering the oligomeric procyanidins were found to prevent lipid oxidation, experiments performed at room temperature, the cocoa extract signifi- interacting with membrane phospholipids (Verstraeten, Hammerston, cantly protected liposomes, showing a maximum activity for the Cocoa Keen, Fraga, & Oteiza, 2005) and enhancing the resistance of LDL from PRE sample (with respect to the control the peroxide formation was in vitro oxidation (Osakabe et al., 2002). Also clovamide and some of reduced for the 56.7%). The different activity observed for clovamide its lipophilic derivatives were studied to test their antioxidative poten- and cocoa extract could be related to the phenolic composition of tials against bulk lipid oxidation (Ley & Bertram, 2003). All these cocoa; in fact, it is known that cocoa beans are particularly rich in cate- findings have great importance, for the numerous biological and nutri- chins (ca. 37%) and proanthocyanidins (ca. 58%) (Wollgast & Anklam, tional implication of lipid oxidative damage in both food preservation 2000). Considering the phenolic composition of the extract investigat- (deterioration of flavor and aroma of food, decay of nutritional and safe- ed, previously characterized and reported in Arlorio et al. (2009),the ty qualities) and human health (cellular damage related to carcinogen- main compound identified was epicatechin (1156±22 mg/kg of esis, premature aging and other degenerative diseases) (Kanner & cocoa, dry weight), while clovamide was only a minor component Rosenthal, 1992). (2.63±0.19 mg/kg, dry weight). Moreover, other methanol-soluble In this work, the study of the interaction of clovamide or cocoa compounds like minor flavonoids and pigments could be co-extracted, extract with phospholipids was investigated by thermogravimetric influencing the antioxidant activity of cocoa extract. In fact, different analysis. Antioxidants changed the PL90G decomposition behavior: constituents of cocoa could variously interact, and clovamide present in particular, the phospholipid degradation temperatures increased, in cocoa extract could contribute to its global antioxidant activity only suggesting a protective activity of clovamide and cocoa extract during in a lesser degree than other compounds. TGA. The modification of the thermal behavior of PL90G was evident In the second part of the present work we evaluated the behavior mainly in the presence of cocoa extract. and properties of liposomes. The results showed that multilamellar PL90G was also used as phospholipidic substrate to prepare lipo- vesicles of 120–210 nm diameter were obtained. The size distribution somes. In fact, liposomes are commonly studied as carriers for drugs was relatively wide (Poly Index 0.505–0.607, Table 2): this may be or bioactive compounds within the human body, as well as a biolog- due to the selection of a single pass HPH treatment. The Poly Index ical model to evaluate in vitro lipid peroxidation. In this work values of all the vesicular systems were comparable and the single 134 M. Locatelli et al. / Food Research International 50 (2013) 129–134 pass homogenization, even if carried out under high pressure (with a Arlorio, M., Locatelli, M., Travaglia, F., Coïsson, J. D., Del Grosso, E., Minassi, A., et al. (2008). Roasting impact on the contents of clovamide (N-caffeoyl-L-DOPA) and relevant energy contribution), did not determine the degradation of the antioxidant activity of cocoa beans (Theobroma cacao L.). Food Chemistry, 106, phospholipids that would occur in more strong HPH treatment condi- 967–975. tions (multiple pass). Moreover, the addition of cocoa extract or Bangham, A. D., Standish, M. M., & Watkins, J. C. (1965). Diffusion of univalent ions across the lamellae of swollen phospholipids. Journal of Molecular Biology, 13, clovamide to the lipid dispersion before the HPH treatment, led to 238–252. an increase of vesicle size. As suggested by Gibis, Vogt, and Weiss Brandl, M., Bachmann, D., Drechsler, M., & Bauer, K. H. (1993). Liposome preparation (2012), this is a first indication that antioxidant molecules interacted using high-pressure homogenizers. In G. Gregoriadis (Ed.), Liposomes Technology, – with phospholipid bilayer; they can partially be incorporated into the vol. 1. (pp. 49 65)Boca Raton, FL: CRC Press Inc. Chatterjee, S. N., & Agarwal, S. (1988). Liposomes as membrane model for study of lipid vesicles or be absorbed onto their surface. Finally, we determined the peroxidation. Free Radical Biology & Medicine, 4(1), 51–72. capacity of phosphatidylcholine liposomes to incorporate clovamide Fallarini, S., Miglio, G., Paoletti, T., Minassi, A., Amoruso, A., Bardelli, C., et al. (2009). fi Clovamide and rosmarinic acid induce neuroprotective effects in in vitro models (encapsulation ef ciency), also evaluating if the homogenization pro- – fi of neuronal death. British Journal of Pharmacology, 157, 1072 1084. cess can affect the quanti cation of clovamide (in terms of recovery); Fang, J. Y., Hwang, T. L., Huang, Y. L., & Fang, C. L. (2006). Enhancement of the transdermal in fact, clovamide added to phospholipid matrix before the liposomes delivery of catechins by liposomes incorporating anionic surfactants and ethanol. preparation is subjected to very high pressure (15,000 psi), and it International Journal of Pharmaceutics, 310,131–138. fi Fraga, C. G., Litterio, M. C., Prince, P. 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