Malvidin 3-GlucosideâFatty Acid Conjugates - American Chemical

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MALVIDIN-3-GLUCOSIDE-FATTY ACID CONJUGATES: FROM HYDROPHILIC TOWARDS NOVEL LIPOPHILIC DERIVATIVES Luis Miguel Cruz, Marta Guimarães, Paula Araujo, Ana Évora, Victor De Freitas, and Nuno Mateus J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.6b05461 • Publication Date (Web): 09 Feb 2017 Downloaded from http://pubs.acs.org on February 9, 2017

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Journal of Agricultural and Food Chemistry

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Malvidin 3-glucoside-Fatty Acid Conjugates: From Hydrophilic Towards Novel

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Lipophilic Derivatives.

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Luís Cruz*, Marta Guimarães, Paula Araújo, Ana Évora, Victor de Freitas, Nuno

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Mateus

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REQUIMTE/LAQV, Departamento de Química e Bioquímica, Faculdade de Ciências,

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Universidade do Porto, Rua do Campo Alegre, 687, 4169-007 Porto, Portugal.

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*Corresponding author (Tel.: +351 220402558; fax: +351 220402658; Email:

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[email protected])

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1 ACS Paragon Plus Environment


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ABSTRACT

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This work describes the lipophilization reactions of malvidin 3-glucoside with different

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saturated fatty acid chain lengths using an enzymatic synthesis and the study of their

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physical-chemical and antioxidant properties. The lipophilic anthocyanins were

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obtained with satisfactory yields (22-40%) after column chromatography purifications

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and they revealed the same appealing chromatic features of the parent anthocyanin. All

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the compounds were characterized by mass spectrometry confirming the regioselective

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acylation on the glucose moiety. The octanol-water partition coefficients and the

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hydrophobicity index of the different derivatives were determined confirming a

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lipophilicity increase concomitant with the fatty acid chain length. The antioxidant

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profile was also evaluated by two in vitro methods (β-Carotene-lineolate method and

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oxygen consumption assay). Overall, a maximum of antioxidant activity was achieved

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when malvidin 3-glucoside was conjugated with caprylic acid (C8). Altogether, the

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results obtained provides a good perspective for the technological application of these

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functionalized anthocyanins in cosmetic and food industries.

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Keywords: malvidin 3-glucoside; fatty acids; enzymatic catalysts; lipophilicity;

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antioxidant features.

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INTRODUCTION

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It is well known that polyphenolic compounds are secondary metabolites of plants and

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they are extensively present in many fruits, vegetables, and in some beverages such as

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tea, berry juices and red wine. Usually, they are associated with organoleptic properties

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of foods and with health-promoting effects because of their extensively reported

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antioxidant and biological properties.1-7 For these reasons, research on the application of

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polyphenols has attracted attention over recent years from the food, cosmetic and

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pharmaceutical industries.

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The main class of polyphenols are the flavonoids which have a 2-phenylbenzopyran

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type structure. Depending on the unsaturation degree of the C ring, flavonoids could be

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divided into several families such as flavan-3-ols, flavonols and anthocyanins.

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However, there are some drawbacks that have limited their industrial applications such

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as high reactivity, the chemical equilibrium due to pH variation (in the case of

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anthocyanins), temperature, light sensitivity and low solubility in lipophilic media. To

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overcome these drawbacks/issues, chemical and enzymatic lipophilization of native

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flavonoids have been widely described in the last decade as a tool for overcoming these

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limitations and consequently to expand their technological applications in food,

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therapeutic and cosmetic products.8

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The main functionalizations reported are alkylations and acylations of carbons and

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hydroxyl groups of flavonoids with hydrophobic molecules such as the fatty acids.9-11 It

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has been reported that the lipophilization of flavonoids may not only enhance their

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physical-chemical properties in a lipophilic environment but could also increase several

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biological effects of native molecules namely antioxidant,12 antifungal,13 antibacterial,14

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anticancer15 and anti-inflammatory16 properties as well as their bioavailability.17,18

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Conversely to other flavonoids, the chemistry involved in anthocyanins is much more



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difficult to handle such as the organic synthesis of anthocyanins and their

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metabolites.19,20 The investigation in the esterification and other derivatization reactions

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of anthocyanin molecules is practically unknown and therefore becomes a more

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challenging task. Anthocyanins are naturally occurring polyphenolic compounds

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widespread in the diet that have been attracted the scientific community due both to

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their health promoting properties and appealing colors (from orange to blue). Recently,

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there have been a few works dealing with the lipophilization of anthocyanin extracts by

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chemical/enzymatic approaches, however, the structural and physical-chemical

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characterization are missing.21,22 Very recently, the synthesis, structural characterization

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and antioxidant properties of malvidin 3-glucoside-fatty acid derivatives have been

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reported.23,24 It is well known that in drug delivery and absorption it is important to

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determine some physical properties of the molecules such as water solubility and

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hydrophilic/lipophilic balance. These two parameters provide for the capacity of the

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molecules to cross biological membranes and to reach a cell target.

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In order to improve the chemical stability, the solubility in lipophilic media and

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bioavailability, the synthesis of various malvidin 3-glucoside-fatty acid conjugates was

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performed using a biocatalyst. The novel molecules were characterized by mass

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spectrometry and their lipophilic parameters (octanol-water partition coefficients, Log P

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and hydrophobicity index, RM) and their antioxidant profile were determined. The

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overall information obtained will allow us to establish a structure-property/activity

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relationship.

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MATERIALS AND METHODS

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Reagents


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Silica gel RP-18 (40-63 µm) LiChroprep was provided from Merck (Darmstadt,

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Germany). Fatty acids, lipase acrylic resin from Candida antarctica lipase B (≥ 5000

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U/g, recombinant, expressed in Aspergillus niger), molecular sieves 4Å, 2-methyl-2-

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butanol, β-Carotene, linoleic acid and L-α-Phosphatidylcholine from soybean

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(lyophilized powder) were obtained from Sigma-Aldrich (Madrid, Spain). Malvidin 3-

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glucoside (Mv3glc) extract was obtained from red wine isolation through the method

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described previously.23

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Synthesis and isolation of Mv3glc-fatty acids derivatives

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To a flask it was subsequently added 10 mg of Mv3glc red wine extract [corresponding

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to 6.5 µmol of pure Mv3glc 1 (34%) (Figure 1)], activated 4Å molecular sieves (100

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g/L) and dry 2-methyl-2-butanol (5 mL). To this solution, the respective fatty acid (from

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C4 to C16) was added (100 eq.). The reactions started with the addition of the enzyme

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(Candida antarctica lipase B, CALB) (20 g/L). The reactions were stirred at 60 ºC over

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a 24 h period and their progression was monitored by HPLC-DAD.23 Each reaction was

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performed in triplicate.

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At the maximum product formation, the reaction was filtered to remove the molecular

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sieves and CALB and the solvent was evaporated under vacuum. The residue was

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redissolved in acidified methanol (with 2% HCl solution) and the excess of fatty acid

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and other impurities were extracted with heptane. Methanol fraction was concentrated,

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diluted in 20% of aqueous acidified MeOH and purified by chromatography on a

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column loaded with reversed-phase C18 silica gel (150 mm × 16 mm i.d.). The starting

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material was recovered with 30% of aqueous MeOH and the desired Mv3glc-fatty acid

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derivatives were isolated from 70-90% of aqueous MeOH. MeOH was removed by

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evaporation and the pigments were lyophilized and stored at -18 ºC until use. 5 ACS Paragon Plus Environment


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HPLC

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The enzymatic reactions were monitored by a Hitachi L-7100 HPLC (Merck) equipped

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with a diode array detector (Merck-Hitachi L-7450A) and a reversed-phase column (150

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mm × 2.1 mm i.d., 5 µm, Vydac 208TP C8, Grace Davison Discovery Sciences)

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thermostatted at 25 ºC. The detection was carried out at 520 nm. The solvents were (A)

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water/formic acid (9:1, v/v) and (B) acetonitrile/formic acid (9:1, v/v), with the

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following gradient: 0-20% B over 5 min, 20-100% over 10 min and isocratic 100% B

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for 15 min at a flow rate of 0.4 mL/min. The sample injection volume was 20 µL. The

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chromatographic column was washed with 100% B during 10 min and then stabilized

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with the initial conditions during another 10 min.

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LC-DAD/ESI-MS

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The samples were analysed on a Finnigan Surveyor series liquid chromatograph

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equipped with a LCQ DECA XP MAX (Finnigan Corp., San Jose, Calif.) mass detector,

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an atmospheric pressure ionization (API) source using an electrospray ionization (ESI)

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interface and a reversed-phase column (150 mm × 2.1 mm, i.d., 5 µm, Vydac 208TP

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C8, Grace Davison Discovery Sciences) thermostatted at 25 ºC using the same solvents,

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gradients, injection volume and flow rate referred to above for HPLC analyses. Double-

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online detection was done by a photodiode spectrophotometer and mass spectrometry.

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The vaporizer and the capillary voltages were 5 kV and 4 V, respectively. The capillary

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temperature was set at 325 ºC. Nitrogen was used both as sheath and auxiliary gas at

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flow rates of 90 and 25, respectively (in arbitrary units). Spectra were recorded in

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positive and negative ion modes from m/z 250-1500. The mass spectrometer was

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programmed to do a series of three scans: a full mass (MS), a zoom scan of the most


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intense ion in the first scan (MS2), and a MS-MS of the most intense ion using relative

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collision energy of 30 and 60 (MS3).

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Lipophilicity characterization

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The lipophilicity of Mv3glc esters 2-8 was evaluated by determining the following

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parameters: partition coefficients (Log P) and hydrophobicity index (RM).

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Theoretical Log P values were achieved using Molinspiration software, which is based

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on fragmental methods.25 Experimental Log P values were determined as octanol-water

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biphasic mixture by the shake-flask method.26,27 This method requires that the solvents,

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acidified water (2% HCl) and n-octanol, need to be previously mutually saturated for

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more than 24 h before use. Briefly, the test compounds were dissolved in presaturated

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n-octanol (3 mL) in a vial and the same volume of acidified water saturated with n-

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octanol was added (final concentration in the vial of 0.12 mM). Then, the flask was

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vigorously stirred for 1 h and kept for 24 h for separation to reach the equilibrium.

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Sampling of both phases was performed by careful use of syringes. The syringe used to

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collect the water-rich phase was filled with air, which was expelled slowly while

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passing through the octanol phase. After that, both phases were injected and analyzed

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by HPLC-DAD and the areas of the compounds were taken at 280 nm. The octanol-

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water partition coefficient (Log P) was calculated using the following equation:

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Log P = Log Aoctanol /Awater

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The hydrophobicity index (RM) values were determined by reversed-phase thin layer

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chromatography (RP-TLC) based on the method described in literature28 with a slight

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modification29. The RM values were calculated by the formula:

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RM = Log (1/RF − 1)



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where RF is the retention time from RP-TLC. For RP-TLC analysis, samples were

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placed on Kieselgel 60 RP-18 F254 plates and eluted with acetonitrile/acetic acid/water

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(1:40:8, v/v/v). Mv3glc and their derivatives were detected under UV light at 254 nm.

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Lipid peroxidation assays

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β-Carotene Bleaching Method

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The effect of Mv3glc-fatty acid conjugates on inhibition of linoleic acid peroxidation

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was performed by the β-carotene bleaching method,29 which included the following:

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400 µL of β-Carotene (0.1 mg/mL in chloroform), 4.4 µL of linoleic acid and 40 mg of

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Tween 40. Then, the chloroform was evaporated and to the mixture was added 500 µL

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of methanol and 4.5 mL of water and the emulsion was shaken vigorously. The

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compounds were dissolved in MeOH (25 µL, final concentration 200 µM) and 225 µL

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of β-carotene solution were added to a 96-well plate. The control was 25 µL of MeOH

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and a blank was performed in the absence of linoleic acid. The experiments were

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performed in triplicate. The samples were exposed to thermal oxidation over 90 min at

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50 ºC in a microplate reader. The zero time absorbance was read at 470 nm immediately

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after the emulsion was added and after 90 min of incubation. The percentage of

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inhibition of lipid peroxidation was calculated according the equation:

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% inhibition = [1-(A0-At)/(A'0-A't)]×100

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where A0 is the absorbance of sample at zero time, At is the absorbance of sample after

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incubation (90 min) at 50 ºC, A'0 is the absorbance of control at zero time and A't is the

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absorbance of control after incubation (90 min) at 50 ºC.

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Liposome Preparation


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Liposomes were prepared as described elsewhere.24,30 The peroxidation of the

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liposomes was induced by peroxyl radicals at a constant rate by thermal degradation of

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the azo compound AAPH in the presence or absence of antioxidants.

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Oxygen Consumption Assay

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Oxygen consumption was measured with a Clark-type oxygen electrode (Hansatech

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Instruments, Pentney, United Kingdom) provided with an automatic recording apparatus as

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previously described.24 In brief, a mixture containing 1350 µL of Hepes buffer, 150 µL of

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liposome solution (340 µM final concentration) and 2 µL of antioxidant (1 mM) in MeOH

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was kept in a 37 °C thermostated bath for 1 h, introduced in a closed glass vessel, protected

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from light, at 37 °C, and the reaction started after the addition of AAPH (10 mM).31 The

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induction periods of the compounds were determined graphically from the profiles of oxygen

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consumption, by the coordinates of the interception of tangents to the inhibited and

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uninhibited rates of oxidation. Results were expressed comparatively to those obtained with

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Trolox.

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RESULTS AND DISCUSSION

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Synthesis

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In this work, reactions between Mv3glc-rich red wine extract and different fatty acids

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were performed by enzymatic catalysis (Figure 1). All reactions were carried out in

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anhydrous 2-methyl-2-butanol and at 60 ºC as usually reported in the literature11 and the

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amount of molecular sieves 4Å (100 g/L) and lipase (20 g/L) used were in concordance

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with a previous work.23

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The progress of the individual reactions was monitored by HPLC-DAD and the

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formation of new peaks was detected (Figure 2). The maximum formation of the

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different products was achieved from 6 h (C4) to 12 h (C16). The reactions were further

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analyzed by LC-DAD/ESI-MS in positive ion mode. The molecular ion [M]+ of the

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chromatographic peak products correspond to the respective Mv3glc-fatty acid

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conjugates as well as the MS2 and MS3 fragments indicate that the esterification

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occurred only on the glucose residue. These reactions would be regioselective to the

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more reactive hydroxyl group of the sugar (primary alcohol) as it was previously

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demonstrated by NMR for the Mv3glc-oleic acid conjugate.23 Furthermore, it was

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observed that the new compounds preserved their maximum absorption wavelength in

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the visible region with only a slight bathochromic shift compared with native Mv3glc

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(Table 1). Afterwards, the individual ester products were purified by C18 gel column

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chromatography and the yields obtained were 22 % (C16) to 40 % (C4 and C8).

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Lipophilic Parameters

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In order to assess the overall properties of the antioxidant compounds, the lipophilicity

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characterization of Mv3glc and all Mv3glc-fatty acid derivatives was determined by two

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parameters: hydrophobicity index (RM) which was calculated taking into consideration

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the retention factor (RF) of the compounds on C18 TLC plates and the partition

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coefficient (Log P) which was calculated either theoretically or by HPLC by measuring

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the distribution of compounds in an octanol-water biphasic system. The octanol-water

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partition coefficient is the classic method to assess hydrophilic/lipophilic balance32,33

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and it is the most used parameter in drug development quantitative structure-activity

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relationship models.


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As we can be observed in Table 2, RM and experimental Log P parameters increased

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with the fatty acid chain length which is an indication of the lipophilicity increase from

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Mv3glc to Mv3glc-C16. Although the theoretical and experimental Log P values are not

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in the same order of magnitude, they follow the same trend. For compounds Mv3glc-

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C12 up to Mv3glc-C16 it was not possible to determine their octanol-water partition

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coefficients since no concentration of the solute was detected in the water phase as

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already described for other amphiphilic molecules with surfactant properties.34

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Antioxidant Activity

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The antioxidant features of the Mv3glc and Mv3glc-fatty acid conjugates were assessed

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by lipid substrate peroxidation assays namely through β-Carotene assay linoleate

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bleaching method and by measuring the O2 consumption.

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β-Carotene Assay

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The occurrence of peroxidation of a lipidic substrate (linoleic acid) was monitored by

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the decrease of the absorbance of the β-Carotene (λmax 470 nm) over 90 min incubation

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at 50 ºC in a control sample (absence of antioxidant) and in the presence of the different

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compounds. In Figure 3, the percentage of antioxidant activity (% AA) for Mv3glc and

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the different Mv3glc-fatty acid conjugates is shown. Overall, it was possible to observe

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that in this lipophilic environment the antioxidant activity of the parent Mv3glc

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increases as the fatty acid chain length attached to the glucose moiety increases up to a

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maximum of 8 carbons and then decreases dramatically. This means that the appropriate

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structural modification is crucial to enhance the antioxidant activity of these pigments in

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lipidic matrices.

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O2 consumption assay

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In order to validate and correlate the results of antioxidant activity obtained by the β-

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carotene-lineolate method, the lipid peroxidation of a biomimetic model of the

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biological membranes (liposomes) in the presence of the different antioxidant

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compounds was measured by the O2 consumption assay (Figure 4). The results seem to

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indicate a relationship between the antioxidant capacity and the hydrophobicity of the

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molecules. The measured antioxidant activity increased with the anthocyanin linked to a

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fatty acid with chain length up to 8 carbons and then decreased significantly. This

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nonlinear phenomenon, also called as “cut-off” effect, was widely studied and reported

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in emulsions, membranes and cultured cells.35-37 In summary, this work focuses on the

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enzymatic synthesis of novel Mv3glc-fatty acid conjugates, structural characterization,

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evaluation of relevant physical parameters such as octanol-water partition coefficients,

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hydrophobicity index and antioxidant activity assessment.

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Overall, the results indicate that a proper structural modification could enhance the

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physical-chemical properties of anthocyanins as well as improve their antioxidant

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capacity in lipophilic environment. In this study, the screening of various Mv3glc-fatty

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acid derivatives with different chain lengths allowed conclusion that a maximum

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antioxidant efficiency was achieved with the lipophilization of Mv3glc with 8 carbons.

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The achievement of these lipophilic anthocyanin-derived pigments using green

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chemistry approaches bring important insights for their future application in lipophilic

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matrices such as lipid-based foods and cosmetic formulations.

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Acknowledgments

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The authors would like to thank Dr. Zélia Azevedo for the MS analysis and Dr. Mariana

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Andrade for the NMR analysis. The financial support was given by a research project


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grant (PTDC/AGR-TEC/3078/2014) from FCT/MEC through national funds and co-

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financed by FEDER, under the Partnership Agreement PT2020 (UID/QUI/50006/2013 -

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POCI/01/0145/FEDER/007265). Luís Cruz acknowledges the Post.Doc.Grant from

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FCT (SFRH/BPD/72652/2010).

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14. Mellou, F.; Lazari, D.; Skaltsa, H.; Tselepis, A. D.; Kolisis, E.; Stamatis, H., Biocatalytic preparation of acylated derivatives of flavonoid glycosides enhances their antioxidant and antimicrobial activity. J. Biotechnol. 2005, 116, 295-304. 15. Mellou, F.; Loutrari, H.; Stamatis, H.; Roussos, C.; Kolisis, F. N., Enzymatic esterification of flavonoids with unsaturated fatty acids: Effect of the novel esters on vascular endothelial growth factor release from K562 cells. Process Biochem. 2006, 41, 2029-2034. 16. Cho, H.; Yun, C.-W.; Park, W.-K.; Kong, J.-Y.; Kim, K. S.; Park, Y.; Lee, S.; Kim, B.-K., Modulation of the activity of pro-inflammatory enzymes, COX-2 and iNOS, by chrysin derivatives. Pharmacol. Res. 2004, 49, 37-43. 17. Caldwell, S.; Bennett, C.; Hartley, R.; McPhail, D.; Duthie, G. Flavonoid compounds as therapeutic antioxidants. Patent US 7601754, 2009. 18. Buchholz, H.; Rosskopf, R.; Carola, C. Flavonoid Complexes. Patent US 20080045478, 2008. 19. Cruz, L.; Basílio, N.; Mateus, N.; Pina, F.; de Freitas, V., Characterization of Kinetic and Thermodynamic Parameters of Cyanidin-3-glucoside Methyl and Glucuronyl Metabolite Conjugates. J. Phys. Chem. B 2015, 119, 2010-2018. 20. Cruz, L.; Mateus, N.; De Freitas, V., First chemical synthesis report of an anthocyanin metabolite with in vivo occurrence: cyanidin-4′-O-methyl-3-glucoside. Tetrahedron Lett. 2013, 54, 2865-2869. 21. De Castro, V. C.; da Silva, P. H. A.; de Oliveira, E. B.; Desobry, S.; Humeau, C., Extraction, identification and enzymatic synthesis of acylated derivatives of anthocyanins from jaboticaba (Myrciaria cauliflora) fruits. Int. J. Food Sci. Technol. 2014, 49, 196-204. 22. Zhao, L.-y.; Chen, J.; Wang, Z.-q.; Shen, R.-m.; Cui, N.; Sun, A.-d., Direct acylation of cyanidin-3-glucoside with lauric acid in blueberry and its stability analysis. Int. J. Food Prop. 2016, 19, 1-12. 23. Cruz, L.; Fernandes, I.; Guimaraes, M.; de Freitas, V.; Mateus, N., Enzymatic synthesis, structural characterization and antioxidant capacity assessment of a new lipophilic malvidin-3-glucoside-oleic acid conjugate. Food Funct. 2016, 7, 2754-2762. 24. Cruz, L.; Fernandes, V. C.; Araújo, P.; Mateus, N.; de Freitas, V., Synthesis, characterisation and antioxidant features of procyanidin B4 and malvidin-3-glucoside stearic acid derivatives. Food Chem. 2015, 174, 480-486. 25. Calculation of Molecular Properties and Bioactivity Score. http://www.molinspiration.com/cgi-bin/properties, Molinspiration, Cheminformatics, Bratislava, Slovak Republic, Accessed in April 2016. 26. Rothwell, J. A.; Day, A. J.; Morgan, M. R. A., Experimental determination of octanol−water partition coefficients of quercetin and related flavonoids. J. Agric. Food. Chem. 2005, 53, 4355-4360. 27. Zhu, S.; Li, Y.; Li, Z.; Ma, C. Y.; Lou, Z. X.; Yokoyama, W.; Wang, H. X., Lipase-catalyzed synthesis of acetylated EGCG and antioxidant properties of the acetylated derivatives. Food Res. Int. 2014, 56, 279-286. 28. Kiraly-Veghely, Z.; Katay, G.; Tyihak, E.; Merillon, J. M., Separation of stilbene isomers from red wine by overpressured-layer chromatography. J. Planar Chromatogr.--Mod. TLC 2004, 17, 4-8. 29. Viskupicova, J.; Danihelova, M.; Ondrejovic, M.; Liptaj, T.; Sturdik, E., Lipophilic rutin derivatives for antioxidant protection of oil-based foods. Food Chem. 2010, 123, 45-50. 30. Rodrigues, C.; Gameiro, P.; Reis, S.; Lima, J.; de Castro, B., Spectrophotometric determination of drug partition coefficients in dimyristoyl-L-α14 ACS Paragon Plus Environment

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370 371 372 373 374 375 376 377 378 379 380 381 382 383 384 385 386 387 388 389 390 391 392 393

phosphatidylcholine/water: a comparative study using phase separation and liposome suspensions. Anal. Chim. Acta 2001, 428, 103-109. 31. Porto, P.; Laranjinha, J. A. N.; de Freitas, V. A. P., Antioxidant protection of low density lipoprotein by procyanidins: structure/activity relationships. Biochem. Pharmacol. 2003, 66, 947-954. 32. Hansch, C.; Dunn, W. J., Linear relationships between lipophilic character and biological activity of drugs. J. Pharm. Sci. 1972, 61, 1-19. 33. Leo, A.; Hansch, C.; Elkins, D., Partition coefficients and their uses. Chem. Rev. 1971, 71, 525-616. 34. Florindo, C.; Araújo, J. M. M.; Alves, F.; Matos, C.; Ferraz, R.; Prudêncio, C.; Noronha, J. P.; Petrovski, Ž.; Branco, L.; Rebelo, L. P. N.; Marrucho, I. M., Evaluation of solubility and partition properties of ampicillin-based ionic liquids. Int. J. Pharm. 2013, 456, 553-559. 35. Laguerre, M.; López Giraldo, L. J.; Lecomte, J.; Figueroa-Espinoza, M.-C.; Baréa, B.; Weiss, J.; Decker, E. A.; Villeneuve, P., Relationship between hydrophobicity and antioxidant ability of “Phenolipids” in emulsion: a parabolic effect of the chain length of rosmarinate esters. J. Agric. Food. Chem. 2010, 58, 2869-2876. 36. Laguerre, M.; López Giraldo, L. J.; Lecomte, J.; Figueroa-Espinoza, M.-C.; Baréa, B.; Weiss, J.; Decker, E. A.; Villeneuve, P., Chain length affects antioxidant properties of chlorogenate esters in emulsion: the cutoff theory behind the polar paradox. J. Agric. Food. Chem. 2009, 57, 11335-11342. 37. Laguerre, M.; Bayrasy, C.; Lecomte, J.; Chabi, B.; Decker, E. A.; WrutniakCabello, C.; Cabello, G.; Villeneuve, P., How to boost antioxidants by lipophilization? Biochimie 2013, 95, 20-26.

394



395

Page 16 of 23

FIGURE CAPTIONS

396 397

Figure 1. Esterification reactions of Mv3glc with different fatty acids by enzymatic

398

catalysts.

399

Figure 2. HPLC chromatograms of individual enzymatic esterification reactions

400

recorded at the maximum wavelength and conversion percentage for each Mv3glc-ester

401

conjugated.

402

Figure 3. Percentage of antioxidant activity for Mv3glc and respective ester derivatives.

403

Columns represent mean ± standard deviation. Columns with the same letter do not

404

differ statistically (*p < 0.05).

405

Figure 4. Inhibition of AAPH-initiated oxidation in soybean PC liposome membranes

406

(LUV) measured by O2 consumption of 100 µM of Mv3glc and Mv3glc-fatty acid

407

derivatives. Columns represent mean ± standard deviation. Columns with the same

408

letter do not differ statistically (*p < 0.05).

409 410 411 412


Page 17 of 23


Table 1. HPLC Retention Times and Mass Data of Malvidin 3-Glucoside, 1, and Ester Derivatives, 2-8. MW

Rt

λmax

Compound

η (%)a

[M]+ m/z

MS2 m/z

MS3 m/z

(g/mol)

(min)

(nm)

Mv3glc, 1

528

9.1

525

-

493

331 (-162)

315 (-162-16)

Mv3glc-C4, 2

598

11.0

530

40

563

331 (-232)

315 (-232-16)

Mv3glc-C6, 3

626

11.8

533

27

591

331 (-260)

315 (-260-16)

Mv3glc-C8, 4

654

12.4

535

40

619

331 (-288)

315 (-288-16)

Mv3glc-C10, 5

682

13.2

536

30

647

331 (-316)

315 (-316-16)

Mv3glc-C12, 6

710

13.9

537

35

675

331 (-344)

315 (-344-16)

Mv3glc-C14, 7

738

14.9

537

24

703

331 (-372)

315 (-372-16)

Mv3glc-C16, 8

766

15.7

537

22

731

331 (-400)

315 (-400-16)

a

after column chromatography isolation.

17

ACS Paragon Plus Environment


Page 18 of 23

Table 2. Physical-Chemical Properties of Malvidin 3-Glucoside, 1, and Ester Derivatives, 2-8, Related to Lipophilicity. RF

RM

Log P theo.a (Log P exp.b)

Mv3glc, 1

0.87

-0.83

-2.47 (-1.31)

Mv3glc-C4, 2

0.85

-0.75

-0.85 (0.56)

Mv3glc-C6, 3

0.83

-0.69

0.17 (1.30)

Mv3glc-C8, 4

0.80

-0.60

1.18 (2.04)

Mv3glc-C10, 5

0.73

-0.43

2.19 (2.77)

Mv3glc-C12, 6

0.63

-0.23

3.20

Mv3glc-C14, 7

0.51

-0.02

4.21

ned with

Mv3glc-C16, 8

0.39

0.19

5.22

Molinspi

Mv3glc-C18:1c

0.35

0.27

5.74

ration

Compound

a

determi

calculation software.23 b

c

determined by HPLC using octanol-water shake-flask method.25

previously obtained.21


Page 19 of 23


Figure 1 OMe

OMe OH

OH

+

O

HO

+

O

HO

OMe

OH

O

O

OH

1

OMe

Fatty acid

HO

OH

CALB 60 ºC 2-Me-2-Bu MS 4A

HO OH

O

O

OH

OH

O OH

R=

O

OR

butyric, 2 hexanoic, 3

O

caprylic, 4 O

decanoic, 5 O

lauric, 6 O

myristic, 7 O

palmitic, 8



Page 20 of 23

Figure 2 4

300

2 250

6

3

mAU

200

5

150

7

1

8

100

50

0

0

0

2

4

6

8

10

12

14

16

18

20

Minutes


Page 21 of 23


Figure 3

30

c c b

20

% AA

b

10 a

a a,d d

0 1

2

3

4

5

6

7

8



Page 22 of 23

Figure 4

1.0

c

Ti/TI Trolox

0.8 d

0.6 0.4 0.2

b

b a

a

a a

0.0 1

2

3

4

5

6

7

8


Page 23 of 23


Table of Contents Graphic


Malvidin 3-GlucosideâFatty Acid Conjugates - American Chemical

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