Enzymatic Acylation of Anthocyanins Isolated from Alpine Bearberry

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Enzymatic Acylation of Anthocyanins Isolated from Alpine Bearberry (Arctostaphylos alpina) and Lipophilic Properties, Thermo-stability and Antioxidant Capacity of the Derivatives Wei Yang, Maaria Katariina Kortesniemi, Baoru Yang, and Jie Zheng J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b05924 • Publication Date (Web): 27 Feb 2018 Downloaded from http://pubs.acs.org on February 28, 2018

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Journal of Agricultural and Food Chemistry

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Enzymatic Acylation of Anthocyanins Isolated from Alpine Bearberry

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(Arctostaphylos alpina) and Lipophilic Properties, Thermo-stability and

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Antioxidant Capacity of the Derivatives

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Wei Yang †, Maaria Kortesniemi †, Baoru Yang †, Jie Zheng ‡,*

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† Food Chemistry and Food Development, Department of Biochemistry, University of Turku, FI-20014 Turku, Finland

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‡ Department of Food Science and Engineering, Jinan University, Guangzhou, 510632, China

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*Corresponding author: (Tel: +86 20 85226630; Fax : +86 20 85226630; E-mail: [email protected])

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1 ACS Paragon Plus Environment


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ABSTRACT

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Cyanidin-3-O-galactoside (cy-gal) isolated from alpine bearberry (Arctostaphylos alpine L.)

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was enzymatically acylated with saturated fatty acids of different chain lengths with Candida

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antarctica lipase immobilized on acrylic resin (Novozyme 435). The acylation reaction was

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optimized by considering reaction medium, acyl donor, substrate molar ratio, reaction

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temperature and reaction time. The highest conversion yields of 73% was obtained by reacting

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cy-gal with lauric acid (molar ratio, 1:10) in tert-butanol at 60 °C for 72 h. A novel compound

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was synthesized, which was identified as cyanidin-3-O-(6ʹʹ-dodecanoyl)galactoside by mass

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spectrometry and NMR. Introducing lauric acid into cy-gal significantly improved both the

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lipophilicity and thermo-stability, and substantially preserved the UV-VIS absorbance and the

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antioxidant properties. The research provides an important insight in expanding the application

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of natural anthocyanins in the cosmetics and food industries.

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Keywords: alpine bearberry; anthocyanins; antioxidative activity; cyanidin-3-O-

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galactoside;enzymatic acetylation; fatty acids; lipophilicity; thermo-stability


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INTRODUCTION

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Anthocyanins are the most common and widely distributed group of flavonoids, naturally

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occurring as glycosides of anthocyanidins in a wide range of plant tissues, principally flowers

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and fruits. Anthocyanins provide a variety of bright colors in plants, where they also play a key

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role in attracting pollinators and mitigating photo-oxidative damage, as well as in protecting

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against herbivores and pathogens.1-3

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In the recent years, natural pigments are increasingly favored by consumers due to concern

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on negative health impacts of synthetic colorants and the increasing knowledge of the health

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benefits of some natural color compounds.4,5 As the most important water-soluble natural

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pigments, anthocyanins have been studied extensively over the past decade. Over 600

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anthocyanins consisting of over 30 aglycones have been isolated from various plants species,

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and these numbers are expected to grow.6,7 The vast majority of anthocyanins are derived from

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six aglycones (cyanidin, delphinidin, pelargonidin, malvidin, peonidin and petunidin). 7

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Anthocyanins are not only responsible for the natural coloration in fruits and vegetables, but

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also for potential biological activity on human health due to their antioxidantive, antimicrobial,

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anti-inflammatory, cytoprotective, neuroprotective and lipidemic activities.8-11 Moreover,

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extensive epidemiological studies have associated the dietary consumption of anthocyanins

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with reduction of the risk of cardiovascular diseases, diabetes, arthritis and cancers. 12-16

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Thereby, anthocyanins are listed as natural colorants for food adopted by the Codex

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Alimentarius Commission and approved by the European Union with the E code as E163.17

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These plant-derived anthocyanin pigments are highly potential natural colors in food industry.

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However, anthocyanins are relatively unstable and they also have a tendency to form condensed

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polymers, which both present the challenge of loss of color during food processing and

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storage.18 The hydrophilic properties also limit the effective application of anthocyanins in 3 ACS Paragon Plus Environment


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lipophilic systems.

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To compensate the limitations in use of anthocyanins, various methods have been applied the

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compounds, such as enzymatic acylation, microencapsulation, and additions of co-pigmenting

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compounds.19 In the nature, some anthocyanins are acylated with aromatic acids bound to sugar

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residues. These acylated anthocyanins are mainly responsible for color stability in plant

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organs.20 The role of acylation in the stability of anthocyanins has recently been reviewed.

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Various studies demonstrate that acylation in general increases the stability of anthocyanins,

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which is further affected by the acylation site, and type and number of acyl groups.21 In the

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recent years, enzymatic acylations of different anthocyanins with different acyl donors have

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been reported. Cruz et al. described the enzymatic acylation of malvidin-3-glucoside with oleic

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acid and saturated fatty acids of different chain lengths, and the physico-chemical and

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antioxidant properties of the fatty acid conjugates.22-24 However, the stability of these acylated

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products were not evaluated empirically. Stability of the acylated anthocyanidin derivatives

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isolated from black rice (Oryza sativa L. subsp. japonica) has been reported.25 Further, acylated

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anthocyanins from jaboticaba (Myrciaria cauliflora) fruit and blueberry (Vaccinium sp.) were

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also synthesized, but the conjugates were not purified and characterized.26,27 Latifa et al.

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summarized the effects of various factors (enzymes, flavonoids, acyl donors, operating

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conditions and reaction media etc.) on enzymatic acylation of flavonoids.28 However, this

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review focused more on flavonols and flavan-3-ols, but less on anthocyanins. In the previous

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studies on enzymatic acylation of anthocyanins, the highest conversion yields obtained reached

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up to 91% with methyl benzoate as an acyl donor,25 and ranged from 21 to 40% with fatty acids

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as acyl donors.22,23 However, optimization based on various factors has not been reported so

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far.

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Alpine bearberry (Arctostaphylos alpina L.), also known as mountain bearberry or ptarmigan


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berry, is a matted or trailing shrub belonging to the family Ericaceae. It is widely distributed in

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high altitude areas of the northern hemisphere. 29 Alpine bearberry is edible with intense color

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in the skin and pulp, and strong taste of astringency and bitterness. The total content of

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anthocyanins in alpine bearberry is up to 870 mg/100 g fresh berries, and more than 95% of

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the anthocyanins is cyanidin-3-O-galactoside (cy-gal).30 The unique anthocyanin composition

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allows cy-gal to be easily purified on a large scale, which makes alpine bearberry an excellent

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source of this natural pigment.

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In the present study, acylation of anthocyanins from alpine bearberry was systematically

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optimized, and the lipophilic, thermo-stability and antioxidant properties of the acylation

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product were investigated for the first time. An anthocyanin-enriched extract from alpine

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bearberry was obtained using aqueous ethanol solution, followed by purification with

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Sephadex LH-20 chromatography. Enzymatic acylation of cy-gal purified from alpine

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bearberry with saturated fatty acids of different chain lengths was carried out. The effects of

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reaction medium, reaction temperature, reaction time, acyl donor, and the molar ratio of

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substrates on conversion yield were evaluated. The acylated derivative was characterized by

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mass spectrometry (MS) and, and for the first time, by nuclear magnetic resonance (NMR)

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spectroscopy (1H,

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antioxidant activity, as well as stability of the acylated derivatives were studied to evaluate the

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application potential of the novel product in food, cosmetics and pharmaceutical industry.

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MATERIALS AND METHODS

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C and standard homo- and heteronuclear 2D NMR). The lipophilicity,

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Materials and Reagents Wild alpine bearberries were picked when optimally ripe at

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Skallovaara located in Utsjoki, Finland during September 2014. The berries were frozen

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immediately after picking and stored at −20 °C until use.

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Fatty acids (lauric acid, myristic acid, palmitic acid and stearic acid), molecular sieves 4 Å, 5 ACS Paragon Plus Environment


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2,4,6-tri(2-pyridyl)-s-triazine (TPTZ), 2,2-diphenyl-1-picrylhydrazyl (DPPH), Candida

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antarctica lipase immobilized on acrylic resin (Novozyme 435) (≥5,000 U/g, recombinant,

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expressed in Aspergillus niger), acetone (HPLC grade), acetonitrile (HPLC grade), tert-butanol

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(reagent grade), and tert-amyl alcohol (reagent grade) were purchased from Sigma–Aldrich Co.

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(St. Louis, MO). The reference compound cyaniding-3-O-galactoside (cy-gal) was purchased

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from Extrasynthese (Genay, France).

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Sample Preparation and Purification. About 400 g of alpine bearberries were thawed in a

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microwave oven (MW201, Whirlpool, China) for 2 min at 160 W and homogenized with a

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Bamix M133 mixer (Bamix, Mettlen TG, Switzerland) when half-melted. The slurry was

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extracted with 2 L of 70% aqueous ethanol by sonicating for 30 min, followed by centrifugation

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at 12,100 ×g for 10 min. The supernatants were collected and evaporated in a rotary evaporator

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(Hei-VAP Advantage, Heidolph, Germany) at 35 °C to remove ethanol. The remaining aqueous

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solution was diluted to 200 mL with MQ-water. The crude extract was centrifuged at 12,100 ×

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g for 10 min and filtered with 0.45 μm regenerated cellulose (RC) filter (26 mm inner diameter,

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Phenomenex, Torrance, CA) before purification by column chromatography.

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A 190 × 27 mm inner diameter plastic column packed with 25 g of Sephadex LH-20

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(Pharmacia, Uppsala, Sweden) was used for purification of crude extracts of anthocyanins. The

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column was activated and rinsed with 200 mL of MQ-water before the crude extract (10 mL)

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was loaded. The column was eluted with 200 mL of water to remove the sugars, and pure cy-

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gal was collected by elution with gradient ethanol in water (10:90, 20:80 ,30:70, v/v, 200 mL

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of each) with a flow rate of 1 mL/min maintained by a Alitea-XV peristaltic pump

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(Bioengineering, Wald, Switzerland). After elution of anthocyanins, the column was cleaned

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with 500 mL of acetone in water (70:30, v/v) and balanced with 500 mL of water. The fractions

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of purified anthocyanins were lyophilized using a freeze dryer (VirTis Wizard 2.0, SP Scientific,

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Stone Ridge, NY) and stored at −80 °C prior to analysis. 6 ACS Paragon Plus Environment

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Synthesis of acylated derivatives. Reaction media (acetone, acetonitrile, tert-butanol, and

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tert-amyl alcohol) were fully dried with the 4 Å molecular sieves (100 mg/mL). The acylation

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reaction was performed in 50 mL screw thread test tubes fixed on the shelf in a shaking

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incubator (GFL GmbH, Germany) with a shaking speed of 250 rpm. For each reaction, 10 mL

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reaction medium, 10 mg of purified cy-gal extract and the respective saturated fatty acid in

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corresponding molar ratio (acyl donor/anthocyanin molar ratio, 1:1, 2:1, 5:1, 10:1) were added

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into the test tube. 4 Å molecular sieves (100 mg/mL) were also added to maintain a low water

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activity in the reaction medium. The substrates were sufficiently dissolved overnight, and the

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solubility of anthocyanins was measured in different reaction media at 40 °C, 50 °C, and 60 °C.

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The acylation reaction was started by addition of Novezym 435, 10 g/L (≥5,000 U/g). Finally,

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the reaction was terminated by filtration through a polytetrafluoroethylene (PTFE) filter (13

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mm inner diameter, 0.22 μm, VWR International, West Chester, PA) to remove the enzyme.

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HPLC-DAD analysis. The anthocyanin fractions purified by column chromatography were

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analyzed and the amount of anthocyanins in acylation reactions was quantitatively monitored

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using a Shimadzu Nexera UHPLC system (Shimadzu Corporation, Kyoto, Japan), which

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consisted of a CBM-20A central unit, an SIL-30AC auto sampler, two LC-30AD pumps, a

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CTO-20AC column oven and an SPD-M20A diode array detector. A Phenomenex Aeris peptide

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XB-C18 (3.6 μm, 150 × 4.60 mm) column combined with a Phenomenex Security Guard

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Cartridge Kit (Torrance, CA) was used. The analysis was carried out by a gradient elution of

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formic acid/water (5:95, v/v) as solvent A and acetonitrile as solvent B at a mobile phase flow

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rate of 1 mL/min. The gradient program of the mobile phase was: 0–5 min, 5–10% B; 5–10

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min, 10% B; 10–17 min, 10–24% B; 17–27 min, 24–90% B; 27–30 min, 90% B; 30–35 min,

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90–5% B; 35–40 min, 5% B. The injection volume was 10 μL, and the column temperature

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was maintained at 40 °C. The peaks were monitored at multiple wavelengths (280 nm, 360 nm

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and 520 nm) for all the phenolic compounds, and their acylated derivatives. Quantitative 7 ACS Paragon Plus Environment


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analysis of anthocyanins was carried out with an external standard method using a calibration

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curve of cy-gal constructed in the concentration range from 0.0005 to 0.1000 mg/mL.

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ESI-MS/MS analysis. A Waters Acquity ultrahigh-performance LC (UPLC) system (Waters

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Corp., Milford, MA) with a sample manager, binary solvent delivery system, Waters 2996 PDA

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detector, and Waters Quattro Premier Tandem Quadrupole mass spectrometer equipped with

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an electrospray ionization (ESI) source was employed to analyze the acylated derivatives. The

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chromatographic conditions were the same as described above in the HPLC-DAD analysis,

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except a split joint was used after the UV detector to direct a flow of 0.4 mL/min to the mass

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spectrometer and the rest to a waste bottle. The mass spectrometer was operated in a positive

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ion mode by scanning ions between m/z 100 and 1000. The ESI inlet conditions were as follows:

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capillary voltage, 0.6 kV; cone voltage, 30 V; extractor voltage, 2 V; source temperature, 120 °C;

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and desolvation temperature, 350 °C.

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Isolation of the acylated derivative with semipreparative HPLC. The acylated derivatives

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were isolated using Shimadzu semi-preparative HPLC system, which consisted of a CBM-20A

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system controller, an SIL 20AC automatic injector, an LC-20AB pump, a CTO-10AC column

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oven, an SPD-20A UV/vis detector, and an FRC-10A fraction collector (Shimadzu Corporation,

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Kyoto, Japan). A Phenomenex Aeris PEPTIDE XB-C18 column (5 μm, 250 mm × 10 mm)

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combined with a Phenomenex Prodigy guard column was employed for isolation of acylated

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derivatives. Formic acid/water (5:95, v/v) was used as solvent A and acetonitrile as solvent B

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with gradient elution at a flow rate of 5 mL/min. The gradient program of the mobile phase

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was: 0–2 min, 5–10% B; 2–7 min, 10–60% B; 7–10 min, 60–65% B; 10–13 min, 65–90% B;

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13–15 min, 90% B; 15–20 min, 90–5% B; 20–25 min, 5% B. The injection volume was 150

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μL, and the column temperature was maintained at 40 °C. The peaks were monitored at the

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wavelength of 520 nm. The collected fractions of acylated derivatives were evaporated at 35 °C

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to remove organic solvents, freeze-dried and stored at −80°C prior to NMR analysis. 8 ACS Paragon Plus Environment

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NMR analysis. The freeze-dried acylated derivatives (1.8 mg) collected by preparative

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HPLC were analyzed with a Bruker AVANCE-III 500-MHz spectrometer (Fällanden,

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Switzerland) operating at 500.13 and 125.77 MHz for 1H and

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(1H, 13C, COSY, HSQC and HMBC) were recorded in CD3OD+TMS (VWR International Oy,

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Espoo, Finland) acidified with 5% CF3COOD (Eurisotop, Saint-Aubin Cedex - France).31 The

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spectra were processed with the TopSpin 3.5pl6 software (Bruker BioSpin GmbH, Rheinstetten,

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Germany).

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13

C, respectively. The spectra

Measurement of lipophilicity. The lipophilicity of the acylated derivatives was evaluated

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by determining the octanol/water partition coefficient (logP)

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HCl) and n-octanol were mutually saturated with each other (1:3, v/v) for 24 h. The samples

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were dissolved in n-octanol (1 mL) saturated with acidified water, and the absorbance (A0) at

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520 nm was determined. Then, 1 mL acidified water (2% HCl) saturated with n-octanol was

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added, and the mixtures were vigorously shaken for 1 h, centrifuged at 2,000 × g for 10 min.

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Absorbance (Ax) of n-octanol in the upper layer was measured at 520 nm. The octanol/water

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partition coefficient was calculated using the equation logP = log Ax / (A0 − Ax).

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. Briefly, acidified water (2%

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Evaluation of thermo-stability. The thermos-stability of cy-gal isolated from alpine

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bearberry and its acylated derivative was investigated in a thermostated water bath at 65, 80,

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and 90 °C. Both samples (100 μM) were dissolved in 0.01 M HCl (pH around 2.1) aqueous

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methanol (methanol: H2O, 1:1, v/v). At time-points 0, 2, 6, 12, 20 h after the heat treatment,

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the absorbance of each sample (100 μL in a 96-well micro plate) was measured using a plate

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reader (Hidex, Turku, Finland) at 520 nm.

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Evaluation of antioxidant capacity. The antioxidant capacity of anthocyanin from alpine

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bearberry and its acylated derivative was evaluated by DPPH radical scavenging capacity assay

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and ferric reducing antioxidant power (FRAP) assay. The DPPH assay was performed 9 ACS Paragon Plus Environment


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according to the method reported in literature with minor modification.22 In brief, 270 μL of

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freshly prepared DPPH radical (60 μM in methanol) was mixed with 30 μL of

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anthocyanin/acylated derivative (1000 μM in DMSO). The reaction was carried out in a 96-

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well micro plate at 25°C. The absorbance values at 515 nm were detected at 30 min of reaction

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by a plate reader (Hidex, Finland). The absorbance values of Trolox in DMSO ranging from 5

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to 300 μM were used to construct the calibration curves. The antioxidant activity was expressed

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as Trolox equivalent (μM).

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The FRAP assay was performed using the method described by Cruz et al. 22. The freshly

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prepared FRAP reagent (300 mM acetate buffer, pH 3.6; 10 mM TPTZ in 40 mM HCl; 20 mM

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FeCl3·6H2O in H2O; 10:1:1, v/v/v) was diluted to one-third with acetate buffer (300 mM acetate

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buffer, pH 3.6), and incubated at 37 °C. 270 μL of diluted FRAP reagent was mixed with 30

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μL of anthocyanin/acylated derivative (1,000 μM in DMSO) in a 96-well micro plate. The

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reaction was undertaken at 37 °C for 4 min, and the absorbance values were measured at 593

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nm against blank, which consisted of 270 μL of diluted FRAP reagent and 30 µL of DMSO.

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The calibration curves were the same as used in DPPH assay. The reduction ability was

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expressed as Trolox equivalents (μM).

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Statistical analysis. All the experiments were conducted in triplicate. Statistical analyses

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were performed using SPSS 16.0.1 (SPSS Inc., Chicago, IL). A one-way analysis of variance

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(ANOVA) and independent-sample t test were performed to compare the differences in the

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conversion yield and antioxidant capacity. Differences were considered statistically significant

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when p < 0.05.

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Results and Discussion

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Purification of Anthocyanins by Column Chromatography. In the present study, cy-gal

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was the clearly dominating anthocyanin in the crude extracts of anthocyanins from alpine 10 ACS Paragon Plus Environment

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bearberry, which was identified using the reference compound with HPLC-DAD analysis and

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HPLC-MS analysis.30 The crude extracts were further purified with Sephadex LH20 column

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chromatography system. The fractions eluted with gradient ethanol in water (10:90 to 30:70,

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v/v) were analyzed by HPLC-DAD at 280 nm, 360 nm and 520 nm. Cy-gal was mainly eluted

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in the fraction with ethanol in water (30:70, v/v), and hardly any traces of non-anthocyanin

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substances were recognized at 280 nm and 360 nm simultaneously. The cy-gal fraction was

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collected and evaporated at 35 °C to remove ethanol, then freeze–dried and stored at −80 °C

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prior to acylation reaction. The purity of the freeze-dried cy-gal reached up to 87%.

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Effect of reaction medium on efficiency of acylation reaction. The effect of reaction

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medium on the reaction efficiency was studied by using four organic media with different

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polarities (acetone, acetonitrile, tert-butanol and tert-amyl alcohol). The solubility and

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conversion of cy-gal in each medium were measured. As shown in supplementary Figure S1,

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higher solubility was achieved at higher temperatures in each medium, and the highest

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solubility of cy-gal was obtained at 60 °C in tert-butanol. The conversion yield in each medium

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was measured under selected conditions (reaction temperature, 60 °C; acyl donor/cy-gal molar

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ratio, 2:1; reaction time, 24h).

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An optimal medium in a non-aqueous lipase reaction is extremely important since the nature

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of the medium can affect the specificity and regioselectivity of substrates, as well as the

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catalytic power of enzymes.33 Correlation between the conversion yield and solubility in

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reaction media at 60 °C is presented in supplementary Figure S2A. The conversion yield

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showed positive association with the solubility in reaction media (R2 = 0.8101). The highest

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conversion yields were obtained in tert-butanol reaction medium with the highest solubility of

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cy-gal. Therefore, tert-butanol was selected as the solvent for further studies. Kontogianni et

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al. also observed that tert-butanol was the most suitable reaction medium for obtaining the



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highest yields for acylation of flavonoids (rutin and naringin) with fatty acids catalyzed by

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Novozyme 435.34

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The properties of reaction medium are usually evaluated by the conductivity constant, dipole

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moment hydrophobicity (log P), and Hildebrand solubility parameter, among which log P is

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one of the most important factors.35 Correlation between the conversion yields and log P of

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reaction media (polarity constant, the partition coefficient of the solvent between octanol and

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water) is presented in the supplementary Figure S2B. The conversion yields were positively

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correlated with log P (R2 = 0.7411). The highest conversion yields were obtained in tert-butanol

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reaction medium. In contrast, the conversion yields in acetone and acetonitrile with low log P

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were 50–58% lower than that in tert-butanol. The higher log P appears to be more favorable

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for the dissolution of the acylated derivative, and thereby facilitating the progress of the

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acylation reaction.

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Effect of different acyl donors on acylation reaction efficiency. Four saturated fatty acids

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with different chain lengths (lauric acid, C12; myristic acid, C14; palmitic acid, C16; stearic

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acid, C18) were employed to study the effect of acyl donor on acylation efficiency under the

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selected conditions (reaction medium, tert-butanol; reaction temperature, 60 °C; reaction time,

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24 h; acyl donor/cy-gal molar ratio, 2:1). The results (Figure 1A) indicate that the conversion

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yields decreased from the 41% acylation efficiency with lauric acid (C12) to 10% with stearic

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acid (C18). Similar results were obtained in enzymatic acylation of flavonoids (isoorientin,

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isovitexin, isoquercitrin) with different fatty acids from C12 to C14, and from C4 to C18.36,37

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Ardhaoui et al. also confirmed that both conversion yield and initial rate dropped slightly in

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acylation reactions with the increase in the length of the carbon chain of the fatty acids. 38 The

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effect of acyl donor chain length on conversion yield may be related to the accessibility of the

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lipase binding site and the solubility of acyl donor in the reaction medium.39,40


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Effect of reaction time and acyl donor/cy-gal molar ratio on acylation efficiency. To

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investigate the effect of reaction time and molar ratio of acyl donor/cy-gal on the conversion

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yield, the selected optimal conditions of other parameters (reaction medium, tert-butanol;

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reaction temperature, 60 °C; acyl donor, lauric acid) were held constant while the conversion

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yields of reactions with varying reaction times and acyl donor/cy-gal molar ratios were

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measured. Figure 1B shows that the conversion yields are influenced by both reaction time and

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acyl donor/cy-gal ratio. With increases in reaction time, the conversion yields increased rapidly

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in the first 24 h (p < 0.01), and only moderate increases in conversion yields were achieved

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when the reaction time was elongated from 24 h to 72 h (p > 0.05). For cost efficiency, 24 h

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was considered as the best reaction time for large-scale preparation with conversion yield from

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26 to 68% for different acyl donor/cy-gal ratio applied. As the acyl donor/cy-gal ratio increased

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from 1:1 to 10:1, the conversion yield increased significantly (p < 0.01). The highest conversion

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yield of cy-gal (73%) was obtained with acyl donor/cy-gal molar ratio of 10:1 at 72 h. The

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similar positive effects of high molar ratio of acyl donor/flavonoid on the conversion yield have

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been reported for the enzymatic acylation of flavonoids (flavonols, flavones, flavanones) with

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a variety of acyl donors.28,37,41,42 The behavior could be attributed to the shifting of

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thermodynamic equilibrium to the synthesis of acylated derivatives due to excess of the acyl

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donors.

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Identification of enzymatically acylated derivatives. According to the optimization results

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of the reaction parameters obtained above, the conditions for synthesis of cy-gal acylated

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derivatives were chosen as follows: reaction medium, tert-butanol; reaction temperature, 60 °C;

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reaction time, 72 h; lauric acid/cy-gal molar ratio, 10:1. Only one new peak around 25 min at

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520 nm was detected in the reaction solution by HPLC-DAD analysis. Full scan function of

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ESI-MS and parent ion scan function of ESI-MS/MS in positive ion modes were used to reveal

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the structure of the new product. The product was identified as a conjugate of a cy-gal molecule 13 ACS Paragon Plus Environment


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with a lauric acid moiety attached according to its full MS spectrum [M + H]+ ion at m/z 632

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(Figure 2A) and the fragment ion at m/z 287 from the parent ion m/z 632 in the MS2 spectrum

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(Figure 2B). The mass spectra preliminarily indicated that the acylation had a regioselectivity

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to the galactoside moiety, preferentially occurring in an OH group of galactose rather than in

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any hydroxyl group of the aglycone.

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Structural elucidation by NMR. To further elucidate the exact structure of the conjugate

314

of cy-gal and lauric acid, the acylated product was isolated by semi-preparative HPLC from

315

the reaction solution. The product was obtained as dark red amorphous powders with a purity

316

of close to 90% based on the comparison of peak areas at 520 nm by HPLC analysis. The

317

detailed structure of the conjugate and the position of the lauric acid moiety attached to cy-gal

318

were determined by the 1H and

319

HMBC experiments (Figure 3). As expected based on earlier reports,22,37 the downfield shift

320

of the corresponding chemical shifts for proton and carbon31 indicated the attachment of lauric

321

acid to the 6ʺ-OH position of the galactose unit. The new derivative (acylation product) was

322

therefore identified as cyanidin-3-O-(6ʹʹ-dodecanoyl)galactoside.

13

C NMR data and the correlations from COSY, HSQC and

323

Properties of the acylated anthocyanin compound. The UV-VIS absorbance, lipophilic

324

property, thermos-stability and antioxidant activity of the acylated anthocyanin, cyanidin-3-O-

325

(6ʹʹ-dodecanoyl)galactoside were systematically studied.

326

UV-VIS absorbance. The UV-VIS absorbance spectra of cy-gal and its lauric acid derivative

327

were compared (supplementary Figure S3). cyanidin-3-O-(6ʹʹ-dodecanoyl)galactoside

328

basically maintained the UV absorbance spectrum of cy-gal, except a small bathochromic shift

329

of the absorption maximum from 517 nm (λmax) to 525 nm (λmax).

330 331

Lipophilicity.

Lipophilicity of the acylated derivative was evaluated by octanol/water

partition coefficient (log P). In general, the higher the log P values are, the better the 14 ACS Paragon Plus Environment

Page 14 of 30

Page 15 of 30


332

lipophilicity. As shown in Figure 4, the partition coefficient (log P) of cy-gal was −1.38, while

333

the value significantly increased to 2.60 after acylation with lauric acid. The enhanced

334

lipophilic properties of an acylated derivative will contribute to penetration into lipid matrices

335

or lipophilic media, and expand the scope of application of anthocyanins as colorants from

336

aqueous to fat-rich food matrices. Violeta et al. suggested that low bioavailability of cyanidin

337

3-O-β-glucopyranoside may be related to its glucose moiety, which is difficult to orientate into

338

the water–lipid interface or between the nonpolar acyl chains of membranes.43 Another study

339

indicated that acylated cyanidin shows the highest affinity to the cell-mimic membrane.44

340

Thermo-stability. During the heating process, degradation occurred in both cy-gal and its

341

lauric acid derivative. The color of samples disappeared or changed to yellow. The logarithms

342

of cy-gal (ln (A/A0)) and its derivative (ln (A/A0)) were plotted versus time of heat treatment (t)

343

incubated at 65, 80, and 90 °C, respectively, which all showed nearly linear relationships. (ln

344

(C/C0) = ln (A/A0), where A0 is the initial absorbance at 520 nm, A is the absorbance at 520 nm

345

after heating at selected temperatures, C0 is the initial content of samples, C is the content of

346

samples after heating at selected temperatures). These phenomena proved that the degradation

347

processes of cy-gal and its lauric acid conjugate followed the first-order reaction kinetics. The

348

rate constant (k) and half-life time (t

349

1/2 =

350

ln k = ln k0–Ea/RT, where R is the universal gas constant (8.314 J mol–1), k0 is the frequency

351

factor, T is the absolute temperature (Kelvin).25

1/2)

were calculated by the equations: ln (C/C0) = –k*t, t

–ln (1/2)*k–1, and the activation energy (Ea) was estimated using the Arrhenius equation:

352

The kinetic parameters of degradation during heating are summarized in Table 1. As

353

expected, the thermo-stability of both cy-gal and its lauric acid conjugate decreased with

354

increasing temperature. The t1/2 values of the lauric acid conjugate at each selected temperature

355

were all higher than that of cy-gal. Compared with cy-gal, the Ea value of cy-gal lauric acid

356

conjugate decreased from 46.6 kJ mol–1 to 45.8 kJ mol–1. Normally, high Ea reactions are more 15 ACS Paragon Plus Environment


Page 16 of 30

357

sensitive to temperature changes,45 thus, the degradation rate of cy-gal is more susceptible to

358

temperature elevation than that of cy-gal lauric acid conjugate. The results of the current

359

thermo-stability study are in agreement with those of previous investigations, anthocyanins

360

acylated with methyl aromatic acid, octenyl succinate anhydride and cinnamic and lauric acids,

361

respectively,

362

anthocyanins.25,27,46,47 The enhanced thermo-stability may be attributed to the acylation through

363

the 6ʺ-OH position of glucose moiety, which allows for the folding of acyl aromatic rings over

364

the planar pyrylium ring. Such sandwich type stacking has a protective effect on anthocyanins,

365

and also contributes to the color stabilization.21,48

showed

higher

thermo-stability

when

compared

with

nonacylated

366

Antioxidant activity. The antioxidant activity of cy-gal and its lauric acid conjugate were

367

assessed by both DPPH and FRAP assays, expressed as Trolox equivalent. The DPPH free

368

radical scavenging ability of cy-gal was slightly but significantly (p < 0.01) lowered by

369

acylation with lauric acid. In contrast, the antioxidant capacity of cy-gal and its derivative were

370

at the same level in FRAP assay (p > 0.05) (Figure 5). The reduction in DPPH radical

371

scavenging activity by the acylation was likely due to the increased volume and structural

372

complexity of the acylation product molecule. Moreover, steric hindrance caused by acylation

373

and the twisted acyl moiety, as well as the decreased electron-inductive effects might also

374

prevent the derivative from reaching the active site of DPPH.49 Similar results were also

375

obtained in a variety of acylated flavones and anthocyanins with long-chain fatty acids.22,37

376

Overall, despite the slight decline (by 7.9 %) in the DPPH radical scavenging ability, the total

377

antioxidant capacity by FRAP assay was not affected. Antioxidant capacities of cy-gal were

378

largely retained after the acylation with lauric acid. It is worth to notice that only hydrophilic

379

antioxidant capacities of cy-gal and its lauric acid conjugate were evaluated. Due to the

380

increased lipophilicity of the lauric acid conjugate of cy-gal, the lipophilic antioxidant activity

381

needs to be evaluated further. 16 ACS Paragon Plus Environment

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382

In summary, cy-gal was the most abundant anthocyanin in anthocyanin-rich extract from

383

alpine bearberry. Cy-gal was enzymatically acylated with saturated fatty acids of different

384

chain lengths as acyl donors (lauric acid, C12; myristic acid, C14; palmitic acid, C16; stearic

385

acid, C18) by Novozyme 435. The influence of reaction medium, acyl donor, reaction

386

temperature, reaction time and acyl donor/ cy-gal molar ratio on the conversion yields were

387

investigated. Decrease in carbon chain length of fatty acid, and increases in temperature and

388

reaction time as well as the acyl donor/ cy-gal molar ratio, all increased the yields of the

389

acylation product. The highest conversion yields (73%) were obtained by acylation of cy-gal

390

with lauric acid (C12) at an anthocyanin/acyl donor molar ratio of 10:1 in tert-butanol at 60 °C

391

for 72 h. In the reaction mixture under the selected condition, only one acylated derivative was

392

detected, which was identified as cyanidin-3-O-(6ʹʹ-dodecanoyl)galactoside by mass

393

spectrometry and NMR analyses. Introducing lauric acid into cy-gal significantly improved its

394

lipophilicity and thermos-stability, and retained its antioxidant activity and UV-vis absorbance

395

property. This research has established an acylation route for the synthesis of thermostable

396

lipophilic anthocyanin-based pigments in a low-toxic medium, and further expands the

397

application of natural anthocyanins in lipophilic foods, cosmetics and pharmaceutical products.

398

Acknowledgment

399

This work was financed by the Foundation for Research of Natural Resources in Finland and

400

the National Natural Science Foundation of China [grant numbers 31701607]. The authors

401

thank Heikki Kallio for the improvement of grammar and readability.

402

Supporting Information Available:

403

Supplementary Figure S1. The solubility of cyanidin-3-O-galactoside in mediums (acetone,

404

acetonitrile, tert-butanol and tert-amyl alcohol) at 40°C, 50 °C and 60 °C.



405

Supplementary Figure S2. The effect of solubility of reaction media at 60 °C and reaction

406

medium partition coefficient (log P) on the conversion yield of acylation of cyanidin-3-O-

407

galactoside with lauric acid.

408

Supplementary Figure S3. UV–VIS spectra of cyanidin-3-O-galactoside (cy-gal) and its

409

derivative (cy-gal derivative).


Page 18 of 30

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410

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411 412 413

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44. Strugała, P.; Dudra, A.; Gabrielska, J. Interaction between mimic lipid membranes and acylated and nonacylated cyanidin and its Bioactivity. J. Agric. Food Chem. 2016, 64, 74147422.

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45. Li, J.; Li, X.; Zhang, Y.; Zheng, Z.; Qu, Z.; Liu, M.; Zhu, S.; Liu, S.; Wang, M.; Qu, L. Identification and thermal stability of purple-fleshed sweet potato anthocyanins in aqueous solutions with various pH values and fruit juices. Food Chem. 2013, 136, 1429-1434.

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46. Lv, X.L.; Sun, H.L.; Ji, Z.Y. Acylation of anthocyanins from black rice and their stability properties. Advanced Materials Research. 2011, 204, 750-754.

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47. Sari, P.; Setiawan, A.; Siswoyo, T. Stability and antioxidant activity of acylated jambolan (Syzygium cumini) anthocyanins synthesized by lipase-catalyzed transesterification. International Food Research Journal. 2015, 22, 671-676.

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48. Giusti, M.M.; Wrolstad, R.E. Acylated anthocyanins from edible sources and their applications in food systems. Biochem. Eng. J. 2003, 14, 217-225.

534 535 536

49. Lue, B.; Nielsen, N.S.; Jacobsen, C.; Hellgren, L.; Guo, Z.; Xu, X. Antioxidant properties of modified rutin esters by DPPH, reducing power, iron chelation and human low density lipoprotein assays. Food Chem. 2010, 123, 221-230.

537 538


Page 22 of 30

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539

Figure captions

540

Figure 1. The effects of different acyl donors (lauric acid, C12; myristic acid, C14; palmitic

541

acid, C16; stearic acid, C18) (A), reaction time and molar ratio of cyanidin-3-O-galactoside

542

(cy-gal)/acyl donor (B) on the acylation conversion yield. Significant differences (p < 0.05) are

543

marked as a, b and c. Error bars represent the standard deviations of the mean of three replicates

544

(n = 3).

545

Figure 2. Mass spectra of the acylated derivative of cyanidin-3-O-galactoside in full scan mode

546

(A) and tandem MS (MS2) mode (B).

547

Figure 3. 1H (500 MHz) and 13C (126 MHz) NMR data of cyanidin-3-O-galactoside acylated

548

derivative in CD3OD:CF3COOD (95:5 v/v, with TMS).

549

Figure 4. Partition coefficient (log P) of cyanidin-3-O-galactoside (cy-gal) and its derivative

550

(cy-gal derivative). Error bars represent the standard deviations of the mean of three replicates

551

(n = 3).

552

Figure 5. DPPH radical scavenging capacity (DPPH) and ferric reducing antioxidant power

553

(FRAP) of 100 μM cyanidin-3-O-galactoside (cy-gal) and its derivative (cy-gal derivative).

554

Data represent mean ± standard deviation (n = 3), ** means statistically significant at p < 0.001.

555



556

Table 1. The Kinetic Parameters of Thermal Degradation at Different Temperatures Samplea

Temperature (℃)

k (h-1)

t1/2 (h)

Ea (kJ mol–1)

cy-gal

65 80 90 65 80 90

0.0639 (0.9043) 0.1061 (0.9987) 0.2489 (0.9918) 0.0440 (0.7913) 0.0736 (0.8850) 0.1671 (0.9470)

10.8 6.5 2.8 15.8 9.4 4.1

46.6 (0.9713)

cy-gal derivative 557 558

Page 24 of 30

a

45.8 (0.9759)

Cy-gal is the abbreviation of cyanidin-3-O-galactoside. Numbers in parentheses are the correlation coefficients


Page 25 of 30


559 560

Figure 1.



561 562

Figure 2.

563


Page 26 of 30

Page 27 of 30


564 565

Figure 3.

566



567 568

Figure 4.

569


Page 28 of 30

Page 29 of 30


570 571

Figure 5.

572



573

Table of contents

574


Page 30 of 30

Enzymatic Acylation of Anthocyanins Isolated from Alpine Bearberry

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