New acylated cyanidin glycosides extracted from underutilized potential sources: Enzymatic synthesis, antioxidant activity and thermostability

Article abstract of DOI:10.1016/j.foodchem.2019.125796

Interest in anthocyanins has increased remarkably in recent decades, although their wider application has been hampered by instability problems. Thus, this study aimed at developing a strategy to gain access to more stable anthocyanins via enzymatic esterification. For that purpose, three cyanidin derivatives were obtained from underutilized, but easily accessible sources, and their total anthocyanin content was quantified. The purity of cyanidins obtained ranged from 40% to 88% depending on their source. Subsequently, the critical enzymatic reaction conditions were established, and the best results were found using tert-butanol as a solvent, 20 g/L of lipase B from Candida Antarctica, and vinyl cinnamate as acyl donor at ratio 250:1 (acyl donor to anthocyanin). Finally, five new acylated anthocyanin derivatives were synthesized with improved antioxidant activity and thermostability, in comparison to the cyanidin-3-glucoside, which is an advantageous feature for industrial applications.

Full text of DOI:10.1016/j.foodchem.2019.125796

Journal Pre-proofs
New acylated cyanidin glycosides extracted from underutilized potential sour-
ces: enzymatic synthesis, antioxidant activity and thermostability
Fernanda Fernandez-Aulis, Andrea Torres, Ernesto Sanchez-Mendoza, Luis
Cruz, Arturo Navarro-Ocana
PII:
S0308-8146(19)31929-6
DOI:
https://doi.org/10.1016/j.foodchem.2019.125796
FOCH 125796
Reference:
Food Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
19 July 2019
23 October 2019
23 October 2019
Please cite this article as: Fernandez-Aulis, F., Torres, A., Sanchez-Mendoza, E., Cruz, L., Navarro-Ocana, A., New
acylated cyanidin glycosides extracted from underutilized potential sources: enzymatic synthesis, antioxidant
activity and thermostability, Food Chemistry (2019), doi: https://doi.org/10.1016/j.foodchem.2019.125796
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New acylated cyanidin glycosides extracted from underutilized potential sources: enzymatic synthesis,
antioxidant activity and thermostability.
Fernanda Fernandez-Aulisa, Andrea Torresa, Ernesto Sanchez-Mendozab, Luis Cruzc, Arturo Navarro-Ocanaa*
aDepartamento de Alimentos y Biotecnología, Facultad de Química, Universidad Nacional Autónoma de México,
04510, Mexico City, Mexico
bDepartmento de Sistemas Biológicos, Universidad Autónoma Metropolitana-Xochimilco, Mexico City, Mexico
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cREQUIMTE/LAQV, Departamento de Química e Bioquímica, Faculdade de Ciências, Universidade do Porto, Rua
do Campo Alegre, 687, 4169-007 Porto, Portugal.
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*Corresponding Author: Arturo Navarro-Ocana
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Departamento de Alimentos y Biotecnología, Facultad de Química, Universidad Nacional Autónoma de México.,
C.P 04510, Mexico City, Mexico
Tel: 52 56225346
E-mail address: arturono@unam.mx
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ABSTRACT
Interest in anthocyanins has increased remarkably in recent decades, although their wider application has been
hampered by instability problems. Thus, this study aimed at developing a strategy to gain access to more stable
anthocyanins via enzymatic esterification. For that purpose, three cyanidin derivatives were obtained from
underutilized, but easily accessible sources, and their total anthocyanin content was quantified. The purity of
cyanidins obtained ranged from 40 % to 88 % depending on their source. Subsequently, the critical enzymatic
reaction conditions were established, and the best results were found using tert-butanol as a solvent, 20 g/L of
lipase B from Candida Antarctica, and vinyl cinnamate as acyl donor at ratio 250:1 (acyl donor to anthocyanin).
Finally, five new acylated anthocyanin derivatives were synthesized with improved antioxidant activity and
thermostability, in comparison to the cyanidin-3-glucoside, which is an advantageous feature for industrial
applications.
Keywords: lipases, enzymatic esterification, dihydrocinnamic acids, acylated anthocyanin.
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1. Introduction
Anthocyanins are water-soluble pigments present in fruits, leaves, flowers, and roots. Due to their antioxidant
activity and biological properties, the research on anthocyanin applications has attracted a considerable interest
in food, nutraceutical, cosmetic, and pharmaceutical industries (Nunez & Magnuson, 2014). For example, it was
proved that they played a role in obesity prevention and cardiovascular protection. Nevertheless, the use of
anthocyanins in the industry is limited because of their instability (Yan, Li, Zhang, Liu, Ou, & Zeng, 2016) and
availability (Cruz, Fernandes, Guimarães, de Freitas, & Mateus, 2016).
Anthocyanins are known to be unstable when subjected to the changes, such as pH, temperature, exposure to
light, and are also affected by the presence of oxygen, enzymes and metals. However, it is well documented that
acylated anthocyanins show improved stability in comparison to non-acylated ones. It has been demonstrated
that acylation with aromatic acids is especially efficient (Li et al., 2013). The observed higher stability is
explained by the intramolecular copigmentation caused by π-π interactions (Trouillas, Sancho-García, De
Freitas, Gierschner, Otyepka, & Dangles, 2016). Therefore, it is important to gain access to those types of
anthocyanin derivatives from natural sources,(Gras, Bogner, Carle, & Schweiggert, 2016), or by esterification of
glycosylated anthocyanins. Esterification can be achieved chemically or enzymatically. Chemical esterification
presents certain disadvantages. It usually involves addition of bases that degrade anthocyanins, and is
characterized by poor selectivity, that is, it takes place both at glycoside and aglycone residues (Grajeda-
Iglesias, Salas, Barouh, Baréa, & Figueroa-Espinoza, 2017). On the contrary, enzyme-catalyzed esterification is
performed at soft reaction conditions and with high selectivity. In previous works, enzymatic esterification of
anthocyanins was carried out using 10 % DMSO in acetone (Sari, Setiawan, & Siswoyo, 2015),(Cruz, Benohoud,
Rayner, Mateus, Freitas & Blackburn, 2018), in pyridine (Yan et al., 2016) or in 2-methyl-2-butanol (Cruz,
Guimarães, Araújo, Évora, de Freitas & Mateus, 2017) as reaction solvents. However, those studies lacked the
solvent assessment and systematic evaluation of factors distinct from flavonoid esterification, such as water
activity, type of solvent, the ratio between acyl donor: acyl acceptor, temperature, type and concentration of acyl
acceptor, acyl donor and enzyme (Koskien & Klibanov, 1996). Factors listed above comprise the critical
conditions to perform esterification in organic media, which is often limited by the solubility of the substrates. For
that reason, in previous studies a range of solvents for flavonoid esterification were tested (hexane, acetone,
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acetonitrile, pyridine and mixture of them) with outcome greatly depending on the substrate properties (Chebil,
Humeau, Falcimaigne, Engasser, & Ghoul, 2006).
Another drawback related to anthocyanin esterification is the reaction yield. Owning to the lack of commercial
sources of these compounds, the inevitable first step involves extraction and purification of anthocyanins, so that
esterification studies could be carried out at all. Although these pigments are distributed throughout nature, it is
necessary to find their accessible sources with a simple profile and high anthocyanins concentration, which
would facilitate more specific esterification studies. Different authors have reported the extraction of malvidin-3-
glucoside from grapes; cyanidin-3-glucoside from jaboticaba, purple rice, and raspberry to prepare their
corresponding esters, the type of donor acyl that has been evaluated is mainly fatty acids which are more
liposoluble products, however, esterification with aromatic acids has been evaluated slightly. In those studies,
only four plant sources were evaluated, and two different anthocyanins were obtained. However, given the great
diversity of anthocyanins and their plant sources, it is necessary to expand the access and characterize new
structures belonging to these biologically active pigments. In Mexico, thanks to its biodiversity, a vast number of
plants can be considered as a new and accessible anthocyanin sources, such as purple corn (Zea mays)
(Deineka, Sidorov, & Deineka, 2016), tiliapo (Sideroxylon palmeri), plum (Prunus domestica)(Usenik, Stampar, &
Veberic, 2009), trueno (Ligustrum japonicum), and bottlebrush flower (Callistemon citrinus)(Goyal, Jain, Jain, &
Sharma, 2012). All of them possess high anthocyanin content and are widely distributed in Mexico. For example,
although the purple corn is used as source of anthocyanins, their use is not taken advantage in the whole plant,
for example purple corn husk is a by-product of purple corn production which is used to prepare traditional food
as atole (Chung-Ying et al., 2008); trueno and Callistemon trees grow abundantly in the streets of Mexico City
(Aldama & Grabinsky, 1994) and additionally are used in traditional medicine since they act not only as
antioxidant, antimutagenic, cytoprotective, and immunosuppressive agents (trueno)(Ngo, Lee, Nguyen, Kim,
Woo & Min, 2017), but also show antibacterial activity (bottlebrush leaves and flowers)(Cock, 2012); tiliapo is
utilized as food in its immature and mature state (González-Soberanis & Casas, 2004) though the knowledge of
its chemical composition is limited; plum is a readily available fruit known for its remarkable antioxidant
properties (Cock, 2012) together with its antibacterial and anticancer activities (El-Beltagi, El-Ansary, Mostafa,
Kamel, & Safwat, 2018).
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Therefore, the main goal of this work was to obtain novel acylated cyanidin glycoside derivatives through
enzymatic synthesis and evaluate their antioxidant activity and thermostability. In order to improve the enzymatic
reaction yields, the effects of solvent, enzyme, acyl donor, and glycosyl moiety of the cyanidin were studied.
2. Materials and methods
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2.1. Materials
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Vinyl cinnamate, cinnamic acid, solvents and commercially available immobilized lipase from Candida antarctica
(  5000 U/g, recombinant, expressed in Aspergillus niger), Amberlite XAD-7HP, Sephadex LH-20, trifluoroacetic
acid were purchased from Sigma-Aldrich. Dihydrocinnamic acid, dihydroferulic acid, and dihydrosinapic acid
were obtained by hydrogenation of cinnamic, ferulic and sinapic acid using a Parr hydrogenator and Pd/C as
catalyst.
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2.1.1. Plant material obtention.
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In the case of the tiliapo and the plum, only the husk of the ripe fruit was used, unlike the thunder in which the
whole fruit was used because this fruit is too small to remove the husk, in the case of corn, the leaves covering
the fruit (husk) were used, and finally only the colorful part of the flower was used.
One of the anthocyanins of the red cabbage was used as anthocyanin control in the antioxidant activity. The red
cabbage were extracted purified until obtained the a natural anthocyanin isolated from red cabbage was used,
which structure tentatively corresponds to cyanidin-3-sinapic-diglucoside-5-glucose (Cy-3-sin-diglc-5glc)(Mizgier,
Kucharska, Sokół- Łętowska, Kolniak-Ostek, & Kidon, 2016)
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2.2. Determination of total anthocyanin content (TAC)
The monomeric anthocyanin pigment content in the extracts from the five different plant sources was
determined using the pH differential method, as previously described(Giusti & Wrolstad, 2001). TAC was
determined spectrophotometrically using the extinction coefficient (Jing & Giusti, 2007) of 26900 L cm-1 mg-1 and
a molecular weight of 449.2 g mol-1, and was expressed as equivalents of cyanidin-3-glucoside per gram of dry
weight of the sample. The buffer system used in the assay was 0.025 M potassium chloride at pH 1.0 and 0.4 M
sodium acetate at pH 4.5. In brief, 200 µL of the extract were mixed separately with 1800 µL of potassium
chloride and sodium acetate buffer, and the absorbance at 510 and 700 nm was measured using a visible-UV
spectrometer (GBC-CINTRAL). The samples were tested in triplicate.
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2.3. Extraction of cyanidin glycosides
Anthocyanins from different plant sources were extracted following the same method, though samples differed
on their initial weight. Cyanidin-3-glucoside was obtained from 200 g of tiliapo husk (Syderoxilon palmeri) and
500 g of corn husk (Zea mays); cyanidin-3-rutinoside was obtained from 20 g of plum husk (Prunus domestica)
and 50 g of trueno fruit (Ligustrum japonicum); and cyanidin-3,5-diglucoside was obtained from 10 g bottlebrush
flower (Callistemon citrinus).
For the extraction process, test samples were suspended in the solvent mixture of methanol: water: formic acid
with volume ratio of 80:19:1, and then were sonicated for 20 min. Next, samples were filtered off and
concentrated under reduced pressure. Additionally, washings with hexane and ethyl acetate were carried out to
extract non-anthocyanin organic compounds from the sample. Following that, the sample was loaded to the
aqueous phase on Amberlite XAD-7HP resin was used to absorbed the anthocyanins in a ration of 1:10 of
sample in weight and resin. Subsequent washings with water were performed until colorless eluent was
obtained, the amount of water used was about from 1 L per 1 gram of material, however this value is variable
and depend of each sample. Then, anthocyanins were removed from Amberlite resins with methanol in a ratio
1:5 and this process was repeated three time and the layers was mixed, and excess of the solvent was
evaporated under reduced pressure till obtained a dark solid.
2.4. Purification of anthocyanins extracts
The crude extracts from different food sources were dissolved in 0.1 % TFA in methanol. Sephadex LH-20
column chromatography was performed using 0.1 % TFA aqueous solution as the mobile phase to separate the
anthocyanins. Anthocyanins fractions were concentrated under reduced pressure until dark solids were
obtained. Resulting anthocyanin trifluorate salts were weighted. All anthocyanins were identified by HPLC-MS.
Additionally, the anthocyanins extracted were confirmed by 1H-NMR and 13C-NMR.
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2.5. Evaluation of enzymatic esterification conditions
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Esterification of anthocyanins was achieved by enzymatic transformation using lipase B from Candida antarctica.
The reaction required a high-purity anthocyanin as acyl acceptor and a carbonyl group as acyl donor. In this
study, sequential experiments were performed to identify critical conditions. All reactions were carried out in a 20
mL vial, and the working volume was 3 mL. Temperature (60 ºC), agitation rate (300 rpm), and incubation time
(48 h) were maintained constant throughout the optimization process.
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Effect of solvent: three organic solvents were tested as reaction media for anthocyanin and vinyl cinnamate
substrates in lipase-catalyzed esterification. Solvents tested were dry pyridine, tert-butanol, and 2-methyl-2-
butanol. 1 mL of a test solvent was added to 5 mg of cyanidin-3-glucoside, and the solution was left stirring for
24, 48 and 72 h at 300 rpm and 60 ºC. After that time, the substrate concentrations were measured by HPLC at
520 nm taking methanol as 100% solubility.
Effect of enzyme: three lipases on immobilized support were evaluated: lipase B from Candida antarctica (≥
5000 U/g, Sigma-Aldrich), C-LECTA (2612 U/g for PNPB at pH 7, 25 °C), and lipase from Candida cylindraceae
(1257 U/g for PNPB at pH 7, 25 °C). 5 mg of cyanidin-3-glucoside and 500 mL of vinyl cinnamate were
transferred to the reaction vials containing 15 mg, 30 mg, and 60 mg of the test enzyme. Reactions were carried
out at 60 C and 300 rpm for 48 hours. After that time, the reaction was stopped by filtration, and the solvent was
evaporated under reduced pressure. Next, vinyl cinnamate was removed with hexane, and the anthocyanin and
respective ester were recovered with acidified water. Reaction products were analyzed by HPLC.
Effect of acyl donor: Three derivatives, namely vinyl cinnamate, dihydrocinnamic acid, and cinnamic acid were
examined as acyl donors at the following concentrations: 50, 100, and 250 equivalents.
Effect of glycosyl moiety. Finally, cyanidin-3-glucoside, cyanidin-3-rutinoside, and cyanidin-3,5-diglucoside were
used in esterification reactions to investigate the effect of the glycosyl moiety. The reaction conditions were as
follows: 20 g/L of the enzyme, 250 equivalents of acyl donor, 100 g/L of molecular sieves 4Å in tert-butanol at 60
ºC, 300 rpm and for 48 h.
2.6. Purification of anthocyanins esters
The monitoring of the reaction was done by HPLC, when it was observed that the concentration of the product
began to decrease, and the reaction stopped. The esterification reaction was stopped when the reaction was
filtered. The solvent was concentrated in a rotavapor and the acidified water was dissolved with HCl to later
wash with ethyl acetate and remove the excess from the acyl donor. The aqueous phase was set aside and led
to dryness, this extract contained cyanidin that did not react with its esters, to separate these compounds was
used a column silica (20 cm x 2 cm) using as hexane eluent to remove the excess pyridine, and then with ethyl
acetate: methanol: formic acid (80:10:10). The analyses of thermostability and antioxidant activity were
performed with the purified products.
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The conversion yield was calculated by HPLC. Once the reaction product was identified by HPLC-MS, the
concentration was calculated using the standard curve of the cyanidin-3-glucoside. The calculation of the yield
was made by dividing the obtained value by the expected value per 100, the expected value being the total
conversion of the cyanidin-3-glucoside.
2.7. HPLC / HPLC-MS
The reaction mixture was evaporated under reduced pressure and dissolved in acidified water. Next, extraction
with hexane was performed to remove the excess of acyl donor. The aqueous phase was further concentrated
under reduced pressure and analyzed by HPLC (Waters 2707) and HPLC-MS (Agilent 1260 Infinity Binary)
using the ODS-Hypersil Gold column (250 mm x 5.4 mm x 5 m). Detection was performed at 280 nm and 520
nm (dual UV-VIS detector, Waters 2847). The binary mobile phase consisted of solvent (A) water/acetic acid
94:6 (v:v) and (B) acetonitrile/acetic acid 94:6 (v:v); gradient elution was applied at flow rate of 0.6 mL/min: 0-25
min, from 4 % to 40 % B; 25-35 min from 40 % to 80 % B; 35-45 min from 80 % to 5 % B; 45-52 min, 5 % B (Yan
et al., 2016) and the injection volume was 10 L.
Mass spectrometry (Agilent MS QQQ) in positive ion mode was selected to record the full mass spectra (m/z 50-
1650) on an electrospray ionization ion-trap mass spectrometer (ESI-MS). The pressure of nebulizer gas (N2)
was set at 40 psi and a flow rate at 10 L/min. The capillary temperature was controlled at 300 ºC, and capillary
voltage was maintained at 4 kV.
2.8 NMR
1H-NMR (600 MHz) and 13C-NMR (150 MHz) spectra were recorded in CD3OD:CF3COOD (1 %) using an Agilent
DD2 600 with One NMR probe and TMS as an internal standard. 1H chemical shifts were assigned using 2D
NMR (COSY), and 13C chemical shifts were assigned using HSQC and HMBC. Obtained data were analyzed
and compared with the available literature on structural analysis of cyanidin-3-glucoside (Oliveira, Fernandes, &
de Freitas, 2016), cyanidin-3-rutinoside (Qin, Li, Niu, Ding, Zhang & Shang, 2010), and cyanidin-3,5-diglucoside,
moreover the recopilation of the different anthocyanins NMR spectrum were use as comparison. (Andersen &
Fossen, 2003)
Cyanidin-3-glucoside: 1H/13C-NMR chemical shifts (ppm) and coupling constants J (Hz): H-4, 9.03 s/135.4; H-6,
6.68 d /101.8; H-8, 6.91 d /93.6; H-2’, 8.07 d (2.3)/116.9; H-5’, 7.03 d (8.7)/115.9; H-6’, 8.25 dd (8.7,2.3)/126.7;
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H-1’’, 5.31 d (7.7)/102.2; H-2’’, 3.64-3.67 m/69.5; H-3’’, 3.58- 3.60 m/77.1; H-4’’, 3.56-3.59 m/76.6; H-5’’ 3.69-
3.72 m/73.2; H-6’’, 3.94 dd (12.1,2.2)/60.7.
Cyanidin-3-rutinoside: H-4, 8.95 s/137.8; H-6, 6.70 d /102.0; H-8, 6.90 d /93.7; H-2’, 8.07 d (2.3)/116.9; H-5’,
7.05 d (8.7)/116.0; H-6’, 8.25 dd (8.7,2.3)/126.9; H-1’’, 5.23 d (7.8)/101.9; H-2’’-H-5’’, 3.44-3.70 m; H-6’’a, 4.09 /
66.2, H-6’’b, 3.59 / 66.2. Ramnosyl moiety: H-1’’’, 4.96 d (1.6)/100.6; H-2’’’-H-5’’’ 3.36-3.83 m; H-6’’, 1.19 d
(6.21)/16.2.
Cyanidin-3-5-diglucoside: H-4, 9.12 s/134.5; H-6, 7.07 bs /104.3; H-8, 7.07 bs /95.9; H-2’, 8.05 d /117.1; H-5’,
7.02 d (8.8)/116.1; H-6’, 8.32 dd (8.8,2.3)/127.6; Glucosyl 1: H-1’’, 5.30 d (7.9)/102.5; H-2’’-H-5’’, 3.43-3.67 m; H-
6’’a, 4.00, H-6’’b 3.70 /60.8. Glucosyl 2: H-1’’’, 5.17 dd (7.7,3.9) /101.2; 3.39-3.70 m; H-6’’’a 3.94; H-6’’’b 3.78 m
/61.0.
2.9. Evaluation of thermostability
The thermostability of anthocyanins was evaluated in water bath at different temperatures: 40 °C, 60 °C, and 80
°C. The samples were dissolved in 0.1 M citrate buffer pH 3, and absorbance was measured periodically at 510
nm to analyze the effect of temperature on anthocyanins stability (Sari et al., 2015), the first-order reaction rate
constants (k) and half-time (t1/2) values were calculated.
2.10. Evaluation of antioxidant activity
The measurement of antioxidant activity was determined by DPPH assay (Brand-Williams, Cuvelier, & Berset,
1995). In brief, 400 μL of 0.05 mM DPPH was added to 40 μL of the extract, and the decrease in absorbance at
515 nm was recorded every 30 s during 30 min in the dark at room temperature. The antioxidant activity of test
compounds was expressed as percentage of DPPH inhibition. The assays were performed on Cary 60 UV-Vis
Agilent Technologies spectrophotometer. Solution of 1 % formic acid (v/v) in methanol/water (80:20) was used
as a negative control, and cyanidin-3-sinapic-diglucoside-5-glucose as positive control. Test compounds were
diluted in 1 % formic acid (v/v) in methanol/water (80:20) to the final concentration of 0.5 mM in accordance to
the availability of these anthocyanins after isolation process.
In order to prepare dose-response curves for the IC50 determination (concentration of compounds that reduces
half the maximum response), a set of standard solutions for each of the compound tested was prepared by serial
dilution, and the following concentrations were obtained: 0.900, 0.600, 0.300, 0.150, 0.075, 0.030 and 0.015
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mM. For the IC50 assay, 40 μL ot the test compound at different concentrations were mixed with 400 μL of 0.05
mM DPPH, and following the 30 min incubation, absorbance was measured at 515 nm.
3. Results and discussion
3.1. Extraction and characterization of cyanidin glycosides
The first part of our studies consisted of the extraction and purification of cyanidin glycosides to perform
enzymatic esterification. Table 1 shows the anthocyanin content and purity following extraction from 5 different
plant sources (measured by HPLC at 520 nm). Results indicate that purple corn husk had the highest content of
anthocyanins among the test samples. Noteworthy, it was found to be richer in anthocyanins than blueberries
when compared to the published data (1.91-4.61 mg/g D.W.)(Castrejón, Eichholz, Rohn, Kroh, & Huyskens-Keil,
2008). Purple corn husk has been known as an important source of anthocyanins, and HPLC analysis showed it
contained a mixture of six such compounds(Chung-Ying et al., 2008), and that cyanidin-3-(6’’-malonyl)glucoside
was the predominant structure. Because the main objective of this work was the esterification of cyanidin-3-
glucoside (C3G), the cyanidin-3-(6’’-malonyl)-glucoside was hydrolyzed with 1 % HCl (v/v) for 48 h. As a result,
the amount of C3G increased from 26.1 % to 64.4 % of total anthocyanin content according to the HPLC
analysis. These results also proved that corn-husk, despite having the most complex anthocyanins profile of all
samples, showed its great potential as C3G source since it could be obtained 16 mg/g of C3G from the
hydrolysis of the first extract with 24 mg/g of total anthocyanin content.
Another source of C3G was tiliapo husk which total anthocyanin content was assessed to be 2.38 mg/g , a
concentration comparable to other fruits (Feng et al., 2016). Due to the fact that anthocyanins found in this fruit
had not been reported before, the NMR spectra were compared with previous reports (Fernandes et al., 2014) to
confirm the presence of C3G in tiliapo fruit. Since C3G extracted from tiliapo husk had higher purity than from
corn husk (88 % versus 64 %, respectively), the tiliapo was considered a better source of C3G for this study as it
provided more controlled reaction medium.
The second test compound, cyanidin-3-rutinoside (C3R), was obtained from trueno fruit and plum husk at 1.98
mg/g and 12.46 mg/g, respectively, of total anthocyanin content. Trueno fruit contained only one type of
anthocyanin while plum husk had four anthocyanins structures, namely cyanidin-3-glucoside, cyanidin-3-
xyloside, cyanidin-3-rutinoside, and peonidin-3-rutinoside (Usenik et al., 2009). The HPLC chromatogram
obtained for trueno fruit matched with the elution time of C3R obtained from the plum husk. Additionally, the
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HPLC-MS analysis proved the presence of a molecular ion of m/z 595 corresponding to cyanidin-3-rutinoside
(Slimestad, Vangdal, & Brede, 2009). C3R from plum husk was used for the propose of this study, owing to the
higher purity and greater total anthocyanin content in comparison to trueno fruit. However, trueno fruit remains
an excellent source of C3R in terms of its availability as it is hardly consumed as a fruit.
Finally, cyanidin-3,5-diglucoside (C35G) was obtained from bottlebrush flowers, and their anthocyanin content
stood at 6.15 mg/g and purity of 67.6 %. Molecular ion of m/z 611 and its MS2 fragmentation to m/z 287 in
positive ion mode confirmed the presence of C35G (Barnes & Schug, 2011); and demonstrated by NMR
analysis. Overall, three cyanidin derivatives were obtained to evaluate the effect of glycosyl moiety on enzymatic
esterification.
3.2. Evaluation of enzymatic esterification conditions
In this work, lipases were used to catalyze reactions between cyanidin glycosides and different aromatic acyl
donors (Figure 1). All reactions were carried out at 60 ºC and 300 rpm for 48 h as usually reported in previous
works (Cruz et al., 2017). The progress of individual reactions was monitored by HPLC, and the formation of new
peaks was detected; anthocyanins at 520 nm and all other phenolic compounds at 325 nm. Figure S1 shows
the chromatogram of the reaction between C3G and dihydrocinnamic acids (dihydrocinnamic and dihydroferulic
acids), in which the retention time of the esters was detected at 25.2 min and 20.4 min, respectively. The
conversion of the esterification was calculated from the area of the reaction product.
3.2.1. Effect of solvent
The solubility of the substrates in the reaction media is a fundamental requirement to perform any chemical
reaction; therefore, in this investigation it was necessary to make an analysis of solvents that were compatible
with each of the substrates participating in the esterification reactions, since anthocyanins are soluble in
aqueous solvents while esterification reactions with lipases are favored in organic solvents and with low aqueous
activities, so it was also necessary to guarantee the removal of water from the medium through molecular
sieves. Table 2, last column, shows the conversions obtained for different reaction conditions tested in this
study, where the importance of the reaction solvent stands out, with tert-butanol with the molecular mesh being
the solvent where a higher conversion of anthocyanin ester was achieved, followed by pyridine and 2M2B. This
is due to the solubility of the substrates in the reaction medium, mainly of the anthocyanins since in the C3G
solubility data described in Figure S2 it is shown that C3G has a greater zero time solubility with pyridine,
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however, when exposed to longer times the C3G begins to degrade. On the contrary with tert-butanol the
solubility of anthocyanin at zero time is less than 40 % and when the agitation time increases the solubility
reaching more than 60 % solubility at 24 hours and even at 72 hours the substrate degradation wasn't detected
in this organic solvent; while with 2M2B the solubility overtime does not exceed 40 %. Therefore, to ensure
greater efficiency of the transformation of anthocyanin to esterified anthocyanin, it must first be ensured that the
products are soluble in the reaction medium and do not degrade.
3.2.2. Effect of the enzyme.
As it was mentioned before, the enzymatic esterification with lipases represented a challenge that needed to be
addressed. In the next step, the effect of different lipases and enzyme concentrations were evaluated.
Therefore, three commercial enzymes were studied: two Candida antarctica lipases and Candida cylindracea
lipase. According to Table 2, seventh column, the Candida antarctica from SIGMA-ALDRICH performed better
than Candida cylindracea.
The results showed in Table 2, eighth column, indicate that enzyme concentration of 10 g/L or 20 g/L had no
effect on the conversion value. However, it was observed a lower conversion when the enzyme concentration
was reduced to 5 g/L. In consequence, a higher enzyme concentration improved the conversion.
3.2.3. Effect of acyl donor
Another parameter studied was the type of acyl donor. The following compounds were included in the study:
vinyl ester (vinyl cinnamate VC), α-β unsaturated carboxylic acid (cinnamic acid CA) and α-β saturated
carboxylic acids (dihydrocinnamic acid DHC, dihydroferuloyl acid DHF, dihydrosinapic acid DHS). The results
showed in Table 2, fifth column, indicate that the usage of vinyl ester improved enzymatic conversion by shifting
the equilibrium of the reaction towards the product formation because of the acetaldehyde evaporation. On the
other hand, the addition of molecular sieves to the reaction media had the same effect when CA and DHC were
used as acyl donors, but the conversion was lower in comparison to the reaction with VC. For α-β unsaturated
and α-β saturated carboxylic acids as acyl donors, it was found that DHC gave higher conversion than CA,
which is in accordance with the results reported (Cassani, Luna, Navarro, & Castillo, 2007). Additionally, the
concentration of acyl donor was evaluated, using an excess of VC: 50, 100, and 250 equivalents. The
conversion was measured after 48 h, and the best results were obtained when 250 eq. of VC was used. For 100
eq of acyl donor, the reaction also occurred although with lower conversion yields, but no progress was
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observed when the concentration was below 50 eq. The results obtained are in agreement with previous reports
on aromatic esterification, where a large excess of acyl donor (Enaud, Humeau, Piffaut, & Girardin, 2004) or
modest excess(Stevenson, Wibisono, Jensen, Stanley, & Cooney, 2006) was used to perform the reaction. To
sum up, enzymatic esterification of cyanidin-3-glucoside required a huge excess of acyl donor, which was the
critical factor in the lipase catalyzed reaction.
3.2.4. Effect of anthocyanin glycoside.
Once the critical factors of the enzymatic esterification of cyanidin-3-glucoside were established, it was decided
to expand the study towards other cyanidin glycosides, such as cyanidin-3,5-diglucoside that contains two
glucoses, one in position 3 and the other in position 5; and cyanidin-3-rutinoside that contains disaccharide in
the position 3 (6-O-α-L-rhamnosyl-D-glucose). The results obtained (+) indicated that the esterification of
cyanidin-3,5-diglucoside had the highest conversion, followed by cyanidin-3-glucoside. The lowest conversion
was obtained for cyanidin-3-rutinoside as it lacks the primary alcohol group and therefore, it exhibits less
reactivity. Oppositely, the success on the esterification of cyanidin-3,5-diglucoside could be explained by the
presence of two primary alcohol groups, although the esterification at both glucosides was not detected.
Finally, Table 3 summarized the HPLC results obtained for the extracted cyanidin-3-glucoside and its esters
together with the results of MS analysis. The molecular ion [M]+ of the chromatographic peak products
corresponds to the respective cyanidin-3-(6’’-dihydrocinnamoyl)glucoside, cyanidin-3-(6’’-cinnamoyl)glucoside,
cyanidin-3-(4’’’-cinnamoyl)rutinoside and cyanidin-3-(6’’-cinnamoyl)glucoside,5-glucoside and MS2 fragments
indicate that esterification occurred only on the glycoside moiety.
3.3. Structural elucidation by NMR.
Five acylated anthocyanin products were isolated by silica gel column using a mixture of ethyl acetate : methanol
: formic acid (80:10:10) as an eluent. Chemical structure of the novel products was determined by means of 1D
and 2D NMR techniques (Table S1 ) (Cruz et al., 2018), ( Pérignon, Lecomte, Pina, Renault, Simonneau-Deve,
& Villeneuve, 2013),(Moloney, Robbins, Collins, Kondo, Yoshida, & Dangles, 2018). The acylation took place at
the position 6’’-OH of glucose moieties, according to the observed downfield shifts of the corresponding chemical
shifts: 3.9 to 4.5 ppm in the 1H spectrum, and from 60 to 63 ppm in the 13C spectrum and also confirmed by the
HMBC spectrum. The NMR spectra of cyanidin-3,5-diglucoside cinnamate ester showed that acylation occurred
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preferentially on the primary alcohol of the glucose in the position 3, according to the observed downfield shift
from 3.70 to 4.5ppm.
These resonance spectra also show us the interactions of stacking π-π between the phenolic nuclei (Trouillas,
2016) in the case of compounds C5 and C8 the chemical displacements of hydrogen corresponding to H6, H4
and H8 of the aglycone move to a low field compared to native anthocyanin (C3G) while in compounds C4, C7
and C9 the chemical shifts of hydrogen corresponding to H6, H4 and H8 of the aglycone move to a high field
compared to native anthocyanin (C3G) . It should be noted that this effect is more noticeable in C9 (anthocyanin
acylated with DHS), so it can be suggested that in this compound the interactions between both phenolic nuclei
are closer than in the rest of the esterified anthocyanins.
3.4. Thermostability evaluation.
Thermostability of C3G and its corresponding purified esters was evaluated at 40, 60, and 85 °C. As shown in
Table 4, the kinetic rate constant (k) and half-life parameter indicated that cinnamoyl, dihydroferuloyl and
dihydrosinapoyl cyanidin esters (4, 8 and 9) demonstrated improved stability than C3G, with the most
pronounced effect observed at 85 °C. However, in case of dihydrocinnamoyl cyanidin ester (7), the kinetic rate
constant (k) was higher than C3G but showed shorter half-life value in comparison to C3G. The obtained data
suggest that both the double bond presented in the cinnamic acids and the substituents in the ring are
responsible for copigmentation and therefore the thermostability. Till present, no studies on the evaluation of the
thermostability of the anthocyanin esters with dihydrocinnamic acids have been reported, however, previous
reports demonstrated that the esterification improves the thermostability (Yang, Kortesniemi, Ma, Zheng, &
Yang, 2019)(Sari et al., 2015). To sum up, compound 9 that corresponds to dihydrosinapoyl cyanidin ester has
improved considerably the thermostability of C3G, becoming an option to stabilize this most abundant
anthocyanin.
3.5. Antioxidant activity
The radical scavenging activity of the purified cyanidin-3-glucoside acylated esters was evaluated. Figure S3
shows that compound 9 and dihydrosinapic acid had the highest inhibition potential, followed by the red cabbage
anthocyanin which exhibited similar percentage of DPPH inhibition to that presented by the compound 4.
Compound 8 showed 30 % less antioxidant activity than compound 1 and dihydroferulic acid. In Table 5, the
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IC50 values of the test compounds were compiled, and the results obtained suggest that the antioxidant activity
depends on the acyl donor linked to the cyanidin-3-glucoside.
Furthermore, compound 9 had the highest radical scavenging activity, exceeding even red cabbage anthocyanin
which possess a sinapic acid moiety and more than one of glycosyl moieties. On the other hand, the
esterification of the cyanidin-3-glucoside with the dihydrocinnamic acid (compound 7) reduced the antioxidant
activity of the cyanidin-3-glucoside. This effect could be explained by the rotation of the dihydrocinnamic moiety
that may interfere with access to DPPH due to steric hindrance (Lue, Skall, Jacobsen, Hellgren, Guo, & Xu,
2010). These results showed that instauration in the acyl moiety has an important role in that antioxidant activity,
and the esterification improved the antioxidant activity in comparison with the cyanidin-3-glucoside.
4. Conclusions
In summary, cyanidin-3-glucoside, cyanidin-3-rutinoside, and cyanidin-3,5-diglucoside were successfully
extracted from underutilized plant sources, purified and identified by HPLC-MS. Furthermore, cyanidin-3-
glucoside was characterized in tiliapo (Sideroxylon palmeri) and cyanidin-3-rutinoside in trueno (Ligustrum
japonicum) fruits. All cyanidin glycosides were enzymatically esterified after establishing the best reaction
conditions. Optimal reaction conditions involved tert-butanol as reaction media, 20 g/L of Candida Antarctica,
and 250 equivalents of vinyl cinnamate as acyl donor which was found to be a critical parameter. Under those
conditions, five new acylated anthocyanins were synthesized, purified and characterized: cyanidin-3-(4’’’-
cinnamoyl)rutinoside (6), cyanidin-3,5-(6’’-cinnamoyl)diglucoside (5), cyanidin -3-(6’’-dihydrocinnamoyl)glucoside
(7), cyanidin-3-(6’’-dihydroferuloyl)glucoside (8), and cyanidin-3-(6’’-dihydrosinapoyl)glucoside (9). Overall, it was
also demonstrated that cyanidin-3-(6’’-dihydroferuloyl)glucoside (8) and cyanidin-3-(6’’-dihydrosinapoyl)glucoside
(9) obtained by enzymatic esterification exhibited better thermostability than cyanidin-3-glucoside, and its
antioxidant activity was improved with dihydrosinapoyl and cinnamoyl residues. This work provided a systematic
study for the preparation of a series of acylated anthocyanins from underutilized sources. In further work, the
stability of these compounds at different pH will be determined.
Notes
The authors declare no competing financial interest
Acknowledgments
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This work was supported by CONACYT (CB-2012/180128) and PAPIIT-IT (202318), and CONACYT fellowship
number 273829 awarded to the PhD student Fernanda Fernandez Aulis.
Authors thank Margarita Guzmán for technical assistance in HPLC-MS experiments during the course of this
research work and Dr. Rafael Castillo (L-122) for his assistance in the use of the Parr hydrogenator.
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FIGURE CAPTIONS
Figure 1. Esterification reactions of cyanidin glycosides
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Table 1. Total anthocyanin content and purity of cyanidin glycosides from different plant sources.
Source
Scientific name
Total
Anthocyanin
Cyanidin
obtained
(mg)
120
Purity
(%)
anthocyanins
(mg/g D.W.)
24.87±1.03
2.38±0.05
Purple corn-husk
Tiliapo
Zea mays
Cyanidin-3-glucoside
Cyanidin-3-glucoside
Cyanidin-3-rutinoside
Cyanidin-3-rutinoside
Cyanidin-3,5-diglucoside
64.4
88.6
40.0
83.1
67.6
Syderoxylon palmeri
Ligustrum japonicum
Prunus domestica
Callistemon citrinus
60
Trueno
1.98±0.57
20
Plum husk
Bottlebrush
flower
12.46±1.58
6.15±0.39
16
13
512
23

513
Table 2. Reaction conditions of enzymatic esterification of cyanidin glycosides.
Reaction
Solvent
Cyanidin
Cyanidin
Acyl
donor
VC
Concentration
AD (eq.)
250
Lipase
Concentration
Molecular
sieves 4A
100
0
Conversion
(%)
(mg)
5
5
(g(/L)
20
20
20
20
20
20
10
5
1
2
Pyridine
Pyridine
C3G
C3G
C3G
C3G
C3G
C3G
C3G
C3G
C3G
C3G
C3G
C3G
C3G
C3G
C3G
C3G
C3G
C3G
C3G
C3G
C3G
C3G
C3G
C3G
C3R
C35D
CAS
CAS
70.3
54.2
59.5
36.4
74.1
38.1
59.9
21.3
59.9
49.1
5.3
VC
250
3
4
2M2B
2M2B
5
5
VC
VC
250
250
CAS
CAS
100
0
5
6
7
8
Tert-butanol
Tert-butanol
Tert-butanol
Tert-butanol
Tert-butanol
Tert-butanol
Tert-butanol
Tert-butanol
Tert-butanol
Tert-butanol
Tert-butanol
Tert-butanol
Tert-butanol
Tert-butanol
Tert-butanol
Tert-butanol
Tert-butanol
Tert-butanol
Tert-butanol
Tert-butanol
Tert-butanol
Tert-butanol
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
VC
VC
VC
VC
VC
VC
VC
VC
VC
VC
VC
VC
DHC
DHC
DHC
DHF
DHS
CA
250
250
250
250
250
250
250
250
250
250
100
50
250
100
50
250
CAS
CAS
CAS
CAS
CLECTA
CLECTA
CLECTA
CYL
CYL
CYL
CAS
CAS
CAS
CAS
CAS
CAS
CAS
CAS
100
0
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
9
20
10
5
20
10
5
20
20
20
20
20
20
20
20
20
20
20
20
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
21.3
9.4
1.8
41.7
24.4
67.3
20.6
1.1
45.0
21.8
18.1
0
5
5
5
5
5
5
250
250
100
50
250
250
CA
CA
VC
VC
CAS
CAS
CAS
CAS
0
45.5
85.7
C3G, cyanidin-3-glucoside; C3R, cyanidin-3-rutinoside; C35D, cyanidin-3,5-diglucoside; VC, vinyl cinnamate; CA, cinnamic acid; DHC, dihydrocinnamic acid;
DHF dihydroferuloyl acid, DHS dihydrosinapoyl acid, CAS, lipase of Candida antartica from SIGMA-ALDRICH, CLECTA lipase of Candida antartica from
C-lecta industry, CYL, lipase of Candida cylindracea
Reaction condition: 300 rpm, 60°C, 48 h.
514
24

515
Table 3. HPLC-mass spectrometry and percentage of conversion.
Retention
Molecular
Ion
Compound
time
(min)
11.6
13.6
9.9
MS2
[M]+ (m/z)
(1) Cyanidin-3-glucoside
(2) Cyanidin-3-rutinoside
449
287
287
595
(3) Cyanidin-3,5-diglucoside
611
449, 287
287
(4) Cyanidin-3-(6’’-cinnamoyl)glucoside
(5) Cyanidin-3-(6’’-cinnamoyl)glucoside-5-glucoside
(6) Cyanidin-3-(4’’-cinnamoyl)rutinoside
(7) Cyanidin-3-(6’’-dihydrocinnamoyl)glucoside
(8) Cyanidin-3-(6’’-dihydroferuloyl)glucoside
(9) Cyanidin-3-(6’’-dihydrosinapoyl)glucoside
24.8
23.8
24.1
25.6
20.4
16.6
579
741
449, 287
287
725
581
287
627
287
657
287
516
25

517
518
Table 4. Kinetic parameters for the thermal degradation of acylated anthocyanins obtained by enzymatic
esterification
Compound
1
Temperature (°C)
k (h)
t 1/2(s)
24.3
10.5
3.6
57.0
52.0
12.3
14.8
4.0
40
60
85
40
60
85
40
60
85
40
60
85
40
60
85
40
60
85
0.0285 (0.9544)
0.0657 (0.9820)
0.1909 (0.9985)
0.0121 (0.9540)
0.0133 (0.9773)
0.0563 (0.9365)
0.0468 (0.9985)
0.1751 (0.9883)
0.4947 (0.9665)
0.0228 (0.9087)
0.0512 (0.8876)
0.1391 (0.9678)
0.0321 (0.9143)
0.0508 (0.9639)
0.0728 (0.9939)
0.0224 (0.8148)
0.0712 (0.8070)
0.1657 (0.9562)
4
7
8
9
1.4
30.4
13.5
5.0
22.2
13.6
9.5
30.9
9.7
4.2
Red cabbage
anthocyanin
Numbers in parentheses are the correlation coefficients
519
520
26

521
522
523
524
525
526
527
528
529
530
531
Table 5. Comparison of the antioxidant activity (IC50) of the anthocyanins and acyl donors.
Compound
IC50 (mM)
0.265 ±0.019
0.119 ±0.008
N/A
1
4
7
8
0.909 ± 0.127
0.027 ± 0.003
0.564 ±0.073
0.151 ± 0.013
N/A
9
Dihydroferulic acid
Dihydrosinapic acid
Dihydrocinnamic acid
Cinnamic acid
Red cabbage anthocyanin
N/A
0.036 ± 0.004
N/A: The compound does not show antioxidant activity
27

532
Figure 1.
533
534
535
536
537
538
539
 Evaluation of five underutilized plants to obtain sources of cyanidin glycosides
 Reaction conditions of enzymatic esterification to anthocyanins for the food industry
 Synthesis of four new anthocyanins derived from dihydrocinnamic acids
 Development of cyanidin esters with more thermostability and antioxidant activity
540
541
542
Declaration of interests
543
544
545
546
547
548
549
☒ The authors declare that they have no known competing financial interests or personal
relationships that could have appeared to influence the work reported in this paper.
☐The authors declare the following financial interests/personal relationships which may be considered as
potential competing interests:
28

550
551
552
553
554
29