Current Research in Biotechnology 4 (2022) 47–57

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Current Research in Biotechnology

journal homepage: www.elsevier.com/locate/crbiot

Effects of salicylic acid on the production of polyphenols and the reducing

power of Theobroma cacao calli
Lillien Fajardo Rosabal a,⇑, Yans Guardia Puebla b, Suyén Rodríguez Pérez c, Ursula M. Rosabal Cordoví d,
Juan J. Silva Pupo a, Stefaan P.O. Werbrouck e
a
Plant Biotechnology Studies Center (CEBVEG) University of Granma. Carretera a Manzanillo km 17, 5 Peralejo, Bayamo, Granma, Cuba. CP 85100
b
Applied Chemistry Studies Center (CEQA) University of Granma. Carretera a Manzanillo km 17, 5 Peralejo, Bayamo, Granma, Cuba. CP 85100
c
LABEX, Molecular Immunology Center, Calle 23 y Carretera del Caney s/n, Santiago de Cuba, Cuba
d
Oral Liquids Pharmaceutical Laboratory (MEDILIP) Granma. Carretera Central vía Santiago de Cuba kilómetro 845, Bayamo, Granma, Cuba
e
Laboratory of Applied in Vitro Plant Biotechnology, Department of Applied Bioscience, Faculty of Bioscience Engineering, Ghent University, Ghent, Belgium

A R T I C L E I N F O A B S T R A C T

Keywords: Cocoa (Theobroma cacao) contains high levels of phenolic compounds with pharmacologically interesting activ-
Theobroma cacao ities. Prior research has not sufficiently investigated obtaining these compounds through in vitro culture by
Salicylic acid using metabolic elicitation and quantifying polyphenols in undifferentiated cell biomass samples in this spe-
Callus cies. In vitro calli obtained from staminodes and petals were elicited with 0.01 and 0.1 mg L−1 salicylic acid
Polyphenols production
(SA). Secondary metabolites were determined in the calli and their culture medium, from the immature and
Reducing power
mature flowers, leaves, and fruits of field‐grown plants. The most abundant compounds were free amino acids,
alkaloids, reducing carbohydrates, phenols, pyrocatecholic tannins, and flavonoids. Resins, triterpenes, ster-
oids, saponins, coumarins, and quinones were absent in the calli. A high concentration of total polyphenols
(58.4 µg g−1) was obtained from staminode‐derived calli elicited with 0.1 mg L−1 SA. With these conditions,
the total polyphenol content was 40.22 µg g−1 greater than that in the control. In general, SA also stimulated
the reducing power activity; a better value (5.87 µmol mL−1 Fe2+) was obtained in callus extracts from petals
with salicylic acid (0.1 mg L−1). This research was based on a full factorial study design with two levels, a
method that allows the desired information to be obtained at the lowest possible cost.

Introduction
Two main methods are currently used to obtain complex secondary
metabolites: direct extraction from plant material or in vitro from plant,
organ and cell cultures. The first method can lead to overexploitation
of natural resources, thus negatively affecting natural biodiversity. In
addition, the content of secondary metabolites can vary according to
environmental factors, including temperature, drought, salinity, and
soil variability. In contrast, the production of secondary metabolites
in cultures in vitro has the following advantages:
(a) independence of biotic and abiotic environmental factors; (b)
higher yield of specific secondary metabolites; (c) increased productiv-
ity through automated process control; (d) uniform and constant qual-
ity; (e) potential for biosynthetic pathway engineering enabling the
redesigning of secondary metabolites; and (f) a more efficient extrac-
tion process (Tripathi et al., 2019). Consequently, the culture of plant
tissues is a viable alternative for the production of secondary metabo-
lites, provided that a suitable elicitor is found that allows for higher
concentrations of these compounds to be obtained (Isah et al., 2018;
Tripathi et al., 2019).
One of the most commonly used elicitors is salicylic acid (SA)
(Patel and Krishnamurthy, 2013). SA initiates the expression of genes
associated with pathogenesis and the synthesis of defensive com-
pounds by activating signals of basal resistance, hypersensitivity, and
systemic acquired resistance (Basit et al., 2017).
Cacao (Theobroma cacao L.) provides raw materials not only for
chocolate, but also for cosmetics and medicines. The plant contains
active substances for the treatment of cardiovascular diseases and
some cancers, and has been demonstrated to have antibacterial activ-
ity (Negaresh and Marín, 2013). Furthermore, the polyphenols present
in the plant have antibiotic and pesticidal effects (Rangel et al., 2010).
According to Duke et al. (2003), these polyphenols include flavonoids,
such as catechins (37%), anthocyanins (4%), and procyanidins (58%),
which also have potent antioxidant effects (Baharum et al., 2016).

⇑ Corresponding author.
E-mail address: lfajardor@udg.co.cu (L. Fajardo Rosabal).

https://doi.org/10.1016/j.crbiot.2021.12.005
Received 9 September 2021; Revised 24 December 2021; Accepted 27 December 2021

2590-2628/© 2022 The Authors. Published by Elsevier B.V.

This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
L. Fajardo Rosabal et al. Current Research in Biotechnology 4 (2022) 47–57

Several factors should be optimized to boost secondary metabolite Table 1

production by in vitro cultures, such as the selection of mother plants Phytochemical screening tests for plant aerial organs and calli.
with high content of the desired substances, growth medium composi- Extract Test Compounds References
tion, acidity, inoculum density, and the culture room conditions (Isah
Ethereal Dragendorff Alkaloids Jones and Kinghorn (2006)
et al., 2018).
extract Mayer
Determination of the culture medium composition is a crucial step Baljet Coumarins Jones and Kinghorn (2006)
that should account for the physiological requirements of the particu- Ethanolic Resins Resins Mir et al. (2013); Al-Kaf et al.(2019)
lar species (Isah et al., 2018). Manipulation of growing conditions is a extract Liebermann- Triterpenes Jones and Kinghorn (2006)
fundamental approach for culture optimization productivity (Murthy Burchard and/or
steroids
et al., 2014; Ochoa‐Villarreal et al., 2016). Foam Saponins Jones and Kinghorn (2006)
To obtain the desired information at the lowest possible cost, this Ninhydrin Free amino Somkuwar and Kamble (2013);
research was based on a two‐level full factorial design (Fukuda acids Yadav et al.(2017)
et al., 2018). The study aim was to examine the individual effects Dragendorff Alkaloids Jones and Kinghorn (2006)
Mayer
and interactions between several factors involved in one or more
Baljet Coumarins Jones and Kinghorn (2006)
responses of interest (Gottipati and Mishra, 2010). Fehling Reducing Sanmugarajah et al.(2013); Banu
Cacao tissue culture research has focused on somatic embryogene- carbohydrates and Cathrine (2015); Shalavadi
sis (Maximova et al. 2002). Research to obtain polyphenols through et al.(2018)
in vitro culture of T. cacao flower explants has been performed in calli FeCl3 Phenols and/ Jones and Kinghorn (2006)
or tannins
with embryogenic structures in studies including Alemanno et al.
Borntrager Quinones Ganatra et al.(2012); Longbap et al.
(2003) and Gallego et al. (2016). However, no studies have been (2018)
reported in T. cacao undifferentiated cell masses with metabolic elici- Shinoda Flavonoids Jones and Kinghorn (2006)
tation to quantify polyphenols and evaluate their response to reducing Aqueous Foam Saponins Jones and Kinghorn (2006)
extract Fehling Reducing Sanmugarajah et al.,(2013); Banu
power.
carbohydrates and Cathrine (2015); Shalavadi
The aims of this study were: (i) to analyze the secondary metabo- et al. (2018)
lites produced by calli that were initiated on staminodes and petals, Shinoda Flavonoids Jones and Kinghorn (2006)
(ii) to identify and quantify the total content of polyphenols produced FeCl3 Phenols and/ Jones and Kinghorn (2006)
in the callus and medium, and (iii) to evaluate their antioxidant activ- or tannins
Dragendorff Alkaloids Jones and Kinghorn (2006)
ity according to the reducing power with and without metabolic
Mayer
elicitation.

Materials and methods
As part of this research, the effects of the addition of SA, chitosan,
and jasmonic acid at different concentrations on the callus formation
and differentiation of T. cacao (UF‐613; UF‐650 and Pound‐7) clones
were analyzed. The biological response with SA was superior in the
three clones (unpublished data). Therefore, subsequent research used
this elicitor and indicated that the best response in callus formation
was achieved with the UF‐650 clone and SA at 15 days of culture. Con-
sequently, a higher cell biomass was obtained in less time (Fajardo
et al., 2015).
For these reasons, the T. cacao UF‐650 clone and SA as the meta-
bolic elicitor were chosen for the present research.
Extraction and phytochemical screening
Plant materials from the T. cacao UF‐650 clone were collected at
the Tercer Frente Experimental Station at Santiago de Cuba. The col-
lection time window was between 9:00 and 10:00 a.m. at 25–26 °C.
Mature and immature flowers, and leaf and fruit samples were sub-
jected to extraction. The seeds were removed from the cocoa pods,
and the pericarp was separated. The materials were dried in a recircu-
lating air oven (model WSU 400, Germany) at a constant temperature
of 60 °C for 3 days, then ground into powder with an electric mill.
The main groups of metabolites present in the plant organs and in
the calli produced from them were identified (Table 1). Extraction was
performed with solvents of increasing polarity. To 5.0 g of powder,
15 mL of ethyl ether was added. After 72 h, the extracts were filtered
through Whatman No. 1 paper. Then 15 mL of 82% (v/v) ethanol was
then added to the remaining sample, which was macerated for another
72 h and then filtered. The third extraction was performed with 15 mL
distilled water (Banu and Cathrine, 2015). The procedure was
repeated for staminodes and petal calli. Table 1 summarizes the anal-
yses performed with these extracts.
In vitro culture
Flower buds were washed with 1.0 % detergent solution for 20 min
and rinsed three times with sterile distilled water before external ster-
ilization with 1.0 % sodium hypochlorite for 20 min and another four
rinses with sterile distilled water. The petals and staminodes were iso-
lated and transferred to Murashige and Skoog (1962) basal salt med-
ium supplemented with 100 mg L−1 myo‐inositol, 10 mg L−1 LB
vitamins (Lopez‐ Baez et al., 1993), 3 mg L−1 glycine, 40 g L−1
sucrose, 0.25 mg L−1 2,4‐D, 2 mg L−1 kinetin, and 6 g L−1 agar (pH
5.7). The culture medium was divided into three equal parts in
250 mL Erlenmeyer flasks (one flask per treatment). After autoclaving,
0; 0.01 or 0.1 mg L−1 SA was added and homogenized in an Erlen-
meyer flask, each with a separate treatment. Ten milliliters of medium
was dispensed into sterilized culture flasks (5.0 cm high and 2.5 cm
wide), with 25 flasks per treatment. In each flask, five staminodes or
petals were transferred. The explants were kept for 60 days in the dark
at 25° C. Fifteen random calli were subcultured in 100 mL conical
flasks (three flasks per treatment) containing 10 mL liquid medium
that were shaken at 110 rpm. The composition of the liquid medium
was the same as that of the semi‐solid medium. The calli grew in total
darkness at 27 ± 1 °C. After 7 days of callus fragmentation, 60 μL was
sampled for microscopic evaluation.
Quantification of total polyphenols
Total polyphenols were quantified by UV/vis spectrophotometry
(Jenway 6305) with Folin‐Ciocalteu reagent (Ainsworth and
Gillespie, 2007). A calibration curve was established with a pyrocate-
chol standard p.a. (Sigma‐Aldrich) in the concentration range of
0–50 µg mL−1. The following solutions were used: 0.7 M sodium car-
bonate solution (CaCO3), pyrocatechol stock solution 500 mg L−1 (in
acetone/water solution 1:1 v/v), and pyrocatechol working solution
50 mg L−1 (10 mL of pyrocatechol stock solution diluted to 100 mL
with acetone/water mixture, 1:1 v/v).

48
L. Fajardo Rosabal et al.
To 1 g of callus, 10 mL of acetone/water (1: 1 v/v) was added. After
48 h of maceration, the extract obtained was filtered over a Millipore
filter (0.45 µm). To 1.0 mL of filtrate, 2.0 mL of Folin‐Ciocalteu (Pan-
reac®) reagent was added. After 2 min, 8.0 mL of sodium carbonate
(0.7 M) was added, and after 30 min, color formation was detected
at λ = 760 nm. The content of phenolic compounds (µg pyrocatechol
per g sample) was determined with the following equation:
Cex  Vex
CPC ¼  1000
Pm
where Pm is the weight of the sample; Vex is the volume of the extract,
and Cex is the concentration in the extract.
Antioxidant activity determined by reducing power
Reducing power was determined according to the method of
Oyaizu (1986), in which the samples of extracts with high concentra-
tions of total polyphenols were chosen. Different concentrations of
each sample (100, 200, 300, 400, and 500 μg mL−1) were used and
mixed with 1.0 mL deionized water, 2.5 mL phosphate buffer
(0.2 M, pH 6.6), and 2.5 mL potassium ferricyanide, K3[Fe (CN)6]
(1.0 % m/v). The mixture was incubated at 50 °C for 20 min, after
the addition of 2.5 mL of trichloroacetic acid (10 % m/v). Then the
samples were centrifuged at 3000 rpm for 10 min. To 2.5 mL of super-
natant, an equal volume of distilled water and 0.5 mL of 0.1 % m/v
ferric trichloride (FeCl3) was added. The absorbance was measured
at λ = 700 nm. An increase in absorbance values of the samples indi-
cated an increase in reducing power. For reducing power quantifica-
tion, a standard curve of Fe2+ (1.0–10.0 µmol mL−1) was used, and
the results are expressed in μmol mL−1 of Fe2+.
Experimental design
The 2k factorial designs (two‐level test for each k factor) is an
experimental design that has been valuable in industry and research
because of its efficiency and versatility. In general, two‐level factorial
designs, whether full or fractional factorial designs, are the set of
designs with the greatest impact on chemical compounds production
at industrial scale, design projection, and diagnostic checking of them
(Montgomery, 2013). Among them, 23 full factorial designs can be
used to study three experimental factors with two levels for each fac-
tor. The experimental matrix of this design can be easily constructed
because it consists of 23 = 2 × 2 × 2 = 8 different combinations,
and it enables studying the three main effects, as well as three double
interactions and a triple interaction of the factors analyzed (Fukuda
et al., 2018).
To evaluate the effects of the SA concentration (X1), explant type
(X2), and extract type (X3) on the production of total polyphenols,
we designed a 23 factorial experiment, involving two levels (low
(−1) and high (+1) of each variable (Table 2).
To validate the hypothesis of the equality of the levels in the eval-
uated factors, we used analysis of variance (ANOVA) with respect to
the average value of the response variable. Regression methods are
often used to analyze data from experiments that were not designed,
such as in studies of uncontrolled phenomena or historical records.
However, regression analysis is also useful in designed experiments.
Table 2
Coded and uncoded levels chosen for the 23 factorial design.
Factors Low level
Coded value
Concentration of SA (X1) −1
Explant type (X2) −1
Extract type (X3) −1
Current Research in Biotechnology 4 (2022) 47–57
Generally, the ANOVA in a designed experiment helps determine
which factors are important but uses the regression method to build
a quantitative model that relates the important factors to the response
(Fukuda et al., 2018).
The experimental results were fitted by a multiple linear regression
according to the following mathematical model:
Y ¼ R0 þ R1 X1 þ R2 X2 þ R3 X3 þ R4 X1 X2 þ R5 X1 X3 þ R6 X2 X3
þ R7 X1 X2 X3 :
where Y is the predicted response, R0 is a constant, and R1 to R7 are the
linear coefficients of the regression model. Coefficients R0, R1,…, R7,
which are usually unknown, were subsequently estimated by regression
analysis. Eight experiments were sufficient to estimate the coefficients
of the multiple regression model. The experiments were performed in
triplicate.
To estimate the antioxidant activity, we used linear regression to
calculate the relationship between the absorbance measured at
700 nm and the concentrations of total phenols in the callus extracts
derived from petals and staminodes, with and without elicitation. Nine
different regression models were evaluated to determine the influence
of the extract concentration measured after reduction with potassium
dichromate (K2Cr2O7) (Table 3). Several input variables were tested to
obtain the highest correlation between the experimental data and pre-
dicted values, and to investigate their effects on the target values.
Hence, t‐ratios and the corresponding p‐values were determined for
better evaluation of the significance of the regression coefficient,
wherein the descriptive statistics of the residual errors improves model
performance. The best‐fit models were determined by application of
the coefficient of determination (R2).
Results and discussion
Identification of secondary metabolites of T. cacao clone UF-650
Table 4 shows the results of the phytochemical screening. Free
amino acids, alkaloids, reducing carbohydrates, phenols, tannins,
pyrocatecholic tannins, and flavonoids were detected in all samples.
The most represented metabolites were alkaloids, which were mainly
found in flowers and calli. In the latter, flavonoids were most abun-
dant. Large amounts of coumarins were found in immature flowers
and leaves. In contrast, quinones were significantly present in the
mature pericarp, leaves, and seeds of the fruits, whereas saponins
and coumarins were detected in all organs of the plant. However,
the presence of these metabolites was not demonstrated in the calli.
Differences in triterpenes and steroid compounds were also
observed in the pericarp and seeds. These differences are attributable
to the developmental stages of the plant organs. According to Pérez‐
Alonso and Jiménez (2011), the biosynthesis of secondary metabolites
is usually restricted to specific developmental stages and to periods of
stress.
The absence of resins, triterpenes, steroids, saponins, coumarins,
and quinones in calli might be attributed to their degree of dedifferen-
tiation. Constantly dividing callus cells, the most abundant cell type,
are characterized by a dense cytoplasm. and give rise to meristematic
centers (Hernández‐Pimentel and Tavera‐Martínez, 1996). Because
High level
Real value Coded value Real value
0.01 +1 0.1
Petals +1 Staminodes
Callus +1 Culture medium
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L. Fajardo Rosabal et al. Current Research in Biotechnology 4 (2022) 47–57

Table 3 ing cells showed a dense cytoplasm and tended to form cell groups.
Regression models evaluated to determine the best mathematical relationship These results were similar to those described by Alemanno et al.
between the experimental data and predicted values. (1996) during the somatic embryogenesis of T. cacao. Strong mitotic
Model Equation Y-Transformation X-Transformation activity was observed in both callus types. Two stages of cell division
were demonstrated: anaphase (Fig. 1 a, b, c, d, and f) and telophase
Linear y ¼ β0 þ β1 x None None
Inverse of X y ¼ β0 þ β1 =x None Reverse
(Fig. 1 b, d, and e).
Inverse of Y y ¼ ðβ0 þ β1 xÞ1 Reverse None
Inverse Y, log X y ¼ β0 þβ11 ln x Reverse Logarithm
pffiffiffi Quantification of total polyphenols from cellular biomass
square root of X y ¼ β0 þ β1 x None Square root
β0 þβ1 x
Exponential y¼e Logarithm None
pffiffi The highest concentration of total polyphenols (58.4 µg g−1) was
log Y, square root X y ¼ eβ0 þβ1 x Logarithm Square root
log X, square root Y y ¼ ðβ0 þ β1 ln xÞ2 Square root Logarithm achieved when calli obtained from staminodes were elicited with
square Y, log X
pffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi
y ¼ β0 þ β1 ln x Square Logarithm 0.10 mg L−1 SA (Table 5). This value was higher than that when an
equal concentration of SA was applied to petal‐derived calli
(49.4 µg g−1). The other concentrations were in the range of 14.0–3
5.3 µg g−1 (Table 5). At the highest concentration, SA increased the
many metabolites are synthesized according to the degree of cellular
concentration of total polyphenols in both callus types to
differentiation, a certain degree of organization in cell culture is nec-
40.2 µg g−1 and 14.1 µg g−1, as compared with the control treatments
essary for metabolite production.
(18.3 ± 0.18; 35.29 ± 0.15 for staminode and petal derived calli).
Secondary metabolites are stored mainly in the vacuoles, which,
Similarly, increases of 8.3 µg g−1 and 16.0 µg g−1 were observed in
from an ultrastructural viewpoint, are located at the adjacent inner
the culture medium extracts (16.21 ± 0.18; 13.93 ± 0.18 for stamin-
periphery of the cell or in the centers of cytoplasmic organelles (Lees
ode and petal culture media, respectively). Greater diffusion of
et al., 1993). Therefore, the low production of secondary metabolites
polyphenols to the culture medium was also observed with 0.10 mg
in cell cultures may be a consequence of limited cell differentiation
L−1 SA from calli derived from petals.
(Espinosa‐Leal et al., 2018).
The ANOVA indicated, with a 95% probability, that all main
High similarity between preliminary phytochemical tests of natural
effects: (X1, X2, and X3) and their interactions significantly influenced
plants' aerial organs and undifferentiated cells was observed. Callus
total polyphenol production. The effects of the treatments were plotted
cells do not yet have a central vacuole for a greater storage capacity.
on a Pareto chart to visualize which effects had greater influence on
However, potential exists to produce compounds of interest, particu-
the response variables. An easy way to determine the effect of each
larly phenolic compounds such as phenols, pyrocatecholic and pyro-
variable is to add a line on the standardized Pareto chart at the height
gallotanical tannins, and flavonoids.
of a calculated critical value. The effects of bars crossing that line are
Moreover, according to Pérez‐Alonso and Jiménez (2011) the
significant. The Pareto diagram is shown in Fig. 2. Effects such as con-
chemical composition of the callus varies according to the origin of
centration, concentration‐explant type interaction, and explant type‐
the explant, the parent tissue, and callus age. The results of this study
extract type, in that order, significantly affected the response variable.
were consistent with those of Nwokonkwo and Okeke (2014), who
In contrast, antagonistic interactions were observed for explant and
have demonstrated the presence of alkaloids, tannins, saponins, glyco-
extract type.
sides, phenols, flavonoids, and carboxylic acids in an ethanolic extract
Finally, the codified mathematical model applied for the 23 facto-
of T. cacao bark. Cádiz‐Gurrea et al. (2014) have identified and quan-
rial design was defined by:
tified 61 chemical compounds in different types of extracts, and simi-
larly have detected several structural classes of chemical compounds,
Y ¼ 30:7308  9:83X1 þ 1:42X2 þ 9:56X3 þ 2:30X1 X2  3:75X1 X3
such as flavan 3‐ols and derivatives (including procyanidins), flavo-
nols, and N‐phenylpropenoyl‐L‐amino acids. þ 0:62X2 X3 þ 4:22X1 X2 X3
The calli generally showed a homogeneous morphology (Fig. 1),
although staminode derived calli sometimes showed hard, compact, where Y represents the total polyphenol content, and X1, X2, and X3 are
or necrotic areas. Embryogenic structures, generally characterized by the three factors considered in this study. The best‐fit models were
the presence of small and rounded cells, were not observed. These fea- determined by application of the coefficient of determination (R2). In
tures are typical of meristematic cells. Moreover, the single and divid- this case, the value of R2 = 0.99 indicated that only 0.01% of the total
variation was not explained by the regression model. Furthermore, the

Table 4
Phytochemical screening of plant aerial organs and calli.

Metabolites Aerial organs of T. cocoa, UF-650 clone Calli

Leaves Pericarp Seeds Flowers Stamin. Petals
Immat. Mature Immat. Mature Immat. Mature Before anthesis After anthesis

Resins + +
Triterpenes and/or steroids + +
Saponins + +
Free amino acids + +
Alkaloids + +
Coumarins + ++
Reducing carbohydrates + +
Phenols and/or tannins + +
Pyrocatecholic tannins + +
Pyrogallotanical tannins − −
Quinones ++ ++
Flavonoids + +
+ + + + − − − −
− + − + − − − −
+ + + + + + − −
+ + + + + + + +
+ + + + +++ +++ +++ +++
+ + + + ++ ++ − −
+ + + + + + + +
+ + + + + ++ + +
+ + + + + + + +
+ + + + + + + +
+ ++ ++ ++ + + − −
+ + + + + + ++ ++

Legend: −, absence; +, presence; ++ and +++, abundance; immat, immature state; stamin, staminodes.

50
L. Fajardo Rosabal et al. Current Research in Biotechnology 4 (2022) 47–57

Fig. 1. Callus aggregates: (a) staminode derived callus without SA, (b) staminode derived callus with SA at 0.01 mg L−1, (c) staminode derived callus with SA at
0.1 mg L−1, (d) petals without SA, (e) petals with SA at 0.01 mg L−1, and (f) petals with SA at 0.1 mg L−1. A, anaphase; T, telophase.

Table 5
Observed and predicted values of total polyphenol content of callus extracts.

No Concentration of salicylic acid (mg L−1) Explant type Extract type Observed Predicted

1 0.01 Petals Callus 35.29 ± 0.15 35.29 ± 0.05

2 0.1 Petals Callus 49.39 ± 0.18 49.39 ± 0.05
3 0.01 Staminodes Callus 18.13 ± 0.18 18.13 ± 0.05
4 0.1 Staminodes Callus 58.35 ± 0.18 58.35 ± 0.05
5 0.01 Petals Medium 13.97 ± 0.18 13.97 ± 0.05
6 0.1 Petals Medium 29.97 ± 0.18 29.97 ± 0.05
7 0.01 Staminodes Medium 16.21 ± 0.18 16.21 ± 0.05
8 0.1 Staminodes Medium 24.53 ± 0.18 24.53 ± 0.05

Fig. 2. Pareto diagram showing the effects of the independent variables and their interactions on the total polyphenol content. The length of each bar indicates the
standardized effect of each factor or interaction.

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L. Fajardo Rosabal et al. Current Research in Biotechnology 4 (2022) 47–57

Fig. 3. Total polyphenol content in petals and staminode derived calli with SA in (a) the callus and (b) the culture medium.

Fig. 4. 3D response surface plots corresponding to statistical analysis for the polyphenols obtained (see Table 2 for codes).

high degree of precision of the results was indicated by the value of the
variation coefficient (4.22%).
The highest polyphenol content was found in calli derived from the
staminodes (Fig. 3a) but petal derived calli secreted more polyphenols
into the medium (Fig. 3b). The staminodes derived from calli had a
hard, compact structure and sometimes showed necrotic zones. This
finding might have resulted from polyphenol accumulation causing
changes in the membranes and cell walls, thus triggering cell death.
In contrast, the calli of petals had a homogeneous cellular composition
and a hyperhydric appearance, which is ideal for promoting the
release of polyphenols to the medium. Optimal concentrations of SA
are important to avoid inducing hypersensitivity reactions that cause
cell death (Namdeo, 2007).
Response surface graphs visually represent the relationship
between two factors and describe the behavior of the average response
at each point in the experimental region, presented as a solid surface
framed in three‐dimensional space; these graphs also aid in under-
standing the main and interaction effects between two factors (Cai
et al., 2019). Fig. 4 shows the 3D surface response graphs of polyphe-
nol concentration as a function of two factors. The highest points in
each corner represent the highest concentration of total polyphenols
when SA (0.1 mg L−1) and staminode callus extracts are combined.

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L. Fajardo Rosabal et al. Current Research in Biotechnology 4 (2022) 47–57

Fig. 5. Desirability function charts of total polyphenols: (a) concentration; (b) explant type; and (c) extract type. Values of −1 and +1 indicate low and high
levels, respectively.

Table 6
Iron reducing potential of undifferentiated derived callus extracts from staminodes and petals expressed as μmol mL−1 of Fe2+.

Extract (µg mL−1) SC T1S T2S PC T1P T2P

a;A
100 0.606 ± 0.031 2.046 ± 0.032b;A 1.253 ± 0.018c; A
1.379 ± 0.018 d; A
1,952 ± 0.032 e; A 4.562 ± 0.048f; A
a; B
200 0.982 ± 0.031 2.318 ± 0.036b; B 3.862 ± 0.032c; B
1.598 ± 0.018 d; B
2.297 ± 0.032b; B 4.989 ± 0.032 e; B
a; C
300 1.410 ± 0.018 2.370 ± 0.018b; B 4.937 ± 0.018c; C
1.837 ± 0.018 d; C
2.349 ± 0.018b; B 5.396 ± 0.032 e; C
a; C
400 1.462 ± 0.018 2.610 ± 0.031b; C 5.396 ± 0.113c; D
2.193 ± 0.018 d; D
2.516 ± 0.031 e; C 5.553 ± 0.031f; D
a; D
500 1.837 ± 0.018 2.787 ± 0.018b; D 5.532 ± 0.018c; E
2.641 ± 0.032 d; E
2.735 ± 0.031b; D 5.866 ± 0.031 e; E

Each value in the table is represented as mean ± SD, (n = 3). Different lowercase letters for the same extract concentration or different capital letters for the same
extract type indicate statistically significant differences according to Tukey (p < 0.05).
Legend: (a–f) Analysis referring to the same concentration of different extracts; A–E) analysis referring to different concentrations of the same extract. SC,
staminode callus control; T1S, staminode callus with SA at 0.01 mg L−1; T2S, staminode callus with SA at 0.1 mg L−1: PC, petal callus control; T1P, petal callus
with SA at 0.01 mg L−1; T2P, petal callus with SA at 0.1 mg L−1.

Table 7
Comparison of the nine mathematical models according to the selection criteria used. The asterisk indicates the highest value of R2 obtained.

Number Salicylic acid (mg L−1) Explant type Values of R2 in the regression models
Linear 1/X 1/Y 1/Y √X Exponential log Y √X log X √Y
log X log X

1 0.01 Petals 97.89 70.10 99.99* 94.63 93.59 99.43 96.56 89.10 82.18
2 0.1 Petals 97.77 89.00 95.94 99.16 99.24* 96.95 99.14 98.42 97.21
3 0.01 Staminode 95.38 87.52 88.84 97.73* 96.95 92.86 96.53 92.41 97.12
4 0.1 Staminode 81.47 99.82* 63.90 83.31 89.38 72.29 81.60 92.82 98.71
5 – Petals 93.89 85.03 91.90 94.83* 94.29 93.11 93.45 93.45 91.11
6 – Staminode 98.58 72.00 99.01* 91.74 95.12 98.88 96.04 89.66 87.29

Meanwhile, the points where the surface has the smallest values, that
is the lowest total polyphenol concentration values, are shown with SA
(0.01 mg L−1), extracts of petal calli, and culture medium.
To determine the best global conditions of the experiment and the
best response variable for a given range, we used the desirability func-
tion to fulfill the study objectives. The method involves transforming
the predicted values on a scale from 0 to 1, to indicate their desirabil-
ity (Amdoun et al., 2018). The response variable was placed on the
same scale and a desirability function di(x) was defined. Fig. 5 shows
the response graphs corresponding to the desirability function of total

53
L. Fajardo Rosabal et al. Current Research in Biotechnology 4 (2022) 47–57

Table 8
Summary of the results of regression analysis for the best-fit model.

Number Salicylic acid (mg L−1) Explant type Model R2 Adj-R2 SE

1 0.01 Petals Y¼ 1
16:840:015X1
99.99 99.98 0.031
pffiffiffiffiffiffi
2 0.1 Petals Y ¼ 0:134 þ 0:0033 X1 99.24 98.99 0.0016
3 0.01 Staminode Y ¼ 57:647:29 ln X1
1 97.73 96.97 0.8147
4 0.1 Staminode Y ¼ 0:23  17:21
X1
99.82 99.77 0.0027
5 – Petals Y ¼ 18:941:54
1
ln X1
94.82 93.10 0.2636
6 – Staminode Y ¼ 12:210:0063X
1 99.00 98.67 0.1153
1

polyphenols from cellular biomass. The defined optimum total
polyphenol concentration was 58.3 µg g−1, with a high global desir-
ability function value of 95%.
In a previous report by Fajardo et al. (2020), the total polyphenols
in calli from three species of explants (petals, staminodes, and young
leaves) were quantified with the Folin‐Ciocalteu method. The results
indicated that polyphenol production is possible in these calli and that
staminode‐derived calli produce the highest concentration
(11.21 µg g−1). However, the yield was lower than that in the present
study, wherein metabolic elicitation with SA was used as a trigger for
polyphenol production.
Several authors (Hii et al., 2009; Sotero et al., 2011; Baharum et al.,
2014; Manzano et al., 2017; Ordoñez et al., 2019; Indirato et al., 2019)
have quantified the production of polyphenols in cocoa, by using

Fig. 6. Reducing power in petal and staminode calli: (a) petal callus without elicitation (control); (b) staminode callus without elicitation (control); (c) staminode
callus and SA concentration of 0.01 mg L−1; (d) staminode callus and SA concentration of 0.1 mg L−1; (e) petal callus and SA concentration of 0.01 mg L−1; and (f)
petal callus and SA concentration of 0.1 mg L−1.

54
L. Fajardo Rosabal et al.
extracts from different plant organs to search for potential pharmaco-
logical and nutritional uses. However, few studies have aimed to
improve the production of polyphenols by in vitro culture. Prior studies
have focused mainly on the distribution of the compounds in relation
to the embryogenic response of the callus (Alemanno et al., 2003;
Gallego et al., 2016). Quiñones‐Galvez et al. (2016) have quantified
phenolic compounds from a callus culture with embryogenic struc-
tures. The authors have evaluated the effects of different culture con-
ditions in liquid medium (shaking speed, lighting conditions, and
glucose concentration) on the accumulation of phenolic compounds
in the callus and their expression in the culture medium. Moreover,
the concentration of polyphenols in the callus is has been found to
be genotype dependent (Quiñones et al., 2013; Fajardo et al., 2015;
Gallego et al., 2016).
Antioxidant activity due to the reducing power of selected extracts
Antioxidant activity due to the reducing power was determined in
selected extracts: non‐elicited calli, petal, and staminode extract with
SA at 0.01 mg L−1 and 0.1 mg L−1 (Table 6). A positive correlation
was observed between the total phenol content and reducing power,
although all samples did not have the same biological response.
An increase in reducing power was observed with increasing
extract concentration. The best result (5. 87 µmol mL −1) was obtained
in petal derived calli, with SA at 0.1 mg L−1 (T2P). The addition of SA
as an elicitor increased the iron‐reducing potential in non‐
embryogenic callus biomass extracts of this clone. We did not find
any prior reports of reducing power evaluation for this sample type
in the literature.
Nine regression models were solved and sorted according to the R2
criterion (Table 7). The predictive models were defined as a function
of two variables (elicitor concentration and type of explant). The high-
est value of R2 was considered the best selection criterion. In contrast,
Table 8 shows the performance criteria parameters from the selected
models. High values of R2 and adj‐R2 were obtained in all models (in-
terval of variation for R2 and adj‐R2 were between 94.83 and 99.99,
and 93.10 and 99.98, respectively). Goodness of fit is often measured
with R2, which shows the proportion of variation in the dependent
variables explained by the independent variables. Nevertheless, a bet-
ter practice is to use adj‐R2 because R2 can sometimes provide overly
favorable regression result. Meanwhile, a significant degree of preci-
sion and a substantial portion of the reliability of the models were indi-
cated by the low values of the standard error. The performance criteria
parameters of a statistical model describe how a data set is adjusted. In
general, the measures of goodness of fit summarize the discrepancy
between the observed values and the expected values in a model.
The best adjustment models are obtained according to the dependent
variables studied, defined as a function of the independent variables
(Guardia‐Puebla et al., 2021).
Fig. 6 shows the reducing power in petal and staminode calli
according to the mathematical models, in controls (Fig. 6 a, b), and
SA at 0.01 mg L−1 and 0.1 mg L−1 for petal and staminode elicited
calli (Fig. 6 c‐f). An increase in reducing power was observed as the
concentration of polyphenols increased in the extracts prepared from
the samples (100–500 µg mL−1). The optical density values with
staminode derived calli were between 0.06 and 0.20 whereas, the val-
ues for petals remained in a narrower interval between 0.17 and 0.21.
Although the petal extract had lower production of total polyphenols,
they had a slightly better reducing power than that of the extract
obtained from staminode derived calli.
According to Paladino and Zuritz (2011), even when the total phe-
nolic concentration is equal, the amounts of cinnamic acid, benzoic
acid, anthocyanins, catechins, tannins and flavonoids, may differ,
because each of these phenolic groups may behave differently in terms
of their reducing power. The results of this study differ from those
reported by Quiñones et al. (2013), who have reported that stamen‐
55
Current Research in Biotechnology 4 (2022) 47–57
derived callus extracts showed a four‐fold higher activity than petal
extracts in T. cacao UF‐654.
Several studies have verified the antioxidant activity of cocoa
(Baharum et al., 2014; Cádiz‐Gurrea et al., 2014; Manzano et al.,
2017; Deus et al., 2018; Ginting et al., 2019), by using extracts from
the natural plant and from products derived from cocoa. However,
no prior evidence has indicated that polyphenols can be obtained from
undifferentiated cells by metabolic elicitation.
In this research, the total polyphenols and the reducing power were
successfully predicted from the optical density values. These models
provide good predictions of the effects of treatments. Other authors,
such as Quiroz‐Reyes and Fogliano (2018), have applied 22 factorial
designs, linear regression, and response surface methods to develop
a model to predict and describe the total phenolic content and antiox-
idant capacity in the roasting process of the Criollo and Forastero vari-
eties of T. cacao.
Beaver et al. (2020) have used mathematical models and linear
regression to predict the phenolic content of red wine; their models
were developed by using data from ultraviolet–visible absorption spec-
tra. However, to our knowledge, this experimental setup had not been
applied to the callus culture of T. cacao.
Conclusions
The eliciting role of SA on the polyphenol content and reducing
power activity in undifferentiated calli biomass of T. cacao was demon-
strated. The roles of concentration, explant type, and extract type on
total polyphenol content in staminode and petal calli, as well as their
culture media was demonstrated with a factorial 2 k experimental
design. The conditions maximizing the polyphenol content were the
use of callus extracts of staminodes elicited with SA (0.1 mg L−1);
under these conditions, the content of total polyphenols increased by
40.22 µg g−1 above that of the control without elicitation. This result
should provide a reference for future research in liquid media. The
antioxidant activity according to the reducing power of the callus
extracts was evaluated, and the best result (5.87 µmol mL−1 Fe2+)
was obtained in callus extracts from petals with SA at 0.1 mg L−1.
Mathematical models were developed, which should allow for the pre-
diction of the polyphenol content, and reducing power responses.
CRediT authorship contribution statement
Lillien Fajardo Rosabal: Conceptualization, Data curation, Inves-
tigation, Methodology, Writing – original draft, Writing – review &
editing. Yans Guardia Puebla: Data curation, Formal analysis,
Methodology, Writing – original draft. Suyén Rodríguez Pérez:
Methodology, Resources, Supervision. Ursula M. Rosabal Cordoví:
Data curation, Investigation, Methodology, Writing – original draft,
Writing – review & editing. Juan Silva Pupo: Funding acquisition,
Project administration, Resources. Stefaan P.O. Werbrouck: Funding
acquisition, Project administration, Resources, Supervision, Writing –
review & editing.
Declaration of Competing Interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influ-
ence the work reported in this paper.
Acknowledgements
This work was supported by the program of the Secretariat of
Higher Education, Science, Technology and Innovation of Belgium
(VLIR), coordinated by the University of Gent, in the framework of
L. Fajardo Rosabal et al. Current Research in Biotechnology 4 (2022) 47–57

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Indirato, R., Pranoto, Y., Santoso, U., Supriyanto, 2019. In vitro Antioxidant Activity and
Profile of Polyphenol Compounds Extracts and their Fractions on Cacao Beans. Pak.
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