Food Analytical Methods

https://doi.org/10.1007/s12161-019-01672-8

Tamarindus indica L. Seed: Optimization of Maceration Extraction

Recovery of Tannins
Aleksandra Cvetanović 1 & Sengul Uysal 2,3 & Branimir Pavlić 1 &
Kouadio Ibrahime Sinan 4 & Eulogio J. Llorent-Martínez 5 & Gokhan Zengin 4

Received: 25 September 2019 / Accepted: 31 October 2019

# Springer Science+Business Media, LLC, part of Springer Nature 2019

Abstract
The seeds of Tamarindus indica L., classified as bio-waste, are powerful sources of bioactive compounds, especially tannins. In
order to use its full potential, extraction of such bioactive constituents should be done under the optimized conditions. In the
frame of this paper, central-composite experimental design and RSM (response surface methodology) were applied in order to
investigate the impact of the maceration parameters on target responses and to optimize extraction process. Extraction was
performed under the different levels of extraction solvent (methanol), temperature, and solvent-to-sample ratio. Obtained extracts
were evaluated in terms of total phenols, flavanols, and tannin yield and in vitro antioxidant activity (DPPH, FRAP, CUPRAC).
Experimental results were fitted to a second-order polynomial model where regression analysis and analysis of variance were
used to determine model fitness and optimal extraction conditions. Optimized extraction conditions determined by RSM were
methanol concentration of 69.99%, extraction temperature of 23.38 °C, solvent-to-sample ratio of 1:20. Chemical characteriza-
tion of the extract obtained under the optimized conditions was done by using HPLC-ESI-MS/MS technique. The study could
provide a scientific baseline for designing novel functional products from T. indica seeds in further studies.

Keywords Tamarind . RSM . HPLC-ESI-MS/MS . Antioxidant . Bioactive compounds

Introduction (Shirmohammadli et al. 2018). Previous research reported that

Tannins are natural polyphenolic compounds present in plants
and are significant for food, leather industry, environmental,
and medicinal applications. These compounds are classified
into two groups namely hydrolysable and condensed tannins
Aleksandra Cvetanović and Sengul Uysal contributed equally to this work.
* Sengul Uysal
senguluysal@erciyes.edu.tr
1
Department of Biotechnology and Pharmaceutical Engineering,
Faculty of Technology, University of Novi Sad, Novi Sad, Republic
of Serbia
2
Erciyes University, Halil Bayraktar Health Services Vocational
College, Kayseri, Turkey
3
Ziya Eren Drug Application and Research Center, Erciyes
University, Kayseri, Turkey
4
Department of Biology, Science Faculty, Selcuk University,
Konya, Turkey
5
Faculty of Experimental Sciences, Department of Physical and
Analytical Chemistry, University of Jaén, Jaen, Spain
Quercus sp. (oak bark), Castanea sativa Mill. (chestnut
wood), Acacia mearnsii De Wild. (black mimosa bark), and
Schinopsis balansae Engl. (quebracho wood) are the main
sources of tannins for industrial purposes (Arbenz and
Avérous 2015; Bele et al. 2010; Lochab et al. 2014; Pizzi
1980). In addition, some agricultural wastes such as tea
(Chowdhury et al. 2016), coffee (Janissen and Huynh 2018),
grapes skin (Ricci et al. 2017), and pomegranate peel (Ben-Ali
et al. 2018) are potential sources of tannins. In this sense, new
studies on unexplored waste products for tannins recovery are
of the utmost importance.
Tamarindus indica L., a member of Fabaceae family also
known as Tamarind, is widely spread through tropical zones
(Dey et al. 2011). Various studies acknowledged that tamarind
possesses numerous pharmacological properties namely anti-
oxidant, antidiabetic, anti-inflammatory, and antimutagenic
properties (Mahomoodally et al. 2016; Maiti et al. 2004;
Martinello et al. 2006; Mootoosamy and Mahomoodally
2014; Ramos et al. 2003; Rimbau et al. 1996). Furthermore,
several uses for Tamarind have been reported. For example,
the leaves can be eaten as salads (Bhadoriya et al. 2011) and
the fruit is used as flavoring and ingredients in food products

including drinks (Van der Stege et al. 2011). However, the
tamarind seeds which are discarded during processing of fruit
(Oluseyi and Temitayo 2015) are a rich source of nutritional
components such as proteins, minerals (e.g., calcium and
magnesium), and amino acids (Olagunju et al. 2018). In addi-
tion to these nutritional components, tamarind seeds are an
excellent source of phenolic compounds (Brain and John
2014), especially tannins (Sudjaroen et al. 2005). Several
studies reported that the fruit wastes including peels and seeds
contained a higher level of bioactive compounds compared to
the edible parts (Ayala-Zavala et al. 2011; Rudra et al. 2015).
Therefore, tamarind seeds have become one of the most at-
tractive natural material in the scientific area (Doughari 2006).
The response surface methodology (RSM) is an effective
technique for optimizing the process parameters (Pasandide
et al. 2017; Uysal et al. 2019b), which is extensively
employed in optimizing the extraction of phytochemical com-
pounds including phenolic, tannin, and flavonoid from several
plant samples (Krakowska et al. 2018; Nabet et al. 2019;
Pandey et al. 2018). Many researches have used tools such
as central composite design (CCD), Box Behnken design
(BBD), Doehlert matrix (DM), and three-level factorial (3K)
for RSM. Among these mentioned tools, central composite
design (CCD) is most commonly used in food analysis
(Ferreira et al. 2019). Inspired by the above observation, the
aim of the present study was to optimize the effect solvent,
temperature, and solvent to sample ratio for maximizing the
extraction of phenolic compound (especially tannin) of tama-
rind seed.
Material and Methods
Plant Material
Tamarindus indica was purchased from the local market in
Ivory Coast. Then, the seed was separated and dried naturally
for 10 days. Dried seed was then powdered with a laboratory
mill (Retsch SM 200). These seed samples (1 g) were macer-
ated by methanol for 24 h (Mollica et al. 2018). Extraction was
done by using three parameters of solvent concentration, tem-
perature, and solvent to sample ratio as independent variables,
while other extraction parameters were held constant. The
values of solvent concentration, extraction temperature, and
solvent to sample ratio are summarized in Table 1. Obtained
extracts were filtered (by Whatman blue band) and evaporated
in vacuum at 40 °C. All extracts were stored in a refrigerator
for further analysis.
Experimental Design and Statistical Analysis
In this study, three levels, three-variable full factorial
design (FFD) was employed to investigate and validate
Food Anal. Methods
Table 1 The independent variables used in response surface
methodology
Independent variable Factor levels
−1 0 +1
Temperature (°C) 20 40 60
SSR (g/mL) 1:20 1:30 1:40
Solvent concentration (%) 30 50 70
the extraction parameters affecting the extraction of
polyphenols content and antioxidant activity from
Tamarindus indica seed. For the optimization of extrac-
tion parameters, design consisted of 31 experiments
with five replicates at central point. The parameters
employed in the experimental design were in the ranges:
temperature from 20 to 60 °C; sample-to-solvent ratio
from 1:20 (w/v) to 1:40 (w/v); methanol concentration
from 30 to 70%. Variables were coded according to the
following Baş and Boyacı equation (Baş and Boyacı
2007). Used independent variables are presented in
Table 1.
Obtained results were fitted to the second order polynomial
model (Eq. (1)):
4 4 3 4
Y ¼ β0 þ ∑ β i X i þ ∑ βii X 2i þ ∑ ∑ βij X i X j ð1Þ
i¼1 i¼1 i¼1 j¼iþ1
where Y is the response variable, Xi and Xj are independent
variables and βi, βii, and βij are the regression coefficients for
intercept, linear, quadratic, and interception terms,
respectively.
Design Expert software v. 10 (State-Ease, Minneapolis,
Minnesota, USA) was used for the analysis of obtained
experimental data. Statistical significance of the used
model and lack of fit was determined by analysis of var-
iance (ANOVA) and response surface analysis with the
significance level of 0.05. Coefficient of determination
(R2) and coefficient of variance (CV) were used as de-
scriptive measure of the model adequacy, while response
surface plot was used to describe the relationship between
the response and independent variables.
Determination of Total Phenol Content
Total phenol content was measured using colorimetric
assay according to the Folin–Ciocalteu reagent method
(Mollica et al. 2017; Zengin et al. 2019). The results
were expressed as milligram gallic acid equivalent per
gram of extract (mg GAE/g extract).
Food Anal. Methods

Table 2 Full factorial design with actual values of extraction conditions and experimentally obtained output values

Independent variables Measured response

Temperature SSR Solvent TPC TFC (mgCE/g) TTC (mgCE/g) DPPH CUPRAC FRAP
(mgGAE/g) (mmolTE/g) (mmolTE/g) (mmolTE/g)

60 1:30 50% 324.84 47.80 134.46 3.17 4.17 2.05

60 1:30 30% 310.66 70.26 219.62 2.10 4.05 2.09
40 1:20 30% 341.30 77.32 232.44 3.24 4.47 2.33
60 1:40 70% 341.32 94.07 270.06 3.42 5.04 2.57
60 1:20 30% 289.93 64.98 222.68 3.17 4.35 2.34
60 1:30 70% 351.08 86.50 282.47 3.74 5.45 2.92
20 1:40 50% 334.45 42.95 137.39 3.16 4.92 2.45
40 1:20 70% 346.84 74.61 274.24 3.67 5.24 2.85
40 1:20 50% 324.59 54.05 153.89 3.12 4.63 2.40
20 1:30 30% 244.55 55.39 181.62 2.63 3.81 2.09
40 1:40 70% 350.32 91.54 248.62 3.47 4.83 2.60
60 1:40 30% 283.83 76.09 188.54 3.04 4.23 2.19
40 1:40 50% 301.81 44.52 109.15 2.95 4.90 2.38
20 1:20 50% 341.08 58.86 160.08 3.38 5.17 2.93
40 1:30 70% 350.77 70.32 259.79 3.59 5.64 2.98
60 1:20 70% 353.22 73.79 279.92 3.87 5.61 2.97
20 1:40 70% 332.39 73.66 243.58 3.53 5.24 2.71
20 1:30 70% 357.34 81.29 263.68 3.78 5.73 2.89
40 1:40 30% 260.67 55.80 164.74 2.75 4.01 2.02
20 1:20 70% 292.95 99.33 149.87 3.86 5.93 3.16
60 1:20 50% 328.37 55.53 163.59 3.52 4.98 2.55
20 1:30 50% 309.29 47.44 123.29 3.14 4.77 2.35
40 1:30 50% 311.45 53.74 124.69 3.10 4.74 2.25
40 1:30 30% 243.87 54.47 163.46 2.10 3.78 1.93
20 1:20 30% 232.44 64.65 165.41 2.67 3.71 2.05
20 1:40 30% 227.16 43.92 89.12 2.31 3.75 1.92
60 1:40 50% 297.56 49.09 134.33 2.97 4.67 2.56
40 1:30 50% 351.77 43.87 177.67 3.64 4.91 2.33
40 1:30 50% 343.92 42.55 169.49 3.77 5.08 2.23
40 1:30 50% 361.25 44.32 190.01 3.64 4.95 2.53
40 1:30 50% 350.12 44.73 179.06 3.52 4.69 2.22

Determination Total Flavanol Content Determination of Antioxidant Activity

Total flavanol content was detected with DMACA
(pdimethylaminocinnamaldehyde) method as described by
(Zengin et al. 2015). The results were evaluated as the equiv-
alents of (+)-catechin equivalents (mg CE/g extract).
Determination of Total Condensed Tannin Content
Antioxidant activity of the obtained Tamarindus indica ex-
tracts was measured by using three different antioxidant as-
says: DPPH (1,1-diphenyl-2-picrylhydrazyl), FRAP (ferric re-
ducing antioxidant power), and CUPRAC assays carried out
according to the (Uysal et al. 2019a) procedure. Antioxidant
activity was expressed as trolox equivalents (mmol TE/g).

Vanillin/H2SO4 method was used to determine total con- HPLC-ESI-MSn

densed tannin content with slight modifications (Zengin
et al. 2015). The results were expressed as equivalents of The phytochemical profile was studied by means of an
(+)-catechin (CEs) according to the equation obtained from Agilent Series 1100 with a G1315B diode array detector and
the standard (+)-catechin graph (mg CE/g). an ion trap mass spectrometer (Esquire 6000, Bruker
 Food Anal. Methods

Table 3 Regression coefficients

of the predicted second-order Regression coeficient TPC TFC TTC DPPH CUPRAC FRAP
model for the response variables
β0 338.33* 45.40* 161.94* 3.32* 4.79* 2.32*
Linear
β1 11.62 2.81 21.20* 0.03 − 0.03 − 0.02
β2 − 6.73 − 2.86 − 12.03 − 0.16** − 0.14** − 0.12*
β3 35.66* 10.12* 35.81* 0.50* 0.70* 0.37*
Cross product
β12 − 6.36 7.27* − 5.67 − 0.02 − 0.01 0.04
β13 − 9.78 − 3.94** − 1.61 − 0.07 − 0.18** − 0.07
β23 10.25 3.56 19.70** 0.00 − 0.09 − 0.04
Quadratic
β11 − 12.13 2.51 − 10.51 − 0.06 0.01 0.07
β22 − 7.50 4.03 − 14.19 0.05 0.03 0.10
β33 − 19.10** 22.91* 71.18* − 0.15 − 0.11 0.04
Descriptive statistics
R2a 0.741 0.910 0.809 0.715 0.864 0.849
CVb 7.61 9.64 15.32 9.70 5.66 6.56

*Highly significant p value (< 0.01)

**Significant p value (0.01 < p < 0.05)
a
Coefficient of multiple determination
b
Coefficient of variance (%)

Daltonics) with an electrospray interface operating in negative
mode. The separation of the compounds was performed using
a Luna Omega Polar C18 analytical column of 150 × 3.0 mm
and 5 μm particle size (Phenomenex). Chromatographic con-
ditions are detailed in a previous paper (Llorent-Martínez et al.
2018). Mass spectrometry was used for identification pur-
poses, whereas quantitation was carried out by UV using suit-
able analytical standards for each chemical family.
Results and Discussion
Influence of Independent Variables on Investigated
Outputs
With the aim to get Tamarindus indica seed extracts rich in
bioactive compounds, in the first place polyphenol, flavanols,
and tannins, and with high antioxidant capacity, the influence
of extraction parameters (solvent concentration, temperature,
and solvent-to-sample ratio) on aforementioned responses
was investigated. For this purpose, f independent experiments
were done, and the obtained results are presented in Table 2.
Depending on the extraction conditions, total phenolic con-
tent was in the range from 227.16 to 361.25 mg GAE/g
representing the richness of the extracts in bioactive com-
pounds. The lowest yield of TPC was achieved at temperature
of 20 °C, using SSR of 1:40 (unit) and methanol in the con-
centration of 30%. Increasing in extraction temperature (40
°C) and methanol concentration (50%) with decreasing in
SSR (1:30) led to obtaining extract with the highest TPC.
Significant and highly significant influence on TPC has qua-
dratic and linear term of methanol concentration, respectively
(Table 3). The fitness of the model was confirmed by relative-
ly low value of the coefficient of variance (CV = 7.61%),
while the proper fitting of experimental and theoretical values
was established by the moderately high value of the coeffi-
cient of multiple determination (0.7413). Adequacy of the
applied model was further suggested by significant p values
(< 0.01) for the model and insignificant lack of fit (p > 0.05)
(Table 4). According to the determined values of regression
coefficients, the polynomial model for TPC was given by the
following equation:
TPC ¼ 338:33 þ 11:62X 1 −6:73X 2 þ 35:65X 3 −6:36X 1 X 2 ð2Þ
−9:78X 1 X 3 þ 10:25X 2 X 3 −12:13X 21 −7:50X 22 −19:10X 23
In order to illustrate the influence on investigated parameters
on TPC, three-dimensional (3D) plots were constructed (Fig. 1).
As can be seen from Fig. 1, increasing in extraction temperature
and solvent concentration has a positive influence on TPC.
However, the influence of temperature is weaker comparing to
the influence of solvent concentration. Positive impact of tem-
perature could be explained by direct improvement of mass trans-
fer by increased diffusion and causing degradation of the com-
pact cell wall structure of raw material, as well as improved
penetration and solubility power of the applied solvent
Food Anal. Methods

Table 4 Analysis of variance

(ANOVA) of the fitted second- Source Sum of squares Degrees of freedom Mean of square F- value p - value
order polynomial model for ex-
traction yields and total phenol Total phenolic content
content Model 34,755.33 9 3861.70 6.687 0.00017
Residual 12,126.89 21 577.47
Lack of fit 10,672.52 17 627.80 0.31826
Pure error 1454.37 4 363.59
Total 46,882.23 30
Total flavanol content
Model 7733.42 9 859.27 23.667 4.74 × 10−9
Residual 762.45 21 36.31
Lack of fit 681.73 17 40.10 1.987 0.266125
Pure error 80.73 4 20.18
Total 8495.87 30
Total tannin content
Model 74,469.09 9 8274.34 9.878 9.15 × 10−6
Residual 17,591.02 21 837.67
Lack of fit 15,013.16 17 883.13 1.370 0.416049
Pure error 2577.86 4 644.47
Total 92,060.12 30
DPPH
Model 5.17 9 0.57 5.868 0.00041
Residual 2.06 21 0.10
Lack of fit 1.79 17 0.11 1.579 0.35434
Pure error 0.27 4 0.07
Total 7.23 30
CUPRAC
Model 9.69 9 1.08 14.854 3.11 × 10−7
Residual 1.52 21 0.07
Lack of fit 1.42 17 0.08 3.384 0.12320
Pure error 0.10 4 0.02
Total 11.21 30
FRAP
Model 3.03 9 0.34 13.109 9.04 × 10−7
Residual 0.54 21 0.03
Lack of fit 0.47 17 0.03 1.685 0.32780
Pure error 0.07 4 0.02

(Zeković et al. 2017). Small changes in solvent concentration led
to sharp increase in TPC which could be explained by improved
selectivity of solvent towards moderately polar polyphenols. On
the other hand, utilization of high SSR offers extracts with lower
yield of phenols which was also confirmed with negative values
of linear term of this parameter. Application of low SSR could
also be beneficial from economical point of view that demands
lower solvent consumption and provides production concentrat-
ed liquid extract leading to further cost-effective processing into
powder form (Pavlić et al. 2017). In the case of total flavanols,
the yield was in the range of 42.55 to 99.33 mg QE/g which was
directly influenced by combination of extraction parameters.
Namely, by combining temperature of 40 °C, SSR of 1:30 and
50% methanol extract with the lowest yield of flavanols was
obtained. On the other hand, utilization of higher solvent concen-
tration (70%) and lower SSR (1:20 m/v) with lower temperature
(20 °C) offers extract with much higher flavanol content.
Statistical analysis showed that content of flavanols was highly
influenced by linear term of solvent concentration, quadratic term
of solvent concentration, and cross product term of temperature
and SSR (p < 0.01), while cross product term of temperature and
solvent concentration expressed significant influence on TF.
Descriptive statistical parameters confirmed fitness of the model
(CV = 9.64%) and good fitting among theoretical and experi-
mental values (R2 = 0.910). ANOVA results confirmed good
accordance between the model and experimental data according

Fig. 1 Influence of investigated inputs on total phenolic content in T. indica seed extracts
to significant p value for the model (< 0.05) and insignificant lack
of fit (p > 0.05) (Table 4). According to the calculated values of
regression coefficients, the polynomial model for TFC was given
by the equation:
TFC ¼ 45:40 þ 2:81X 1 −2:86X 2 þ 10:12X 3 þ 7:27X 1 X 2 ð3Þ
−3:94X 1 X 3 þ 3:56X 2 X 3 þ 2:51X 21 þ 4:03X 22 þ 22:91X 23
Additionally, the influence of all three investigated parameters
on TFC was presented graphically in Fig. 2. It could be observed
that 3D plots were noticeably different comparing to TPC; how-
ever, influence of linear terms of extraction parameters of TFC
was similar (Table 3). Linear and quadratic term of solvent con-
centration impact was the most prominent suggesting that higher
TFC would be achieved by application of 70% methanol.
However, it should be highlighted that temperature-solvent con-
centration cross products exhibited significant negative impact
on this response (Table 3).
The third observed group of bioactive constituents in
Ta m a r i n d u s i n d i c a s e e d e x t r a c t s w e r e t a n n i n s .
Spectrophotometrically measured minimal content of total
Fig. 2 Influence of investigated inputs on total flavanols content in T. indica seed extracts
Food Anal. Methods
tannins was 89.12 mg CE/g, and it was measured in the extract
obtained at 20 °C, by using methanol in concentration of 30%
applying SSR of 1:40 which was the same point where minimal
TPC was achieved as well. Higher temperature and solvent con-
centration with lower SSR led to extract in which maximal con-
tent of tannins was measured, even 282.47 mg CE/g. Calculated
p values of regression coefficients showed that total tannins yield
was highly significant influenced by linear terms of temperature
and solvent concentration as well as by quadratic term of meth-
anol concentration (p < 0.01), while cross product term of SSR
and solvent concentration had significant influence on TTC (0.01
< p < 0.05).
Behaviors of the system could be described by the polyno-
mial model given in Eq. (4):
TTC ¼ 161:94 þ 21:20X 1 −12:03X 2
þ 35:81X 3 −5:67X 1 X 2 −1:61X 1 X 3
þ 19:70X 2 X 3 −10:51X 21 −14:19X 22 þ 71:18X 23 ð4Þ
The values of two calculated descriptive parameters—
coefficients of variance (15.32%) and coefficient of
Food Anal. Methods
Fig. 3 Influence of investigated inputs on total tanins content in T. indica seed extracts
determination (0.809)—showed the confirmed fitness of
the model and good matching of theoretical and exper-
imental values, respectively.
Simultaneous influence of different parametric combina-
tions is graphically described in Fig. 3. It was proven that
increasing the methanol concentration up to certain level of
degree had negative impact on TTC, but further increases
have opposite effect. In the same time, it can be seen that by
increasing SSR, the lower TTC could be obtained. On the
other hand, increasing the temperature had positive impact
on TTC increasing their yield. Since tannins contributed with
high ratio in TPC, it could be expected that extraction param-
eters would similarly affect these responses and that was the
case with linear terms (Table 3). Besides solvent concentra-
tion, significant positive impact of temperature on TTC was
observed which could be potentially explained by release of
bound tannins due to rupture of plant matrix which occurs at
elevated temperatures.
Bioactivity of the tested extracts was estimated by
three different antioxidant tests: DPPH, CUPRAC, and
FRAP whereby the influence of operational conditions
on the activity was observed and established. Minimal
Fig. 4 Influence of investigated inputs on DPPH activity of T. indica seed extracts
scavenger activity of the tested extracts against DPPH free
radicals (2.10 mmol TE/g) was obtained in two points
where under the SSR and methanol concentration had
the values of 1:30 and 30%, respectively. On the other
hand, the maximal ability of the extract to neutralize
DPPH radicals was 3.87 mmol TE/g and was expressed
by the extract obtained under the temperature of 60 °C,
using SSR of 1:20 and methanol concentration of 70%. In
the case of reduction capacity measured by CUPRAC as-
say, the lowest activity (3.71 mmol TE/g) was expressed
by the sample obtained under the following conditions:
temperature 20°, SSR 1:20, and methanol concentration
30%. By keeping the same extraction temperature and
SSR and applying higher methanol concentration (70%),
the sample with the highest reduction capacity was ob-
tained (5.93 mmol TE/g). These conditions also provide
the extract with the highest reduction ability measured by
FRAP assay (3.16 mmol TE/g). Simultaneously, in this
point, the highest flavanol content was measured as well.
On the other hand, using higher SSR (1:40) in a combi-
nation with lower solvent concentration (30%) offers ex-
tract with minimal FRAP activity (1.92 mmol TE/g).

Fig. 5 Influence of investigated inputs on CUPRAC activity of T. indica seed extracts
The parameters of statistical significance of the
models for DPPH, CUPRAC, and FRAP are given in
the Tables 3 and 4. ANOVA results suggested good
adequacy between the quadratic model and experimental
results due to significant p value for the model (< 0.05)
and insignificant lack of fit (p > 0.05) in all three cases.
The p values of calculated regression coefficients
showed that linear product terms of methanol concentra-
tion and SSR had highly significant and significant influ-
ence on DPPH extract’s activity, respectively. Appropriate
matching of theoretical and experimental values was con-
firmed by high value of coefficient of determination
(0.715), while calculated value of coefficient of variance
(9.70%) supports fitness of the model. The polynomial
model for DPPH activity was described by the equation:
DPPH ¼ 3:32 þ 0:03X 1 −0:16X 2
þ 0:50X 3 −−0:02X 1 X 2 −0:07X 1 X 3
þ 0:06X 21 −0:05X 22 −0:15X 23
Fig. 6 Influence of investigated inputs on FRAP activity of T. indica seed extracts
Food Anal. Methods
Combination of each two extraction parameters on DPPH
activity of the obtained extracts is presented by 3D plots in
Fig. 4.
The plots from the Fig. 4 clearly show that increasing in
methanol concentration and temperature led to increasing of
DPPH activity of the extracts. Significant negative impact of
liner term of SSR singled out in the case of DPPH, CUPRAC,
and FRAP (Table 3) suggesting that low SSR would be opti-
mal for improved activity. It is assumed that increase of SSR
could cause extraction of concomitant compounds with low
bioactivity reducing the content of antioxidants in obtained
extracts.
The coefficients of determination of both reduction capac-
ity assays (CUPRAC and FRAP) were high (0.864 and 0.849,
respectively) which confirms good fit among experimental
and theoretical values. In both cases, quite low values of co-
efficients of variance (5.66 and 6.56%, respectively) confirm
the fitness of the model. According to the results from the
ANOVA test, polynomial models for CUPRAC and FRAP
ð5Þ were obtained and are given in Eqs. 6 and 7:
Food Anal. Methods

-1 1 -1 1 -1 1

A:Temperature = -0.830884 B:SSR = -0.999978 C:% Solvent = 0.999998

227.16 357.34 42.95 99.33 89.12 282.47

TP [mg GAE/g] = 330.814 Flavonol [mg CE/g] = 90.2333 Tannin [mg CE/g] = 221.56

2.1 3.87 3.71 5.93 1.92 3.16

DPPH [mmol TE/g] = 3.9029 CUPRAC [mmol TE/g] = 5.83743 FRAP [mmol TE/g] = 3.16

Fig. 7 Numerical multi-response optimization results obtained to achieve maximal response values

CUPRAC ¼ 4:79−0:03X 1 −0:14X 2 þ 0:70X 3 −0:01X 1 X 2 ð6Þ FRAP ¼ 2:32−0:02X 1 −0:12X 2 þ 0:37X 3 þ 0:04X 1 X 2 −0:07X 1 X 3 ð7Þ

−0:18X 1 X 3 −0:09X 13 þ 0:01X 21 þ 0:03X 22 −0:11X 23 −0:04X 13 þ 0:07X 21 þ 0:10X 22 þ 0:04X 23

Table 5 Characterization of the compounds found in the analyzed extract of T. indica

No. tR (min) [M-H]− m/z m/z (% base peak) Assigned identification

1 1.7 377 MS2 [377]: 341 (100) Disaccharide (HCl adduct)

MS3 [377→341]: 179 (100), 161 (34), 143 (20), 131 (14), 113 (10)
2 2.0 191 MS2 [191]: 173 (38), 111 (100) Citric acid
3 2.5 405 MS2 [405]: 191 (100) Citric acid derivative
MS3 [405→191]: 111 (100)
4 3.5 431 MS2 [431]: 299 (13), 137 (100) Hydroxybenzoyl-hexose-pentose
5 4.3 321 MS2 [321]: 303 (100), 259 (65), 187 (35), 173 (33) Unknown
6 5.3 353 MS2 [353]: 191 (100), 179 (39), 135 (12) CQA
7 5.6 153 MS2 [153]: 109 (100) Protocatechuic acid
8 6.7 879 MS2 [879]: 727 (100), 709 (69), 547 (32), 439 (10) Procyanidin dimer digallate
MS3 [879→727]: 709 (100), 575 (13), 557 (12)
MS4 [879→727→709]: 691 (43), 557 (100)
9 6.7 577 MS2 [577]: 451 (51), 425 (94), 407 (100), 289 (33), 287 (31) Procyanidin dimer
10 7.3 303 MS2 [303]: 259 (100), 241 (48), 187 (40) Unknown
11 8.8 353 MS2 [353]: 191 (100), 173 (6) CQA
12 10.6 865 MS2 [865]: 847 (35), 739 (58), 713 (27), 695 (100), 577 (44), 575 (31), Procyanidin trimer
425 (28), 407 (47), 289 (14), 287 (22)
13 10.7 577 MS2 [577]: 451 (36), 425 (100), 407 (76), 289 (32), 287 (15) Procyanidin dimer
14 12.0 289 MS2 [289]: 245 (100), 205 (43), 203 (23) Epìcatechin
15 13.4 543 MS2 [543]: 319 (100) Unknown
MS3 [543→319]: 273 (100), 255 (39), 137 (23)
16 14.2 865 MS2 [865]: 739 (36), 713 (35), 695 (100), 577 (80), 451 (27), 407 (44), 287 (41) Procyanidin trimer
17 15.5 1153 MS2 [1153]: 983 (100), 865 (97), 863 (57), 739 (25), 577 (17), 575 (25), 451 (8), 407 (16) Procyanidin tetramer
18 19.6 609 MS2 [609]: 301 (100) Rutin
MS3 [609→301]: 179 (100), 151 (51)
19 20.9 463 MS2 [463]: 301 (100) Quercetin-O-hexoside
MS3 [463→301]: 271 (66), 255 (100), 179 (34), 151 (95)
20 21.4 575 MS2 [575]: 449 (92), 423 (100), 407 (27), 289 (43) Procyanidin dimer
21 23.6 623 MS2 [623]: 315 (100), 300 (33) Isorhamnetin-O-rutinoside
22 23.9 433 MS2 [433]: 271 (100), 151 (10) Naringenin-O-hexoside
23 24.3 575 MS2 [575]: 449 (71), 423 (100), 287 (49) Procyanidin dimer
24 39.1 327 MS2 [327]: 291 (92), 229 (76), 211 (90), 209 (57), 171 (100) Oxo-dihydroxy-octadecenoic acid
25 40.5 329 MS2 [329]: 311 (28), 293 (31), 229 (100), 211 (26), 209 (12) Trihydroxy-octadecenoic acid
 Food Anal. Methods

Illustration of the influence of each parameter as well as
their combination on CUPRAC and FRAP activities is given
in Figs. 5 and 6, respectively.
Data from the Fig. 5 shows that in the beginning, the in-
creasing in SSR led to slight decreases in reduction activity, but
after some points, the opposite effect has occurred; thus, the
further increase in SSR led to increasing in reduction activity
measured by CUPRAC assay. In the case of solvent concen-
tration, it was noticed that increasing in methanol concentra-
tion led to increase in the activity of the extracts. Statistical
analysis implies that linear term of methanol concentration
had highly significant impact on CUPRAC activity, which is
illustrated in Fig. 5 where it can be seen that by increasing the
solvent concentration, the CUPRAC activity sharply increases.
Additionally, ANOVA results showed that cross product term
of temperature and methanol concentration possess significant
influence on CUPRAC. Similar cross product effect was ob-
served in the case of TFC suggesting that elevated temperature
combined with high solvent concentration would decrease of
bioactivity and milder temperature should be used to prevent
this.
In the case of FRAP activity, highly significant influence
was expressed by both SSR and solvent concentration (p <
0.01). Both influences are clearly visible in 3D plots at Fig. 6,
where it can be also seen a positive impact of temperature on
FRAP activity.
pattern typical of a disaccharide compound of two hexoses, most
probably glucose. Compound 2, with [M-H]- at m/z 191 and base
peak at m/z 111, corresponded to citric acid, whereas compound
3 was a derivative. Compound 4 displayed neutral losses of
132 Da (431 → 299) and 162 Da (299 → 137), so it was tenta-
tively characterized as hydroxybenzoyl-hexose-pentose.
Compounds 6 and 11 presented deprotonated molecular
ions at m/z 353 and base peak at m/z 191, characteristic of
caffeoylquinic acids. Their mass spectra and retention times
were compared with those of analytical standards, unequivo-
cally identifying compounds 6 and 11 as neochlorogenic acid
and chlorogenic acid, respectively. Compounds 7, 14, and 18
were also identified by comparison with the corresponding
analytical standards of protocatechuic acid, epicatechin, and
rutin, respectively.
Eight proanthocyanidins, all of them procyanidins (com-
posed of (epi)catechin units), were characterized in the ana-
lyzed extract of T. indica. Compound 8 was characterized as a
procyanidin dimer digallate (Rockenbach et al. 2012); com-
pounds 9, 13, 20, and 23 were procyanidin dimers, whereas
compounds 12 and 16 were trimers (Sarnoski et al. 2012) and
compound 17 a tetramer (Kajdžanoska et al. 2010).
Compounds 19, 21, and 22 were flavonoid O-glycosides,
displaying neutral losses of 162 Da (hexoside) and 308 Da
(rutinoside) to yield quercetin (m/z 301), isorhamnetin (m/z

Table 6 Quantitation of the main phytochemicals in the analyzed

Optimization of Extraction Process extract of T. indica (mg g−1 DE; n = 3)

In order to find extraction conditions which will ensure extract No. Assigned identification Concentration
with the highest of TPC, TFC, and TTC but also with the
Procyanidins
highest DPPH, CUPRAC, and FRAP ability, the optimization
8+9 Dimers 0.82 ± 0.06
process was done. Previously used range of the parameters’
12+13 Trimer + dimer 6.0 ± 0.3
values was used for the optimization process as well. The
16 Trimer 4.3 ± 0.2
obtained results suggest the following conditions: temperature
17 Tetramer 2.7 ± 0.2
of 23.38 °C, SSR of 1:20, and methanol concentration of
20 Dimer 0.36 ± 0.02
69.99%. Predicted values of the responses are given in Fig. 7.
Total 14.2 ± 0.5
Flavonoids
Phytochemical Characterization 14 Epicatechin 3.2 ± 0.2
18 Rutin 0.083 ± 0.005
Mass fragmentation data, used for individual phytochemicals 19 Quercetin-O-hexoside 0.16 ± 0.1
characterization, is shown in Table 5. Twenty-one compounds Total 3.4 ± 0.2
were identified or tentatively characterized in the obtained Other compounds
optimum conditions. Most of the identified compounds were 4 Hydroxybenzyl-hexose-pentose 3.7 ± 0.2
procyanidins, along with epicatechin and glycosides of quer- 6 Neochlorogenic acid 0.31 ± 0.02
cetin, isorhamnetin, and naringenin. These results are in agree- 7 Protocatechuic acid 1.56 ± 0.09
ment with previous studies in T. indica, in which a small 11 Chlorogenic acid 1.06 ± 0.04
number of phenolics have been previously reported (Razali Total 6.6 ± 0.2
et al. 2012; Sudjaroen et al. 2005). A brief explanation of TIPC* 24.2 ± 0.6
the identification follows.
Compound 1 suffered the neutral loss of 36 Da (HCl) to yield TIPC total individual phenolic content.
the base peak at m/z 341, which exhibited the fragmentation Italic values indicate total amounts of main phytochemical groups.
Food Anal. Methods
315), and naringenin (m/z 271). The mass fragments were
compared with both analytical standards and scientific
literature.
Finally, compounds 24 and 25 were characterized as
oxylipins (Van Hoyweghen et al. 2014), a group of biologi-
cally active compounds coming from the oxidative metabo-
lism of polyunsaturated fatty acids.
Phenolic Quantification
Calibration graphs (0.5–100 mg mL−1) were prepared with the
following analytical standards: chlorogenic acid, epicatechin,
neochlorogenic acid, procyanidin B2, procatechuic acid, querce-
tin, and rutin. Repeatability (n = 9) and intermediate precision (n
= 9, three consecutive days) were lower than 3 and 8%, respec-
tively. Thirteen compounds were individually quantified, and
TIPC (total individual phenolic content) was defined as the sum
of all the quantified compounds. Results are shown in Table 6.
Procyanidins (dimers and trimers) accounted for approxi-
mately 59% of TIPC, whereas compounds 4 (hydroxybenzyl-
hexose-pentose) and 14 (epicatechin) were also abundant,
representing 15 and 13% of TIPC, respectively. In a previous
report in seeds and pericarp of T. indica fruits (Sudjaroen et al.
2005), authors also reported epicatechin and procyanidins as
the main compounds, although in lower amounts than the ones
here found: approximately 44% and 10% of the concentra-
tions here observed for procyanidins and epicatechin, respec-
tively. This result is due to the different morphological parts
analyzed, although the trend in the main compounds is the
same.
Acknowledgments Technical and human support provided by CICT of
Universidad de Jaén (UJA, MINECO, Junta de Andalucía, FEDER) is
gratefully acknowledged.
Compliance with Ethical Standards
Conflict of Interest Cvetanović, A. declares that she has no conflict of
interest. Uysal, S. declares that she has no conflict of interest. Pavlić, B.
declares that he has no conflict of interest. Sinan, K.I. declares that he has
no conflict of interest. Llorent-Martínez, E.J. declares that he has no
conflict of interest. Zengin, G. declares that he has no conflict of interest.
Ethical Approval This article does not contain any studies with human
or animal subjects.
Informed Consent Informed consent is not applicable in this study.
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