Combined High-Pressure Extraction Process to Obtain

Phenolic Compounds From Biquinho Pepper (Capsicum

chinense)
Ana Carolina de Aguiara*, Ana Paula Machadoa, Julian Martíneza
a
Department of Food Engineering, School of Food Engineering, University of Campinas, UNICAMP,
13083-862, Campinas, SP, Brazil
*aguiarea@gmail.com

ABSTRACT
It was recently discover by our research group that biquinho (Capsicum chinense), a pepper
native of Brazil is rich in capsinoids (compounds similar to capsaicinoids, but do not present
their characteristic pungency) and phenolic compounds. This work proposes a combined
extraction method to recover capsinoids and phenolic compounds through supercritical fluid
extraction (SFE) followed by pressurized liquid extraction (PLE) from biquinho peppers as an
alternative to add value to this cultivar. SFE was carried out using carbon dioxide as solvent
under the previously optimized condition of 15 MPa and 50 °C. After the extraction of the
nonpolar fraction by SFE, the defatted biquinho pepper (DBP) was subjected of PLE using
mixtures of ethanol and water (50, 75 and 100% of ethanol, w/w) as solvent at different
temperatures (45, 55 and 65 °C) and 10 MPa. The extracts were evaluated in terms of global
yield and total phenolic content by the Folin-Ciocalteu’s method. Regarding the total phenolic
extraction, the condition of 65 °C and 75% ethanol resulted in the highest phenolic
compounds concentration (214±6 mg GAE/g DBP). These preliminary results suggest that the
combined process of SFE+PLE can be an alternative of toxic solvent free processes to obtain
phenolic compounds of economic interest from biquinho peppers.

INTRODUCTION
Biquinho peppers (Capsicum chinense) is a sweet pepper native to the Brazilian
territory and presents considerable concentrations of capsinoids in its composition [1].
Capsinoids (found in some varieties of sweet peppers) have physiological effects equivalent
to those of capsaicinoids, without however expressing the characteristic of pungency [2].
Our research group studied the extraction process of capsinoids with supercritical CO2
(SC-CO2) and conventional methods from freeze-dried biquinho peppers and the results
indicated that the SC-CO2 at 15 MPa and 50 °C was selective for these compounds [1]. Since
SC-CO2 preferentially extracts nonpolar compounds, the defatted biquinho pepper (DBP),
which is the residue of the SFE process, presents a great potential to obtain polar bioactive
compounds, as the phenolic compounds.
Obtaining phenolic compounds from natural sources is of great interest to the food,
cosmetic and pharmaceutical industry. The extraction process is a crucial step for the
successful isolation, identification and subsequent application of these compounds [3].
Pressurized liquid extraction (PLE) is suitable for polar compounds, among then, the
phenolic compounds [4]. The major advantages of employing PLE are related to higher
extraction yields, minimized use of solvents (usually GRAS, such as ethanol, water and their
mixtures), reduced extraction times and the possibility of automation and process scale-up [5].
Thus, the aim of this study was first obtain capsinoids rich extracts from lyophilized
biquinho pepper using SFE and then apply sequentially the PLE process with ethanol, water
and their mixtures as solvents in defatted biquinho pepper to recover bioactive phenolic
compounds.

MATERIALS AND METHODS

Chemicals
Ethyl alcohol P.A. (Synth, SP, Brazil), water obtained from a Milli-Q water
deionization system (Millipore, Bedford, MA, USA) and CO2 (99% purity, White Martins,
Brazil) were used as solvents. For the total phenolic content, the Folin-Ciocalteu reagent and
gallic acid were purchased from Sigma-Aldrich (SP, Brazil).

Sample preparation
Ripe fruits of biquinho pepper were purchased on the market CEASA (Centrais de
Abastecimento de Campinas S.A), Campinas, Brazil. The fruits were selected considering
their physical integrity and ripeness. The fresh peppers were subjected to freeze-drying in a
bench lyophilizer (L101-LioTop/LIOBRAS, SP, Brazil) for 72 h at approximately -42 °C.
After drying, all samples were ground in order to homogenize them and reduce the resistance
to mass transfer during the later stages of extraction.

Supercritical Fluid Extraction (SFE) process

The lyophilized biquinho peppers was submitted to SFE (in a home-made SFE unit) in
order to remove the nonpolar compounds from the sample. In each SFE assay, approximately
15 g of and glass spheres was used to form a fixed bed inside a 54.37 cm3 stainless steel
column. The SFE process conditions were chosen based on previous work, in which the best
condition to recover capsinoids were identified was 15 MPa and 50 °C [1]. The CO2 flow rate
was 2.43 10-4 kg/s and the extraction time was 120 min, resulting in a mass ratio between
solvent and feed (S/F) of 117 kg CO2/kg feed.

Pressurized Liquid Extraction (PLE)

PLE was performed using the dynamic extraction method with pure ethanol and
mixtures of ethanol and water as solvents. Approximately 3.0 g of DBP was used to form a
fixed bed inside a 10.9 cm3 stainless steel column. The experimental design was based on full
factorial with two variables and three levels in duplicate (2 × 32 = 18 experiments). The
studied factors were ethanol percentage in the solvent and temperature. The ethanol
percentages were 50, 75 and 100% and the temperatures were 50, 60 and 70 °C. Temperature
levels were selected above the room temperature and the upper limit was 65 °C to prevent
thermal degradation. The chosen ethanol percentages were based on previous laboratory
studies [6]. Pressure was kept constant at 10±0.5 MPa and the solvent flow rate was 2.30, 3.52
and 3.82 mL/min for 50, 75 and 100% ethanol, respectively (mass flow rate constant at 3.0
g/min). The extraction time was 60 min defined by preliminary tests. The extracts were
collected in glass flasks and stored under freezing (−18 °C) for further analyses. All extraction
assays were carried out in duplicate.
Extract evaluation
Global yield (X0): global yield was calculated by the mass ratio between dried extract (mext)
and mass of dry sample (F), as shown in Equation (1). To determine the mext, 4.0 mL of
extract were dried at 70 °C in stove (Fanem, 320-SE, São Paulo, Brazil) until constant weight.
m (1)
X 0  ext 100
F
Total phenolic content (TPC): TFC was determined according to the Folin–Ciocalteu's
method [7], with modifications. About 0.5 mL of extract solution (diluted in ethanol) and 2.5
mL Folin-Ciocalteu reagent solution (10%, v/v) were mixed and incubated for 3 min. Then,
2.0 mL of saturated sodium carbonate solution was added and the mixture was kept in dark
for 2 h. The absorbance was read at 760 nm in spectrophotometer (AquaMate, Orion 8000,
Thermo Scientific). The results were expressed in mg gallic acid equivalent (GAE) per g of
DBP.

PLE kinetic experiments

The PLE kinetic experiments were performed at the extraction condition that presented
the highest total phenolic content: 65 °C and 75% ethanol. Kinetic data were performed by
measuring the global yield and total phenolic content as a function of the extraction time.

RESULTS
The supercritical extraction process at 15 MPa and 50 °C from lyophilized biquinho
pepper showed a global yield of approximately 5% (g extract / 100 g lyophilized pepper).
This extraction yield value was about four times higher than that obtained by Aguiar et al. [1]
and this difference can be attributed due to the high variability characteristic of peppers of the
genus Capsicum and possible differences in moisture content and the granulometry of the
extracted material.
6

5
SFE Extraction Yield (%)

0
0 10 20 30 40 50 60 70 80 90 100 110 120 130
Time (min)
Figure 1. Kinetic SFE curve for biquinho pepper at 50 °C, 15 MPa and CO 2 flow rate of 2.43 10-4 kg/s.

Table 1 presents the results of global yield (X0) and total phenolic content (TPC) for
the PLE experiments performed under different operating conditions. Among the operating
conditions studied, the highest values of X0 were obtained with ethanol concentration of 50%
for all temperatures studied (~ 40%). It was not possible to establish a relation between
temperature and extraction yield, although it was phenomenologically expected that the
increase in temperature would result in higher extraction yields [6].
Regarding the influence of the ethanol percentage in the mixture, it is well establish
that the use of solvent mixtures can increase the extraction yields due to improvement of
solubility and increasing the interaction of target compound with the extraction solvent [8].
The highest extraction yields were obtained with the 50% ethanol mixture. At this
composition, the high concentration of water present in the solvent mixture is able to help
breaking of matrix and matrix-solute (hydrogen) bonds, which may ultimately favor the
desorption of matrix-bound compounds [8].

Table 1. PLE extraction conditions, global yields (X0, %) and TPC (mg GAE/ g DBP) of the different extracts
obtained from DBP.
T (°C) % Ethanol in water (w/w) X0 (%, w/w) TPC (mg GAE/g DBP)
45 50 40.8±0.9 189±6
75 37±3 201±8
100 19±1 142±3
55 50 40±3 190±4
75 39±2 210±5
100 12±1 85±3
65 50 39±3 190±9
75 40±2 214±6
100 25±2 171±2

With regard to the TPC extraction by PLE, the condition that provided the highest
yield was 75% ethanol and 65 °C (214 mg GAE/g DBP), whereas the lowest yield was
obtained at 100% ethanol and 55 °C (85 mg GAE/g DBP). This result are in agreement with
the observed by Viganó et al. [6], that the mixture of ethanol and water at 75% ethanol
changes the solvent polarity in such way that improves significantly the extraction of phenolic
compounds. Since the PLE conditions of 75% ethanol and 65 °C provided the highest
concentration of phenolic compounds, this was the condition chosen for the extraction kinetic
tests.
Figure 2 displays the global yield curves (X0) (left) and TPC (right) for PLE at 75%
ethanol and 65 °C.

50 250
TPC (mg GAE/ g DBP)

40 200

30 150
X0 (%)

20 100

10 50

0 0
0 20 40 60 0 20 40 60
Extraction time (min) Extraction time (min)
Figure 2. Experimental PLE (75% etanol and 65 °C) curves from defatted biquinho pepper: (left) global yield,
(right) TPC.
 A visual analysis of the extraction curves allows to infer that the extraction kinetic of
the phenolic compounds follows the same behavior of the kinetics of the global yield. For
both curves, from the 30 min of extraction, there is a significant decrease in the mass transfer
rate, which suggests that the diffusion mechanism is predominant from this point.

CONCLUSION
The results obtained so far indicate that the proposed combined high-pressure
extraction process (SFE + PLE) to obtain capsinoids and phenolic compounds from biquinho
pepper may be an interesting alternative for the industrial exploitation of this cultivar. With
respect to the PLE process, the highest extraction yield was obtained with 50% ethanol (~
40% for all temperatures studied) and in terms of total phenolics extraction, the condition of
75% ethanol and 65 °C was more effective (214 mg GAE/g DBP). A better understanding of
the influence of the parameters in the PLE process is expected after the statistic analysis of
data, in addition to separation, identification and quantification by HPLC-MS/MS of the
phenolic compounds present in the extracts. The quantification of the capsinoids in the
different points of the SFE kinetics will also be of extreme importance for a more detailed
evaluation of the proposed process.

ACKNOWLEDGEMENTS
The authors wish to thank FAPESP for the PD-BR scholarship (Process 2015/18119-
0) and for the financial support.

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