The Journal of Supercritical Fluids 147 (2019) 205–212

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The Journal of Supercritical Fluids

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Extraction from Leaves of Piper klotzschianum using Supercritical Carbon T

Dioxide and Co-Solvents
⁎
Rafaely N. Limaa, , Adauto S. Ribeirob, Lucio Cardozo-Filhoc, Diógenes Vedoyd, Pericles B. Alvesa
a
Department of Chemistry, Federal University of Sergipe, São Cristóvão, Sergipe, Brazil
b
Department of Biology, Federal University of Sergipe, São Cristovão, Sergipe, Brazil
c
Departament of Chemistry Engineering, State University of Maringá, Maringá, Paraná, Brazil
d
Department of Chemical and Materials Engineering, University of Alberta, Edmonton, Alberta, Canada

G R A P H I C A L A B S T R A C T

A R T I C LE I N FO A B S T R A C T

Keywords: The objective of this study was to investigate the supercritical carbon dioxide extraction of dry leaves of Piper
Piper klotzschianum klotzschianum using a factorial experiment (32) and the Taguchi L-9 method to optimize the extraction yield of
Pipercallosidine secondary metabolites with pharmaceutical applications. In the first part of this study where only temperature
Piperovatine and pressure were investigated, the highest extraction yield (1.36%) was obtained at 353 K 220 bar. In the
Herbal plant
second part, the addition of organic co-solvents as 5% of methanol significantly improved the extraction yield to
Supercritical fluid extraction
2.18% representing an increase of 40 % compared to extraction using carbon dioxide alone. Chemical analysis of
the extracts revealed that germacrene D, pipercallosidine, 14-oxy-α-muuroleno, bicyclogermacrene and (E)-
caryophyllene were the major constituents present at the supercritical extract. The extraction of bioactive ses-
quiterpenes and amides as pipercallosidine and piperovatine, demonstrate the great potential of supercritical
technique to extract valuable chemical compounds, including ones with phytotherapeutic properties.

1. Introduction first studied by Nascimento et al., [1] who described the phytochemical
composition of its essentials oil and larvacidal activity against Aedes
Piper klotzschianum has been recently cataloged in Brazil and is aegypti and Artemia salina.
known to have anesthetic and spicy effects when tasted. This plant was The P. klotzschianum species belongs to the genus Piper and is known

⁎
Corresponding author. Av. Marechal Rondon, S/N, Campus Universitário, Federal University of Sergipe, São Cristóvão, 49100-000, SE, Brasil
E-mail address: raffinha_lima@hotmail.com (R.N. Lima).

https://doi.org/10.1016/j.supflu.2018.11.006
Received 15 April 2018; Received in revised form 1 November 2018; Accepted 3 November 2018
Available online 07 November 2018
0896-8446/ © 2018 Elsevier B.V. All rights reserved.
R.N. Lima et al. The Journal of Supercritical Fluids 147 (2019) 205–212

Table 1 Table 3
SFE parameters based on factorial experiment 32. ANOVA analysis of variance.
Entry Variable 1 Variable 2 Extract SS Df MS F P
T (K) P (bar) Yield (%)
Co-solvent 1.13 2.00 0.57 288.95 0.00
1 333 220 1.24 Temperature 0.02 2.00 0.01 4.76 3.9E-02
2 353 200 0.94 Pressure 0.09 2.00 0.05 23.58 2.6E-04
3 313 180 1.07 (%) Co-solvent 0.27 2.00 0.14 69.95 3.0E-06
4 313 200 1.22 Residual 0.02 9.00 0.00
5 353 220 1.36
6 333 180 1.26 SS, total quadratic sum; df, degrees of freedom; MS, quadratic mean; F, esti-
7 353 180 0.89 mated variance; p, critical value.
8 313 220 1.30
9 333 200 1.27

Analysis made in 120 min, flow rate of 2 mL min-1 and pump temperature of
278 K.

Table 2
SFE parameters based on Taguchi L-9 method.
Entry Variable 1 Variable 2 Variable 3 Variable 4 Extract
Co-solvent T (K) P (bar) (%) Co-solvent Yield (%)

1 Methanol 313 180 1 1.68 ± 0.03

2 Methanol 333 200 3 1.81 ± 0.05
3 Methanol 353 220 5 2.18 ± 0.04
4 Ethanol 313 200 5 1.32 ± 0.04
5 Ethanol 333 220 1 1.24 ± 0.00
6 Ethanol 353 180 3 1.26 ± 0.02
7 Isopropanol 313 220 3 1.64 ± 0.04
8 Isopropanol 333 180 5 1.74 ± 0.02
9 Isopropanol 353 200 1 1.43 ± 0.02

Analysis made in 120 min, flow rate of 2 mL min-1 and pump temperature of Fig. 3. Variable influence of extractions using temperature, pressure, co-sol-
278 K. vents, and (%) co-solvents as factors.

from the Piper klotzschianum leaves. This extraction can be accom-

Fig. 1. Yield as a function of time for SC-CO2.
Fig. 2. Yield versus time for SC-CO2 extractions using different co-solvents,
pressures and temperatures.
for having diverse biological properties, including psychotropic, anti-
microbial, antioxidant, and cytotoxic effects [2]. In order to fully realize
these biological applications, the organic compounds must be extracted
plished using a number of different techniques. For example, hydro-
distillation uses water as a solvent and enables extraction of essential
oils. Organic solvents can also be used (e.g. hexane, methanol,
chloroform, ethanol, and ethyl acetate) in a temperature independent
fashioned to obtain plant extracts [3,4]. However, these extraction
techniques produce large volumes of waterwaste and/or use of toxic
solvents.
Supercritical fluid extraction (SFE) is the main alternative technique
to replace these traditional solvent extraction techniques. In fact, the
extraction of essential oils and/or extracts at high pressure using SFE
has sparked interest among the food, cosmetic, and pharmaceutical
industry as an environmentally friendly alternative to conventional
extraction methods [5].
Furthermore, the thermal degradation and decomposition of labile
components can be avoided in supercritical carbon dioxide extraction
(SC-CO2) because low temperatures are used during the extraction. The
SC-CO2 technique also allows selective extraction of a single compound
or class of compounds, as a result of changes in solvent density and co-
solvent use. The final product is an extract free of any solvent or other
contaminants from the extraction process [6].
SFE technique has been tested for different species of Piper such as P.
nigrum [8–12], P. regnellii [12,13], P. amalago [14], P. methysticum
[3,15,16], and P. piscatorum [17], showing great results. However, ex-
traction studies using P. klotzschianum are very limited in the literature
and, to the best of our knowledge, SFE technique has not been used to
extract chemical compounds from P. klotzschianum [1,7].
Therefore, the main objectives of the present work were to evaluate
the composition and mass percent yields of P. klotzschianum extracts
obtained from dried leaves using the SFE method as well as to evaluate
the effect of the co-solvents on the yield and type of the extracts re-
covered from the leaves. To reach this goal, two methodologies were
applied, (1) SC-CO2 using a factorial design of 32 where three levels of

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R.N. Lima et al. The Journal of Supercritical Fluids 147 (2019) 205–212

Table 4
Compounds identified in SC-CO2 of P. klotzschianum extracts.
180 bar 200 bar 220 bar 180 bar 200 bar 220 bar 180 bar 200 bar 220 bar
313 K 313 K 313 K 333 K 333 K 333 K 353 K 353 K 353 K

IRR exp. IRR lit. Compound (%) (%) (%) (%) (%) (%) (%) (%) (%)
Non-oxygenated sesquiterpenes
1381 1374 α-copaene 0.68 0.83 0.80 0.58 0.63 ± 0.04 0.68 0.65 0.74 0.69
1391 1387 β-cubebene - - - - 0.59 ± 0.11 0.84 0.89 0.79 0.67
1421 1417 (E)-caryophyllene 7.67 8.09 8.49 7.15 6.48 ± 0.06 6.19 7.08 7.57 6.94
1438 1432 α-trans-bergamotene - - - - 0.73 ± 0.02 0.93 0.89 0.73 0.79
1466 1452 α-humulene 0.68 0.71 0.65 0.68 0.60 ± 0.04 0.58 0.63 0.72 0.73
1482 1478 γ-muurulene 0.83 0.76 0.79 0.81 0.65 ± 0.02 0.64 0.77 0.92 0.91
1491 1484 germacrene D 18.51 18.46 19.07 18.93 15.53 ± 0.39 13.09 16.47 17.06 15.10
1506 1500 bicyclogermagrene 10.88 11.70 11.49 11.62 9.57 ± 0.31 8.75 9.68 10.21 9.44
1520 1513 γ-cadinene 0.81 0.80 0.82 0.92 0.69 ± 0.02 0.66 0.85 1.44 0.82
1522 1511 δ-amorphane 1.28 1.36 1.39 1.46 1.16 ± 0.07 1.13 1.35 1.16 1.30
1567 1559 germacrene B 1.19 1.29 1.16 1.26 1.17 ± 0.19 0.93 1.11 0.84 1.15
Oxygenated sesquiterpenes
1583 1577 spathulenol 6.53 4.91 6.30 7.01 5.93 ± 0.46 5.09 7.81 7.43 6.74
1590 1582 caryophylene oxide 4.38 4.05 4.80 4.88 3.99 ± 0.25 3.85 5.27 4.98 4.79
1777 1767 14-oxi-α-muurolene 13.79 12.26 13.56 14.92 12.86 ± 1.00 11.59 14.20 14.11 12.90
1866 1863 cis-acid thujopsenic - - - - 0.50 ± 0.12 - 0.87 1.23 -
Other classes
1500 1500 n-pentadecane 2.49 2.33 2.44 2.50 1.99 ± 0.09 1.88 2.31 2.41 2.59
1699 1700 n-heptadecane 1.74 1.56 1.61 1.94 1.48 ± 0.15 1.36 1.75 1.96 1.70
2816 2818 squalene 4.11 3.64 4.01 4.41 11.00 ± 1.04 4.50 4.90 4.62 4.39
2093 2095 methyl linoleate - - - - 1.35 ± 0.30 1.30 - - -
2140 2132 linoleic acid 4.24 5.45 3.42 5.15 7.40 ± 1.63 9.60 7.42 5.25 5.33
2552 piperovatine 1.14 1.83 1.35 0.80 0.74 ± 0.30 1.54 - 0.61 0.89
2663 pipercallosidine 12.84 14.20 12.95 9.64 5.29 ± 0.84 16.08 7.88 9.45 13.41
3142 3149 α-tocopherol 2.07 2.22 2.29 1.92 2.85 ± 0.26 3.41 1.49 1.87 2.42
Not identified compounds
2146 NI [MS: 80 (100), 53 (66), 41 0.70 - - - - - - - -
(61),
43 (56), 96 (49), M+. 179
(42)]
2155 NI [MS: 41 (100), 55 (84), 79 1.24 - - - 0.99 ± 0.17 2.40 1.18 1.39 2.56
(67),
95 (39), 43 (35), M+. Abs.]
2901 NI [MS: 43 (100), 57 (91), 71 0.56 0.65 0.52 0.83 3.14 ± 0.73 0.98 0.75 0.67 0.79
(59),
41 (25), 85 (23), M+. Abs.]
3001 NI [MS: 43 (100), 57 (93), 71 - - - - 0.45 ± 0.09 - - - -
(61),
41 (47), 85 (36), M+. Abs.]
3100 NI [MS: 57 (100), 43 (91), 71 1.18 1.78 1.26 1.65 1.45 ± 0.22 1.37 1.57 1.23 1.44
(53),
85 (35), 41 (29), M+. Abs.]
3300 NI [MS: 57 (100), 43 (87), 71 0.46 1.12 0.83 0.94 0.81 ± 0.11 0.63 2.23 0.61 1.51
(58),
41 (27), 85 (27), M+. Abs.]
Total identified compounds 95.86 96.45 97.39 96.58 93.18 94.62 94.27 96.10 93.70

RRIexp.,Experimental retention index; RRIlit.,literature retention index; (%),average relative area (triple) ± standard deviation; NI, not identified; MS, Mass spectra,
Abs, Absent.

Fig. 4. Total ion chromatogram of P. klotzschianum extract by SC-CO2 at 333 K and 200 bar.

temperature and pressure were tested and (2) a multivariate analysis 2. Materials and Methods
using the Taguchi model (L-9), where three levels of experimental
conditions were tested, naming co-solvent type and percentage, tem- 2.1. Plant material
perature and pressure.
P. klotzschianum (ASE 29960) was collected in the Ecological Park
Serra de Itabaiana SE, the sample was identified in partnership with

207
R.N. Lima et al.
Fig. 5. Comparison of terpene classes identified by GC-MS of extracts obtained
using SC-CO2.
Prof. Dr. Adalto Ribeiro (UFS). A voucher specimen was deposited in
the herbarium of the Federal University of Sergipe-UFS. The leaves
were dried in oven (Marconi MA0351336 model) with continuous air
flow at 322 K for 48 h. The dried leaves were triturated using four
knives mill (Marconi, MA680 model) and the particle size of 0.85 mesh
was selected for screening samples since this particle size presented the
best performance during preliminary tests performed in our laboratory.
2.2. Supercritical fluid extraction procedure
The extraction experiment was conducted in a laboratory bench
scale supercritical extraction unit, using carbon dioxide-(CO2) as sol-
vent and modifier agents (methanol, ethanol and isopropanol).
Additional details about procedure apparatus have been already de-
scribed [12–14].
2.3. Experimental conditions
The factorial design 32 was applied totaling nine experimental
conditions (Table 1), the optimization of extraction process from dried
leaves of P. klotzschianum used the variables, pressure (180 bar, 200 bar,
220 bar) and temperature (313 K, 333 K, 353 K). The center point 333 K
and 200 bar was performed in triplicate.
Tests with four variables were applied to the Taguchi method re-
sulting in nine experimental conditions-(L-9) [18]. The variables used
Scheme 1. Fragmentation proposition of main fragments on piperovatine electron impact cleavage (70 eV).
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The Journal of Supercritical Fluids 147 (2019) 205–212
were pressure (180 bar, 200 bar, 220 bar), temperature (313 K, 333 K,
353 K), type of co-solvent (methanol, ethanol, isopropanol) and per-
centage of co-solvent (1%, 3%, 5%) (Table 2). The nine conditions were
tested in duplicate. The supercritical fluid extraction SFE operating
conditions were chosen based on the work available in the literature
[5].
It should be highlighted that Taguchi design was selected instead of
others experimental designs because this design allows the study of the
interactions between the 4 variables at 3 different levels, resulting in
fewer experiments (9 conditions). This type of design is recommended
when a limited amount of sample is available and one seeks to under-
stand the interaction between more than three variables in order to
achieve the optimal conditions [5].
2.4. Gas Chromatography / Mass Spectrometry (GC/MS)
Analyses were performed in GC/MS (QP 5050 A model, Shimadzu,
Japan) equipped with an automatic sampler injection AOC-20i
(Shimadzu). Separations were performed on a fused silica capillary
column Restek Rtx®-5SiMS (5% diphenyl-95% -dimethilpolisiloxane)
30 m x 0.25 mm internal diameter, 0.25 mM of film thickness at a
constant flow 5.0 helium at a rate of 1.0 mL min-1. The injection tem-
perature was 523 K, 1.0 μL (10 mg ml-1) of sample was injected with a
split ratio of 1:70. The oven temperature program started from 383 K,
with an increment of 5 K min-1 until 513 K, then at 10 K min-1 until
573 K, lasting for 13 min.
The molecules were ionized by electrons with energy of 70 eV and
the fragments analyzed by a quadrupole analyzer programmed to filter
fragments ions from m/z 40 to 600 Da and detected by an electron
multiplier. The data processing was performed by the GC/MS software.
2.5. Identification of extracted compounds
Relative retention index (RRI) calculation was performed to identify
the constituents of supercritical extract. The RRI was obtained based on
the equation [18], which uses the retention time of an n-alkane
homologous series (nC8 -nC38, AccuStandard®) and predicts the RRI of
unknown compounds. Having the RRI of no identified compound, it
was possible compare the experimental RRI with RRI from Adams book
[19] or from libraries as WILEY8, NIST105, NIST105 s, NIST21,
NIST107 and program NIST MS Search 2.0 identifying compounds that
R.N. Lima et al.
Scheme 2. Fragmentation proposition of main fragments on pipercallosidine electron impact cleavage (70 eV).
presented similarity index above 80% with already reported in litera-
ture.
3. Results and Discussion
3.1. Analysis of extraction yield obtained by SC-CO2
The extractor was packed with 16 g of dried P. klotzschianum leaves
at a relative humidity of 5.33%. The extractions were performed over
120 minutes, which was sufficient to extract most of the organic com-
pounds present in the P. klotzschianum leaves as indicated by the
straight slopes in the Figs. 1 and 2.
In the first part of this study where only temperature and pressure
were investigated, the lowest yield of pipercallosidine (5.29%, GC-MS
analysis) and piperovatine (0.74%, GC-MS analysis) were obtained for
the extract acquired at 333 K and 180 bar (1.27% extract yield).
However, the highest amides pipercallosidine (16.08%, GC-MS ana-
lysis) and piperovatine (1.54%, GC-MS analysis) percentages were
achieved at this same temperature, but when pressure was increased to
220 bar (1.24% extract yield).
It is important to highlight that similar extraction yields were ob-
tained at lower temperatures (313 K, 220 bar = 1.30%; 333 K,
200 bar = 1.27%; and 333 K, 180 bar = 1.26%). However, this simi-
larity did not follow the pipercallosidine and piperovatine yield when
the extracts were analyzed by GC-MS ranging from 5.29 to 12.95% and
from 0.74% to 1.35%, respectively.
These results suggest that the more reproducible yields of pi-
percallosidine (12.84-14.20%, GC-MS analysis) and piperovatine (1.14-
1.83%, GC-MS analysis) were obtained at 313 K regardless of the
pressure used (180, 200 and 220 bar) whereas the yield of these com-
pounds did not show a consistent trend when temperature was 333 or
353 K. Please note that the optimal point (i.e., the experimental con-
ditions that lead to the maximum extract yield of pipercallosidine and
piperovatine) could be beyond the selected maximum pressure and
temperature used in our study. We were not able to increase the pres-
sure and temperature beyond the values reported in this study due to
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The Journal of Supercritical Fluids 147 (2019) 205–212
limitations in the equipment used.
3.2. Analysis of extraction yield obtained by SC-CO2 using co-solvents
In the second part of this study, type and quantity of organic co-
solvent were also studied together with temperature and pressure. The
results show that the addition of organic co-solvents significantly im-
proved the extracts yield (Fig. 2). The highest yield of extract con-
taining pipercallosidine and piperovatine amides (2.18 %) was ob-
tained using 5% of methanol (353 K and 220 bar). In this case, the
addition of 5% of methanol increased the yield by 40 % compared to
extraction using carbon dioxide alone. The highest yields for the other
two co-solvents used were 1.74% - (333 K, 180 bar, isopropanol 5%)
and 1.32% - (313 K, 200 bar, ethanol 5%).
In addition, F test results (Table 3) show that the addition of co-
solvent and percentage of co-solvent were the most important factors in
the extraction yield (F = 288.95, F = 69.95), emphasizing the im-
portance of these co-solvents in the extraction yield. On the other hand,
pressure and temperature variables were the least influential factors in
the extraction process (Fig. 3). The low p values indicate that these
results do not occur indiscriminately.
3.3. Analysis of compounds obtained by SC-CO2
Composition and percentages of compounds identified in super-
critical fluid extracts using CO2 were similar in most conditions
(Table 4). The levels of germacrene-D (19.07%, 313 K, 220 bar), pi-
percallosidine (16.08%, 333 K, 220 bar), 14-oxy-α-muurolene (14.92%,
333 K, 180 bar), bicyclogermacrene (11.62%, 333 K, 180 bar), and (E)-
caryophyllene (8.49%, 313 K, 220 bar) were the highest present in the
extracts. Squalene (11.00%, 333 K, 200 bar) and linoleic acid (9.60%,
333 K, 220 bar) also presented major yields. The Fig. 4 shows the
chromatographic profile of SC-CO2 extract obtained at 333 K and
200 bar analyzed by GC-MS. This profile was similar to obtained with
the others conditions presented at Table 4.
Identification of the compounds obtained with SC-CO2 revealed
 R.N. Lima et al.

Table 5
Compounds identified in SC-CO2 using co-solvents of P. klotzschianum extracts.
333 K 353 K 313 K 353 K 313 K 333 K 313 K 333 K 353 K
220 bar 180 bar 200 bar 200 bar isopropanol 220 bar 180 bar 180 bar methanol 200 bar methanol 220 bar methanol
ethanol ethanol ethanol 1% isopropanol 3% isopropanol 1% 3% 5%
1% 3% 5% 5%

IRR exp. IRR lit. Compound (%) (%) (%) (%) (%) (%) (%) (%) (%)
Non-oxygenated sesquiterpenes
1381 1374 α-copaene 0.78 ± 0.02 - - 0.83 ± 0.00 1.14 ± 0.02 0.66 ± 0.17 - 0.80 ± 0.10 0.67 ± 0.06
1398 1387 β-cubebene - - - - - - - - 0.90 ± 0.09
1421 1417 (E)-caryophyllene 7.20 ± 0.07 5.85 ± 0.10 5.30 ± 0.26 8.77 ± 0.12 11.66 ± 0.05 6.59 ± 0.66 7.88 ± 0.07 8.46 ± 0.61 7.28 ± 0.39
1041 1432 α-trans- bergamotene - - - - 1.16 ± 0.02 0.61 ± 0.04 0.90 ± 0.11 - 0.87 ± 0.19
1460 1452 α-humulene 0.61 ± 0.02 - 0.63 ± 0.06 0.75 ± 0.04 1.00 ± 0.05 0.63 ± 0.11 0.67 ± 0.07 - 0.71 ± 0.05
1482 1478 γ-murrulene 0.83 ± 0.06 - 0.98 ± 0.09 0.74 ± 0.04 0.94 ± 0.07 0.60 ± 0.15 0.60 ± 0.07 - 1.06 ± 0.05
1491 1484 germacrene D 18.40 ± 0.05 9.75 ± 0.06 13.01 ± 0.08 19.16 ± 0.96 25.00 ± 0.11 15.64 ± 0.56 16.32 ± 0.08 20.22 ± 1.01 16.53 ± 0.70
1506 1500 bicyclogermagrene 13.35 ± 0.25 8.01 ± 0.03 10.20 ± 0.20 12.71 ± 0.62 15.41 ± 0.23 10.58 ± 0.68 11.03 ± 0.05 13.72 ± 0.42 11.79 ± 0.31
1522 1513 γ-cadinene 0.92 ± 0.04 - - 0.75 ± 0.01 0.95 ± 0.02 0.56 ± 0.22 0.62 ± 0.01 0.78 ± 0.01 0.99 ± 0.14
1520 1511 δ-amorfene 1.40 ± 0.05 - - 1.26 ± 0.10 1.47 ± 0.03 1.08 ± 0.25 0.99 ± 0.03 1.14 ± 0.02 1.33 ± 0.10
1567 1559 germacrene B 1.26 ± 0.07 - 1.90 ± 1.02 1.15 ± 0.15 1.52 ± 0.05 1.10 ± 0.27 1.08 ± 0.09 1.30 ± 0.11 1.25 ± 0.10
Oxygenated sesquiterpenes
1584 1577 spathulenol 4.50 ± 0.04 5.07 ± 0.09 3.42 ± 0.09 5.14 ± 0.05 4.82 ± 0.12 4.93 ± 0.32 3.57 ± 0.13 4.81 ± 0.21 4.57 ± 0.35
1590 1582 caryophyllene oxide 4.30 ± 0.06 3.96 ± 0.08 2.35 ± 1.39 4.29 ± 0.28 4.26 ± 0.05 4.03 ± 0.53 3.38 ± 0.20 4.57 ± 0.15 4.33 ± 0.28

210
1777 1767 14-oxi-α- muurolene 15.31 ± 0.06 21.23 ± 0.10 13.96 ± 0.32 13.59 ± 0.85 10.18 ± 0.02 12.99 ± 0.08 9.59 ± 0.15 14.46 ± 0.64 12.45 ± 0.79
1865 1863 cis-thujopsenic acid - 3.65 ± 0.03 6.09 ± 0.46 - - - - - -
Other classes
1500 1500 n-pentadecane 1.90 ± 0.01 - - 2.09 ± 0.22 2.98 ± 0.05 1.67 ± 0.79 1.54 ± 0.12 2.27 ± 0.09 2.12 ± 0.08
1699 1700 n-heptadecane 1.41 ± 0.03 1.67 ± 0.04 - 1.32 ± 0.28 1.46 ± 0.05 1.29 ± 0.40 0.77 ± 0.03 1.60 ± 0.09 1.24 ± 0.02
2817 2818 squalene 4.52 ± 0.20 4.99 ± 0.12 5.35 ± 0.09 4.22 ± 0.02 9.15 ± 0.78 4.54 ± 1.07 2.94 ± 0.08 4.25 ± 0.35 2.93 ± 0.18
2093 2095 methyl linoleate - - - - - - 1.56 ± 0.05 1.72 ± 0.20 1.09 ± 0.52
2143 2132 linoleic acid 2.48 ± 0.04 2.78 ± 0.34 3.10 ± 0.25 5.20 ± 0.35 1.24 ± 1.24 9.97 ± 0.53 4.04 ± 0.11 4.28 ± 0.51 2.25 ± 0.61
2552 piperovatine 0.67 ± 0.17 2.45 ± 0.12 3.27 ± 0.17 1.24 ± 0.27 0.69 ± 0.14 1.72 ± 0.82 3.15 ± 0.01 1.31 ± 0.38 2.75 ± 0.03
2664 pipercallosidine 7.91 ± 0.60 20.75 ± 0.22 19.74 ± 1.37 12.72 ± 2.35 0.94 ± 0.19 15.55 ± 6.00 22.06 ± 0.01 11.94 ± 2.18 19.62 ± 0.02
3142 3149 α-tocopherol 2.69 ± 0.23 5.21 ± 0.33 5.79 ± 0.04 2.92 ± 0.51 1.67 ± 0.15 3.82 ± 1.71 3.93 ± 0.11 2.43 ± 0.47 3.34 ± 0.48
Not identified compounds
2205 NI [MS: 43 (100), 67 (94), 55 (90), 41 (79), 79 5.16 ± 0.09 4.66 ± 0.18 4.94 ± 0.28 - - - 3.44 ± 0.49 - -
(62), M+. Abs.]
2594 NI [MS: 173 (100), 41 (88), 96 (82), 152 (79), 0.79 ± 0.09 - - - - - - - -
M+. Abs.]
2900 NI [MS: 43 (100), 57 (91), 71 (59), 41 (25), 85 0.59 ± 0.02 - - - - - - - -
(23), M+. Abs.]
3100 NI [MS: 57 (100), 43 (87), 71 (58), 41 (27), 85 1.12 ± 0.00 - - 1.20 ± 0.10 2.41 ± 0.17 - - - -
(27), M+. Abs.]
Total identified compounds 90.44 95.37 94.11 98.85 97.64 98.56 93.68 100.00 100.00

RRIexp.,experimental retetion index; RRIlit.,literature retention index; (%), average relative area (double) ± standard deviation; NI, not identified; ; MS, Mass spectra.
The Journal of Supercritical Fluids 147 (2019) 205–212
R.N. Lima et al.
Fig. 6. Comparison of total ion chromatograms obtained using 5% co-solvents in SC-CO2 extractions.
Fig. 7. Comparison of terpene classes identified by GC-MS of extracts obtained
using SC-CO2 and co-solvents addition.
high levels of non-oxygenated sesquiterpenes such as 44.66% at 313 K,
220 bar and 44.00% at 313 K, 200 bar. Other classes (e.g. pipercallo-
sidine, piperovatine, squalene, linnoleic acid, α-tocopherol) represent a
second group extracted using SC-CO2, with 39.67% at 333 K, 220 bar
and 35.20% at 353 K, 200 bar. Oxygenated monoterpenes and non-
oxygenated monoterpenes were not found in these analyses (Fig. 5).
The literature relative retention indexes (RRIlit.) for the two uni-
dentified compounds with the experimental relative retention indexes
(RRIexp.) of 2552 and 2663 were not found in the database [20].
However, their mass spectra were compared in NIST MS Search 2.0 and
it was determined that these substances were likely piperovatine
(C17H23NO2) and pipercallosidine (C18H25NO3), both cited for their
anesthetic effects in previous articles [21,22]. Their molecular ions
were M+• 273 and M+• 303, with bases peaks at m/z 173 and m/z 135
respectively. The spectra of these amides were consistent with those
described in the literature for other species from Piper genus or Piper-
aceae family: P. callosum [23], P. piscatorum [17], Ottonia martiana
[24], and Ottonia corcovadensis [25]. McFerren and Rodriguez [22] also
reported the presence of piperovatine and pipercallosidine in their
study of P. piscatorum and the native species of the Venezuelan Amazon,
as well as in their studies with P. ovatum [26] and P. klotzschianum [7].
For the mass spectrum of piperovatine, the proposed mechanism of
fragmentation is presented in Scheme 1. With nitrogen as the initial site
of ionization, the alpha cleavage of the amide group formed m/z 201
and losing the CO gave a base peak at m/z 173. Methyl radical loss
formed a fragment at m/z 158 and a cleavage of the beta bond to the
aromatic ring formed at m/z 121. Considering the initial ionization site,
the double bond m/z 152 fragment was formed followed by the in-
ductive cleavage that gave m/z 57.
For the mass spectrum of pipercallosidine, the cleavage of the beta
bond of the aromatic ring originated the base peak at m/z 135. The
fragment at m/z 203 was formed by inductive cleavage of the carbonyl
to form the fragment at m/z 68. The fragment at m/z 168 is formed on
211
The Journal of Supercritical Fluids 147 (2019) 205–212
the assumption that ionization removes an electron from the aromatic
ring, which cleaves inductively to restore ring stability (Scheme 2).
3.4. Analysis of compounds obtained by SC-CO2 using co-solvents
The addition of a co-solvent increased the mass percent yield of the
extracts. However, some conditions were distinguished from others in
terms of chemical composition or selectivity (Table 5).
(1) 353 K, 180 bar, ethanol 3% presented 14-oxy-α-muuroleno
(21.23%) and pipercallosidine (20.75%) in higher percentages, but
bicyclogermagrene (8.01%) and germacrene D (9.75%) in lower per-
centages compared the other conditions.
(2) 313 K, 200 bar, ethanol 5% and 353 K, 180 bar, ethanol 3%
enabled obtaining cis-thujopsenic acid (6.09% and 3.65% respectively).
(3) 313 K, 220 bar, isopropanol 3% resulted in the largest percen-
tage of germacrene-D (25.00%), bicyclogermacrene (15.41%), (E)-car-
yophyllene (11.66%), squalene (9.15%), but pipercallosidine was only
0.94%.
(4) 333 K, 180 bar, isopropanol 5% generated the highest percen-
tage of linoleic acid (9.97%).
Despites the selectivity differences observed using co-solvents,
Fig. 6 shows that the chromatographic profile of SC-CO2 extracts ob-
tained using methanol, ethanol and isopropanol as co-solvents were
similar to the ones obtained with the others conditions presented in
Table 5.
Independent of singularities observed in the conditions above, ger-
macrene D (25.00%, 313 K, 220 bar, isopropanol 3%), pipercallosidine
(22.06%, 313 K, 180 bar, methanol 1%), 14-oxy-muuroleno-α (21.23%,
353 K, 180 bar, ethanol 3%), bicyclogermacrene (15.41%, 313 K,
220 bar, isopropanol 3%), and (E)-caryophyllene (11.66%, 313 K,
220 bar, isopropanol 3%) were the main constituents present in all
extracts.
Fig. 7 shows the similarities in the profiles described in Fig. 5. High
levels of non-oxygenated sesquiterpenes were observed in the extract
obtained at 313 K, 220 bar, and isopropanol 3% (60.25%) and this ef-
fect was observed at most conditions tested. For other classes, it was
superior to the non-oxygenated sesquiterpenes in three of the nine
conditions tested; at 313 K, 200 bar, and 5% ethanol, they represented
43.34% of the extract content.
4. Conclusions
This work reports the use of supercritical carbon dioxide for the
extraction of valuable chemical compounds from dry leaves of Piper
klotzschianum. The results show that addition of organic co-solvents
increased the extraction yield by 40 % compared to extraction using
carbon dioxide alone. The sesquiterpenes were the most representative
compound class in the extracts obtained by SFE. Chemical analysis of
the extracts presented germacrene D, pipercallosidine, 14-oxy-α-
muurolene, bicyclogermacrene and (E)-caryophyllene as the main
compounds present in the extracts varying temperature and pressure
and applying different co-solvents and co-solvents percentage.
The increased yield of some compounds when using co-solvent was
R.N. Lima et al.
related to a slight decrease in the percentage of hydrocarbons extracted,
for example, n-pentadecane, n-heptadecane, methyl linoleate, linoleic
acid, and squalene. This change was related to increased polarity
caused by co-solvent addition at supercritical CO2.
The extraction of pipercallosidine and piperovatine as well as ger-
macrene D represent a great biological potential of SC-CO2 P. klotz-
schianum extract, since these compounds are well known for presenting
different biological activities (larvicidal, anti-cancer, anti-in-
flammatory, antinociceptive, among others). The extraction of these
compounds could still be improved in future studies to increase the
potential of P. klotzschianum extract as phytotherapeutic and anesthesic
products.
Acknowledgments
The authors thank the Federal University of Sergipe, Capes for fi-
nancial support and Project PROMOB-Fapitec/Capes.
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