Food Chemistry 173 (2015) 1203–1206

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Food Chemistry
journal homepage: www.elsevier.com/locate/foodchem

Short communication

Comparison of the physico-chemical and phytochemical characteristics

of the oil of two Plukenetia species
Rosana Chirinos a, Romina Pedreschi b, Gilberto Domínguez a, David Campos a,⇑
a
Instituto de Biotecnología (IBT), Universidad Nacional Agraria La Molina – UNALM, Av. La Molina s/n, Lima, Peru
b
Pontificia Universidad Católica de Valparaíso, School of Agronomy, Calle San Francisco s/n, La Palma, Quillota, Casilla 4-D, Chile

a r t i c l e i n f o a b s t r a c t

Article history: A physico-chemical and phytochemical characterisation of the oil of two rich sources of polyunsaturated
Received 23 December 2013 fatty acids, tocopherols and phytosterols is presented for two close species of Plukenetia, endemic to the
Received in revised form 6 October 2014 Amazon Region of Peru. Plukenetia huayllabambana presented approximately 9% more oil yield than Pluk-
Accepted 21 October 2014
enetia volubilis. Fatty acid profiles were pretty similar for both species but P. huayllabambana presented a
Available online 29 October 2014
significantly higher content of a-linolenic acid than P. volubilis (51.3 and 45.6 g/100 g oil, respectively).
Important contents of c- and d-tocopherol were evidenced in both oils (127.6 and 84.0 and, 93.3 and
Keywords:
47.5 mg/100 g oil, for P. volubilis and P. huayllabambana, respectively). b-Sitosterol was the most impor-
Plukenetia volubilis
Plukenetia huayllabambana
tant and representative phytosterol in both oils (127 mg/100 g oil). The results of this study indicate P.
Fatty acids huayllabambana as an important dietary source of health promoting phytochemicals.
Tocopherols Ó 2014 Elsevier Ltd. All rights reserved.
Phytosterols

1. Introduction endemic to rocky patches in the cloud forest province of Rodriguez

The pantropical genus Plukenetia L. (Euphorbiaceae) is com-
posed of 16 species of lianas and scrambling vines (Bussman,
Téllez, & Glenn, 2009). Eleven out of the 16 occur in the Neotropics
and the majority occurs in humid tropical forests at altitudes up to
around 1000 m. One species P. volubilis Linneo, also known as
sacha inchi, is an oleaginous plant that grows in the lowlands of
the Peruvian Amazon at altitudes between 200 and 1500 m and
has been cultivated for centuries by the indigenous population
(Guillén, Ruiz, Cabo, Chirinos, & Pascual, 2003; Hamaker et al.,
1992). Sacha inchi seeds (P. volubilis L.) are considered to be a rich
source of proteins and oil. The oil is characterized by its high
content of polyunstaturated fatty acids mainly of a-linolenic
(C18:3) and linoleic (C18:2), representing 82% of the total oil
content (Fanali et al., 2011; Guillén et al., 2003; Hamaker et al.,
1992). Higher tocopherol content compared to other oleaginous
seeds (e.g., peanut, palm, soybean, corn, sunflower) has been
reported in sacha inchi oil. Important amounts of phytosterols
have also been reported in sacha inchi oil (Bondioli, Della Bella, &
Rettke, 2006).
A new species of Plukenetia from the Peruvian Region of
Amazonas, Plukenetia huayllabambana sp. nov. has been recently
described (Bussman et al., 2009). This new species seems to be
⇑ Corresponding author. Tel./fax: +51 (1) 3495764.
E-mail address: dcampos@lamolina.edu.pe (D. Campos).
de Mendoza. It has only been found in the upper Amazon region, at
altitudes above 1200 m. It is similar to P. volubilis and P. stipellata
L.J. Gillespie but distinctly differs in its small number of stamens,
stylar column length, and very large fruits and seeds of 3–4  4–
6 cm and with pronounced ridges (Bussman et al., 2009). Not only
morphological but also molecular differences have been reported
between P. volubilis and P. huayllabambana by using inter simple
sequence repeats (Rodriguez et al., 2010). Correspondance factorial
analysis supported by fixation index, genetic distance and a
dendogram showed strong differentiation between both species
(Rodriguez et al., 2010). The morphological differences between
P. volubilis and P. huayllabambana might also imply differences in
the phytochemical composition. Thus, the main aims of this work
were: (i) to determine the physico-chemical characteristics and
phytochemical composition of the non-polar fraction: fatty acids,
tocopherols and phytosterols of P. huayllabambana oil and (ii) to
compare this characterisation of the oil with its close species P. vol-
ubilis. The results of this work provide relevant information con-
cerning the phytochemical characterisation of a new rich source
of polyunsaturated fatty acids and other non-polar compounds
for P. huayllabambana. These results aim to promote the consump-
tion of this new species in local and international markets as an
alternative and promising source of unsaturated fatty acids, phy-
tosterols and tocopherols as to improve the income and well-being
of the poor Amazonian communities where this species grows. This
new species presents desirable morphological (bigger seed size),

http://dx.doi.org/10.1016/j.foodchem.2014.10.120
0308-8146/Ó 2014 Elsevier Ltd. All rights reserved.
1204 R. Chirinos et al. / Food Chemistry 173 (2015) 1203–1206
higher seed yields and desirable phytochemical characteristics in
order to be sustainable exploited as an alternative source of
income for the native population.
2. Materials and methods
2.1. Sample material and chemicals
Seeds of Plukenetia volubilis and Plukenetia huayllabambana
were sourced from Province of Lamas (Region of San Martín) and
from Province of Rodríguez de Mendoza (Region of Amazonas)
from Peru, respectively. Seeds (5 kg of each species) were manually
cleaned and selected. The skin was removed and the almond was
further grinded using a coffee mill (1 mm). Almonds were sub-
mitted to a 6 h extraction with petroleum-ether using a Soxhlet
equipment. After the extraction, the almond extract was filtered
and the filtrate was rapidly concentrated under nitrogen flow in
a 30 °C water bath and then the oil was kept at 20 °C until
analysis.
Fatty acid methyl ester standards were provided by Restek
(Bellefonte, PA, USA). Tocopherol standards (a-, ß-, c- and d-
tocopherol) and phytosterol standards (b-cholestanol, b-sitosterol,
campesterol and stigmasterol) were purchased from ChromaDex™
(Santa Clara, CA). All solvents and other chemicals of analytical
grade were purchased from Merck (Darmstadt, Germany) and
Fischer Scientific (Fair Lawn, NJ, USA).
2.2. Quantitative analysis
2.2.1. Physico-chemical characteristics
The acid value, free fatty acid (FFA) and peroxide value (PV) were
determined by standard procedures (AOAC (Association of Official
Analytical Chemists), 1995). The p-anisidine (PA) value, conjugated
dienes (CD) and conjugated trienes (CT) were determined according
to the recommended methods of IUPAC (IUPAC, 1987).
2.2.2. Fatty acid content and composition
The FA composition was determined by gas chromatography
according to the method proposed by Meurens, Baeten, Yan,
Mignolet, and Larondelle (2005) with slight modifications. The
FAs of the oil samples were converted into methyl esters (FAMEs).
FAMEs were separated by injecting 1 ll of the solution into a
GC-2010 plus Shimadzu (Kyoto, Japan) equipped with a flame
ionisation detector FID-2010 and an autoinjector AOC-20i. The
column used was a Restek Rt-2560 (Bellefonte, PA) (0.2 lm,
100 m  0.25 mm ID). The oven temperature was programmed as
follows: initially at 100 °C (for 4 min), increased to 240 °C at 3 °C/
min and an isothermal period of 25 min at 240 °C. The injector
and detector temperatures were set at 225 and 245 °C, respec-
tively. High purity helium was used as carrier gas. FAMEs were
identified and quantified by comparing their retention times to
known previously injected standards. Results were expressed as
g of fatty acid per 100 g of oil.
2.2.3. Tocopherol content and composition
The samples were prepared following the methodology
reported by Amaral, Alves, Seabra, and Oliveira (2005) with slight
modifications. Briefly, 300 mg of sacha inchi (SI) sample and
100 ll of butylated hydroxytoluene (BHT) (10 mg in 1 ml of n-hex-
ane) were homogenised for 1 min by vortex mixing, after the addi-
tion of each of the following reagents: ethanol (2 ml), extracting
solvent (n-hexane, 4 ml), and saturated NaCl solution (2 ml). Then,
the mixture was centrifuged at 4000g for 4 min at 1 °C and then
the clear upper layer was recovered. The sample was re-extracted
twice using 2 ml of n-hexane. The combined extracts were taken to
dryness under nitrogen, and the residue was reconstituted in a vol-
ume of 1.5 ml with n-hexane. The extract was dried with anhy-
drous sodium sulphate (0.5 g), centrifuged at 4000g for 20 min at
1 °C and transferred to a dark vial for the subsequent HPLC
analysis.
Samples were separated using a normal phase HPLC column on
a Waters 2695 Separation Module (Waters, Milford, MA) equipped
with a Waters 2475 multifluorescence detector and the Empower
software. A YMC-Pack Silica Col (3 lm, 250  4.6 mm column
(Kyoto, Japan) and a 4.0  2.0 mm guard column were used for
tocopherol separation at 35 °C. The mobile phase was composed
of n-hexane/2-propanol/acetic acid (1000/6/5, v/v/v). A solvent
flow rate of 1.4 ml/min under isocratic conditions was used. Ten
ll of sample were injected. The fluorescence detector was pro-
grammed at the excitation and emission wavelengths of 290 and
330 nm, respectively. Tocopherols were identified and quantified
by comparing their retention time to known previously injected
standards. Results were expressed as mg per 100 g of oil.
2.2.4. Phytosterol content and composition
Samples were prepared using the methodology reported by
Duchateau et al. (2002). Briefly, 100 mg of SI oil was saponified
with ethanolic KOH solution (1 ml) at 70 °C for 50 min. The inter-
nal standard (1 ml of b-cholestanol, 10 mg/l in n-heptane) was
added to each sample. The unsaponifiable fraction was extracted
using liquid–liquid partitioning into 1 ml of distilled water and
5 ml of n-heptane. The organic phase was transferred to a test tube
containing Na2SO4, and the extraction was repeated two times
with 5 and 4 ml of n-heptane, respectively. The n-heptane extracts
were combined and homogenised before injection into a gas chro-
matography system.
The phytosterol composition was determined by GC. Phytoster-
ols were separated by injecting 2 ll of the extract to a GC-2010
plus Shimadzu (Kyoto, Japan) equipped with a flame ionisation
detector FID-2010. The column used was a Supelco SAC TM–5
(St. Louis, MO, USA) (0.2 lm, 30 m  0.25 mm ID). The oven tem-
perature was programmed as follows: initially at 250 °C (for
2 min), increased to 285 °C at 25 °C/min, an isothermal period of
285 °C for 32 min and a split ratio of 10. The injector and detector
temperatures were set at 300 °C. Helium was used as carrier gas.
Phytosterols were identified and quantified by comparing their
retention times to known previously injected standards. Results
were expressed as mg per 100 g of oil.
2.3. Statistical analysis
All the analyses were carried out in triplicate and the results
were expressed as mean values ± S.D. A two independent sample
t-test was used to account for significant differences (p < 0.05)
between both species for all the analysis. All statistical analysis
were carried out in IBM SPSS Statistics 20 (New York, USA).
3. Results and discussion
The physico-chemical characterisation of P. volubilis and P.
huayllabambana oils is reported in Table 1. Higher oil yields (9%
more) were obtained for P. huayllabambana than for P. volubilis.
Thus, P. huayllabambana seeds represent an important oil source.
P. huayllabambana oil yields are higher than those reported for
other seeds such as soybean (19%), cotton (16%) and safflower
(35.8%) and close to those reported for peanut (45%), canola
(48%) and flaxseed (33.6–44.8%) (Bodwell & Hopkins, 1985;
Carvalho, Miranda, & Pereira, 2006).
Acid and FFA values for both species were low. Carvalho et al.
(2006) reported an acceptable maximum value of acidity for oil
 R. Chirinos et al. / Food Chemistry 173 (2015) 1203–1206 1205

Table 1 (x-3) > linoleic acid (x-6) > oleic acid. No differences were
Physico-chemical characterisation of Plukenetia volubilis and Plukenetia huayllabam- observed in the content of palmitic, stearic and oleic acids in both
bana oils.
oils. Both oils are a rich source of polyunsaturated fatty acids
Characteristic Plukenetia Plukenetia (PUFA). P. huayllabambana presented a higher content of a-linole-
volubilis huayllabambana nic acid (51.3%) but lower content of linoleic acid (26.6%) com-
Yield (%, w/w)a 35.4 ± 1.2 44.1 ± 4.8 pared to P. volubilis. However, total PUFA in both species was
Acid value (mg/g)a 0.37 ± 0.01 0.31 ± 0.03 very similar 78%. Similar quantities of a-linolenic and linoleic
Free fatty acids (as oleic acid, %)a 0.19 ± 0.0 0.15 ± 0.02
Peroxide value (meq. O2/kg)a 2.90 ± 0.2 1.83 ± 0.04
FAs as well as total PUFA for P. volubilis and P. huayllabambana
p-anisidine value 0.29 ± 0.04 0.21 ± 0.02 has been reported previously (Fanali et al., 2011; Hamaker et al.,
Conjugated dienes 1.22 ± 0.11 1.07 ± 0.03 1992; Maurer, Hatta-Sakoda, Pascual-Chagman, & Rodriguez-
Conjugated trienes ND ND Saona, 2012; Muñoz et al., 2013; Ruiz, Díaz, Anaya, & Rojas,
ND: not detected. 2013). The content of x-3 in P. huayllabambana (51%) is close
a
Stand for significant differences assessed by a two independent sample t-test to the values reported for flawseed oil 52%, but flawseed oil pre-
(p < 0.05). sents a lower content of x-6 and PUFA (16.6 and 52.4%, respec-
tively) (Michotte et al., 2011). The significant contents of x-3, x-6
and PUFA found in P. huayllabambana offer important health and
used as food product of 0.6% (of oleic acid). FFA for both Plukenetia nutritional benefits, such as providing protection against cardio-
oils were far below the limit with values corresponding to 0.15% vascular disease (Guillén et al., 2003). The x-6/x-3 ratio for P.
and 0.19%, respectively. The oxidative state in both oils was huayllabambana and P. volubilis oils were 0.52 and 0.72, respec-
assessed through PV, p-anisidine (PA), CD and CT values. PV is tively. These ratios were much lower than the values reported
indicative of the oxidation status of fats or oils mainly as evidence for other important food oils such as canola (1.8–2.2), olive
of primary oxidation. The PV value for P. huayllabambana was (7.69), soybean (6.66), sunflower (8.5) and safflower (78.6)
lower than the value obtained for P. volubilis (1.83 and 2.9 meq oils (Belitz & Grosch, 1999; Carvalho et al., 2006).
O2/kg oil, respectively). A limit of 10 meq O2/kg oil for refined edi- Gamma (c)- and d-tocopherols were the most predominant toc-
ble oil is recommended by the Codex Alimentarius; thus both eval- opherols in both oils. P. volubilis oil presented 36 and 76% more
uated oils were far below the maximum recommended limits. PA is c- and d-tocopherols, respectively than P. huayllabambana. High
used to detect secondary oxidation products produced from pri- values of c- and d-tocopherols in sacha inchi oil have been reported
mary oxidation (Choo, Birch, & Dufour, 2007). CD and CT are good by Follegati-Romero, Piantino, Romero-Grimaldi, and Cabral (2009)
indicators of the oxidant state of oils (Yoon, Kim, Shin, & Kim, and Fanali et al. (2011) (1.14 and 1.25 g/kg oil and 1.25 and 0.86 g/
1985). PA and CD values for both species were extremely low kg, respectively). Chirinos et al. (2013) also reported higher quan-
and close to each other with values of 0.21 and 0.29 for PA and tities of c- and d-tocopherols than a- and b-tocopherols in the
1.07 and 1.22 for CD, respectively. No CTs were detected in both seeds of 16 cultivars of sacha inchi. In flaxseed oil, a rich source
species. The low values of acidity (%) together with the low values of tocopherols as well as in x-3 and -6, c-tocopherol (main tocoph-
of PV, PA and CD are indicative of non-detrimental oxidation reac- erol present in this oil) reached values of 75.6 mg/kg oil (Bozan &
tions in the oils during picking, grinding and extraction processes. Tenelli, 2008), lower than the values found in the oil of both
Fatty acid (FA), tocopherol and phytosterol profiles and evaluated species. Tocopherols are recognised as potent lipophilic
contents for P. volubilis and P. huayllabambana oils are displayed antioxidant compounds. The antioxidant activity of tocopherols
in Table 2 and in Supplementary material (Figs. 1, 2 and 3 in lipids follows this order: c > d > a > b (Schmidt & Pokorný,
respectively). Six different FA were found in both oils. In order of 2005). Thus, the significant quantities of c- and d-tocopherols
abundance, the most important ones were: a-linolenic acid found in both Plukenetia oils constitute an antioxidant protection
mechanism for these oils rich in PUFAs, if proper extraction tech-
Table 2 niques are used as to maximise tocopherol recovery.
Phytochemical characterisation: fatty acids, tocopherols and phytosterol composition Three phytosterols were studied in these two Plukenetia
of Plukenetia volubilis and Plukenetia huayllabambana oils. sources: campesterol, stigmasterol and b-sitosterol. Significant dif-
Characteristic Plukenetia Plukenetia
ferences (p < 0.05) were found in the content of two of these three
volubilis huayllabambana phytosterols for both species. b-sitosterol, stigmasterol and cam-
Fatty acids (%)
pesterol have been reported to represent 92% of total phytosterols
Palmitic (C16:0) 6.30 ± 0.16 6.61 ± 0.45 in sacha inchi oil (Bondioli et al., 2006), with values very close to
Stearic (C18:0) 3.81 ± 0.02 3.75 ± 0.04 the values found in this study (138.9; 68.9 and 17.5 mg/100 g oil,
Oleic (C18:1 x-9) 9.47 ± 0.08 9.38 ± 0.51 respectively). Phytosterols are important for human health due
Linoleic (C18:2 x-6)a 32.66 ± 0.15 26.67 ± 0.24
to their potential to decrease the levels of human serum choles-
a-Linolenic (C18:3 x-3)a 45.62 ± 0.29 51.34 ± 0.34
Polyinsaturated fatty acids 78.15 ± 0.44 78.01 ± 0.53 terol, thus being attractive sources for the development of enriched
(PUFA) foods with these plant sterols (Lagarda, García-Llatas, & Farré
x6/x3 ratioa 0.72 ± 0.00 0.52 ± 0.00 2006; Schröder & Vetter, 2012). Higher levels of b-sitosterol than
Tocopherols (mg/100 g oil) in both Plukenetia oils have been reported in vegetable oils such
a-Tocopherola 0.08 ± 0.0 0.20 ± 0.02 as corn, oat and linseed (586, 411 and 226 mg/100 g) (Normén,
ß-Tocopherol 0.02 ± 0.0 0.01 ± 0.0 Ellegård, Brants, Dutta, & Andersson, 2007); but lower amounts
c-Tocopherola 127.6 ± 8.8 93.3 ± 7.8
d-Tocopherola 84.0 ± 5.3 47.5 ± 3.1
have been reported in olive, palm, soy and sunflower oils (141,
Total Tocopherola 211.8 ± 14.2 141.0 ± 4.8 24, 56–59 and 47 mg/100 g, respectively) (Normén et al., 2007).
Phytosterols (mg/100 g oil)
Both evaluated Plukenetia oils are important sources of
Campesterola 15.3 ± 0.8 11.6 ± 1.3 phytosterols.
Stigmasterola 58.7 ± 1.4 52.1 ± 3.8 Finally, not only the genetic characteristics of P. huayllabambana
b-Sitosterol 127.4 ± 9.3 127.2 ± 10.6 might have influenced the reported results in comparison to P. vol-
a
Stand for significant differences assessed by a two independent sample t-test ubilis, but also the source of origin, growing and environmental
(p < 0.05). conditions (e.g., climate, harvest time and agricultural practices)
1206 R. Chirinos et al. / Food Chemistry 173 (2015) 1203–1206
as well as processing steps prior to oil extraction (e.g. grinding,
roasting) and the method of oil extraction.
4. Conclusions
P. huayllabambana is a rich source of oil, containing 9% more oil
and a-linolenic acid than P. volubilis, but lower tocopherol content.
Important and similar quantities of phytosterols were present in
both oils. Oxidative stability analysis was indicative of non-detri-
mental effects due to oxidative processes during the steps prior to
oil extraction. The commercial value of P. volubilis is not only due
to food, cultural and historical aspects but in its profitability and
huge possibilities of industrialisation. The same characteristics are
foreseen for P. huayllabambana based on the results of this research.
Acknowledgements
The authors thank Rosa Olivera and Daniella Zorrilla for their
technical assistance. This research was supported by CUI project
of the Belgian Coopération Universitaire au Développement (CUD,
Belgium).
Appendix A. Supplementary material
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.foodchem.2014.
10.120.
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